JPH08504824A - Prevention and treatment of sepsis - Google Patents

Abstract

  (57) [Summary] Disclosed are compositions and methods for preventing and treating sepsis in humans and other animals. Surgical patients, infants of low birth weight, victims of burns or trauma, and other individuals at risk can be treated prophylactically. Provided is a method for treating an acute infectious disease, which has many advantages over conventional treatment methods.

Description

Detailed Description of the Invention                       Prevention and treatment of sepsisField of the invention   The present invention provides for the prevention and treatment of blood-borne toxin-mediated diseases, In particular, it relates to methods for preventing and treating sepsis in humans and other animals.Background of the Invention I. Sepsis   Sepsis is the leading cause of morbidity and mortality in humans and other animals. Rice in 1991 In the country alone, 400,000 to 500,000 cases of sepsis killed 100,000 to 175,000. Estimated. Among patients with non-traumatic disease, sepsis is associated with intensive care units. It is the main cause of death [G. W. Machiedo et al., Surg. Gyn. & Obstet. 152: 75 7-759 (1981)]. Sepsis is also the leading cause of death in young livestock and 7.5-29% of live calves are affected [D. D. Morris et al., Am. J. Vet. Res. 47: 2554 -2565 (1986)], it has become a common medical problem in newborn horses [A. M. Hoffm an et al. Vet. Int. Med. 6: 89-95 (1992)]. Overtreatment in the treatment of serious infections Despite significant advances over the last decades, sepsis incidence and mortality rates have increased. Continues [S. M. Wolff, New Eng. J. Med. 324: 486-488 (1991)].   Sepsis is hypotension, metabolic acidosis, decreased systemic vascular resistance, A systemic reaction characterized by tachypnea and organ dysfunction, including bacteremia That is, sepsis such as the presence of bacteria in blood (ie, organisms in the bloodstream, The presence of its metabolic end products or toxins) and endotoxemia (ie, Toxemia such as the presence of endotoxin in the blood (ie, the presence of toxin in the blood) Can occur). The term "bacteremia" is the absence of an infected lesion Includes latent bacteremia observed in children with fever. Also, the term "sepsis" , Fungalemia (ie the presence of fungi in the blood), viremia (ie The presence of virus or virus particles in the blood) and parasitemia ( That is, the presence of parasites or protozoa in blood). Therefore, Sepsis and septic shock (septic often with multiple organ failure and high mortality Acute circulatory failure resulting from the disease is caused by many organisms.   Systemic microbial invasion poses two distinct problems. First, the growth of microorganisms The substance may cause direct damage to the functions of tissues, organs and blood vessels. Second, the microbial Toxic components can lead to a rapid systemic inflammatory response, which is immediately vital. Circulatory collapse (ie, septic shock) that damages various vital organs Often leads to death.   Sepsis is mainly classified into three types depending on the type of infectious organism. Gram-negative bacteria Sexual sepsis is the most common and has a mortality rate of approximately 35%. Most of these infections Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Caused by a fungus (Pseudomonas aeruginosa). Like Staphylococcus or Streptococcus Gu Lamb-positive pathogens are the second leading cause of sepsis. The third major group is fungi Including, this fungal infection accounts for a relatively small proportion of sepsis, but the mortality rate is high.   Many of these infections are acquired in the hospital and some types of surgery (eg, peritoneal surgery) , Immunosuppression by cancer therapy or transplantation therapy, immunodeficiency and intravenous catheter Can be caused by contact with. Sepsis also includes trauma, neonatal dystocia and volvulus ( Intestinal torsion in dogs and horses in particular) may also occur.   The established mechanism of sepsis is related to the toxic component of Gram-negative bacteria. G A common cell wall known as lipopolysaccharide (LPS) that is widely shared by negative bacteria Structure exists. The "endotoxin" produced by Gram-negative bacteria has three main structures: It is believed to be involved in lipoproteins and most of the biological properties of endotoxins. Obtained lipid (lipid A) and polysaccharide structure specific to each species and strain of bacteria Made of [D. C. Morrison, Rev. Infect. Dis. 5 (Supp 4): S733-S747 (1983)]. Studies over the last decade have shown that purified endotoxin is completely gram-negative It has been demonstrated that it can induce all features of the disease. In addition, host response to endotoxin Some of the answers were confirmed. Tumors are two key mediators of septic shock Necrosis factor (TNF) and interleukin-1 (IL-1), these substances Is released by macrophages and causes physiological changes leading to circulatory collapse and organ failure. It appears to act synergistically in triggering the cascade of activation [R. C. Bone, An n. Intern. Med. 115: 457-469 (1991)]. In fact, high doses of TNF [K. J. Trace y et al., Science 234: 470-474 (1986)] and / or IL. -1 [A. Tewari et al., Lancet 336: 712-714 (1990)] show symptoms and consequences of sepsis. Can be imitated.   The distinct cell wall material of Gram-positive bacteria and fungi is likely to trigger similar phenomena. It is generally believed that the structures involved are not as well studied as endotoxins of Gram-negative bacteria. Not investigated.   24 patients with or suspected sepsis, regardless of etiological agent, Shows a sudden weakness within 48 hours. Thus, for effective treatment of the patient Prompt diagnosis and therapeutic dosing regimens are essential. Unfortunately, the type of infection Established diagnostic methods for inoculation are blood culture inoculation, incubating for 18-24 hours. Solution, inoculation of causative microorganisms on solid medium, further incubation period , And traditionally require microbiological analysis with final confirmation after 1-2 days . Therefore, without knowing the type and species of the pathogen, and knowing the extent of infection, No wonder treatment must be started. II. Prevention and treatment   A. Antibiotics   Antibiotics of extremely diverse structure [Berdy, A to prevent and prevent infections] dvances in Applied Microbiology, (edited by D. Perlman), Academic Press, New  York, 18: 309-406 (1974)] is widely used. Nevertheless, the disease Up to half of patients who develop bacteremia in the hospital die (ie, in-hospital or physician). Primary bacteremia) [D. G. Maki, Am. J. Med. 70: 719-732 (1981)]. There are many reasons There are many. First, regarding many commonly used antibiotics, Antibiotic resistance has become common in various bacterial species. This is especially a hospital True to the microbiota present within, where the selection of resistance by the microorganism It is constantly exposed to pressure. In addition, transmission of antibiotic resistant bacteria within hospital facilities Due to the high density of potentially infected patients, staff-to-person contact and staff-to-patient contact It is promoted by the degree. Second, it is the most economical, safe, and easy to administer. Fecal antibiotics have a broad spectrum of antibacterial properties to control several infectious diseases I haven't got together. For example, many broad spectrum antibiotics are taken orally Cannot be administered to the patient and the doctor may Do not want to put the patient in the room. Third, antibiotics can cause allergies and other Adverse drug interactions or direct damage to major organs (eg kidney, liver) And can be toxic to varying degrees. Many potent antibiotics have therapeutic doses Removed from daily use as it can cause adverse reactions. Fourth, Many antibiotics alter the normal gut flora, often causing diarrhea and malabsorption cause. Some can cause life-threatening gastrointestinal (GI) tract infections Usuwami like Clostridium difficile It may even release and free the microbes. For example, Clostridium diff Antimicrobial-Associated Pseudomembranous Colitis Caused by Isil It is a serious complication associated with. Therefore, doctors are Of Preventive Use of Antibiotics on the Global Health Status and Economics of Health Care in Children You have to consider the sound.   Many infections are protected by antibiotics but some Gram-negative bacteremia Presents a special challenge for. Bacterial treatment with antibiotics disrupts bacterial cell walls Is known to catalyze the release of endotoxin from dead cells. In the rabbit Antibiotics reduce levels of bacteremia in experimental E. coli sepsis It nevertheless increases endotoxin levels by 10-2,000 fold [J. L. Shenep and K. A. Morgan, J. Infect. Dis. 150: 380-388 (1984)]. Therefore, once Once rum-negative bacteremia is established, antibiotic therapy increases symptoms but reduces infection There is a justifiable relationship to do.   Conveniently, certain antibiotics are known to neutralize the action of endotoxin. ing. Polymyxin antibiotics, especially polymyxin B and polymyxin E Also known as Listin) is Bacillus polymyxa ) Is a cyclic polypeptide compound produced by a certain strain. These antibiotics The substance binds to the lipid A portion of endotoxin [D. C. Morrison and D.D. M. Jacobs, Im munochem. 13: 813-818 (1976)], animal lethality test [D. Rifkind and J. D ． Palmer, J.M. Bacteriol. 92: 815-819 (1966)], activation of serum complement [D. C. Mor rison and D. M. Jacobs, Infect. Immun. 13: 298-301 (1976)], and the rim Ruth (Horse head crab) modified cell lysate (LAL) test [M. S. Cooperstock, Ant imicrob. Agents Chemother. 6: 422-425 (1974)], its activity Neutralize. Unfortunately, polymyxins are not absorbed through the GI tract and are parenteral Must be administered to. Recommended therapeutic dose for systemic infection with Pseudomonas aeruginosa (1-2.5 mg / kg body weight / day), there is a risk of renal dysfunction [Ph ysicians' Desk Reference, 47th Ed., pp. 818-819 (1993)]. This thing This is a major concern for patients with kidney disease in the United States. In addition to nephrotoxicity, God A toxic reaction was observed and was given immediately after the anesthetic and / or muscle relaxant Respiratory paralysis at that time is the most severe. Polymyxin B, in its intravenous form, It is only given to inpatients with constant control and monitoring of renal function. like this Thus, polymyxins are not used routinely for systemic infections ( However, they are very common as ingredients in topical ointments).   Several measures have been taken to reduce the toxicity of polymyxins. Colistin Shows relatively low systemic toxicity, and when methanesulfonate was formed, muscle The local pain experienced at the internal injection site disappears. Moreover, the toxicity of polymyxin B is high. It is reduced by binding to dextran which is a molecular weight carrier [D. A. Handle y, European Patent Application Publication No. 428486]. Binding to dextran reduces drug toxicity To lower and / or increase the circulating half-life of the drug Often used [P. E. Hallaway et al., Proc. Natl. Acad. Sci. USA 8 6: 10108-10112 (1989); J. Poznansky and L.L. G. Cleland, Drug Delivery Systems: Characteristics and Biomedical Applications, (edited by RL Juliano ), Oxford University Press, New York, pp. 253-315 (1980); Molteni, D rug Carriers in Biology and Medicine, (edited by G. Gregoriadis), Academic Pre ss, New York, pp. 107-125 (1979); C.I. Larsen, Adv. Drug Delivery Rev. 3: 103-154 (1989); D. Virnik et al., Russian Chem. Rev. 44: 588-602 (1975); Oh And Hager et al., French Patent No. 2,342,740 (1977)]. Polymyxin B alone Has a half-life of only a few hours [G. Brownlee et al., Brit. J. Pharmacol. 7 : 170-188 (1952)], but dextran (molecular weight 70,000) depends on the injected dose. Has a half-life of approximately 1 day in humans [Reynolds et al., Martindale-The Extra Pharmacop oeia, 28th Ed., The Pharmaceutical Press, London, pp. 512-513 (1982); Oh And W. A. Gibby et al., Invest. Radiol. 25: 164-172 (1990)].   Polymyxin B has been used in both animal models and human studies for the past 20 years. Researched as a specific therapy for Gram-negative sepsis or endotoxemia However, the result was confused. For example, dissemination induced by endotoxin Intravascular coagulation (DIC) administered polymyxin B 15 minutes after endotoxin challenge It was not prevented in the rabbits that were killed [J. J. Corrigan, Jr. And B. M. Bell, J. Lab. Clin. Med. 77: 802-810 (1971)]. In fact, most experimental studies To reduce or eliminate the effects of endotoxin, premixing endotoxin with polymyxin B , Or that polymyxin B should be administered prior to endotoxin challenge. I made it clear [D. Rifkind and J. D. Palmer, J.M. Bact. 92: 815-819 (1966); J ． J. Corrigan, Jr. And B. M. Bell, J. Lab. Clin. Med. 77: 802-810 (1971 ); B. Hughes et al., Br. J. Pharmac. 74: 701-707 (1981); J. Corrigan, Jr ． And J. F. Kiernat, Pediat. Res. 13: 48-51 (1979); G. Ziv and W. D. S chultze, Am. J. Vet. Res. 44: 1446-1450 (1982); and G. Baldwin et al. J. Infect. Dis. 164: 542-549 (1991)]. Some studies did not include pretreatment Well, almost no efficacy of polymyxin B was found [A. H. L. From et al, Inf ect. Immun. 23: 660-664 (1979)]. Significantly, endotoxin in human obstructive pox Clinical Studies on Blood Loss Reap No Benefits for Polymyxin B Therapy That is [C. J. Ingoldby et al., Am. J. Surgery 147: 766-771 (1984)], this This is consistent with the results of animal experiments [C. J. H. Ingoldby, Br. J. Surg. 67: 565-567 (1980)].   Low dose polymyxin B therapy has also been studied in animals and humans. To immature rats Infecting live bacteria Haemophilus influenzae type B Sub-inhibitory doses of polymyxin B administered 12 hours later were administered alone or in large amounts. In combination with picillin, mortality from infection was significantly reduced. Where the theory Is an endotoxin released from a microorganism whose polymyxin B has been killed by another antibiotic Will be neutralized [J. W. Walterspiel et al., Pediat. Res. 20: 237-241 (1986)]. The design of this experiment was such that live bacteria would challenge instead of free endotoxin. Note that it was different from the endotoxin challenge experiment in that it was the starting material Should. In humans, subtherapeutic doses of polymyxin B (10-50% of usual dose) Continuous infusion reduces levels of endotoxin, restores some immune function, and burns Seems to reduce wound infection in patients (ie, results are not statistically significant) Was found [A. M. Munster et al. Burn Care Rehab. 10: 327-330 (1989)].   B. Immunization   In addition to research and development of antibiotics, efforts to prevent bacterial infections are necessary. It focuses on the main defense, especially the role of the humoral immune system. Of active immunity against bacterial components The role and use of passive immunization with plasma or antibodies from the immunized donor This is a very controversial area. Specific antibodies from infection and toxin challenge There is ample experimental evidence that it can protect laboratory animals, but the body of this protection and The extent and relationship to in vivo infection is well documented on this subject. However, it is not clear [J. D. Baumgartner and M.M. P. Glauser, Rev. Infect. Dis. 9: 194-205 (1987); and E. J. Ziegler, J. Infect. Dis ． 158: 286-290 (1988)]. Disease progression and its prevention in critically ill patients A myriad of factors are involved that complicate the planning and interpretation of the clinical trials of G.   In Gram-negative bacteremia and endotoxemia, septic shock is associated with bacterial LPS It was found to be inversely related to the titer of antibodies that cross-react with covalent antigens [W. R. Mc Cabe et al., New Eng. J. Med. 287: 261-267 (1972)]. With this correlation, endotoxicity Means to raise the titer and / or from donor to experimental subjects and patients Endotoxin Expect a lot of efforts to develop means to passively transfer antibodies.   Antibodies to endotoxin have two important functions. First, free endotoxin By binding to, the antibody blocks endotoxin activity, that is, it circulates Remove from system. Second, the complement binding and binding to Fc receptors on phagocytes is Immunoglobulin effector function mediates bacterial death and opsonophagocytosis You can Thus, attacks of endotoxemia, bacteremia and sepsis Blocked by antibodies.   i) Active immunity   One approach to protecting animals and humans from endotoxin-mediated consequences is Immunization with bacteria or LPS. For example, lacking certain polysaccharide side chains A mutant Escherichia coli strain (J5) carrying a widely shared core lipid A structure Immunizing rabbits with Protected Animals from Challenge with Live Pseudomonas It turned out that [A. I. Braude et al., J. Infect. Dis. 136 (Supp): S167-S17 3 (1977)]. The J5 vaccine is Pseudomonas pneumoniae. umonia) model guinea pigs, but showed only weak protection, but a species-specific strain Pseudomonas LPS showed high protection [J. E. Pennington and E. Menkes, J ． Infect. Dis. 144: 599-603 (1981)]. These results show that the species-specific vaccine Better than cross-protective antigens for immunizing humans and other animals against endotoxin Suggests that. Unfortunately, the vast diversity of LPS antigens is species-specific It is blocking the possibility of a vaccine.   Active immunity to endotoxins is still being studied, but how many are in this approach? There are serious limitations. First, endotoxin is weakly immunogenic, effectively boosting the immune response. It only elicits a 3-5 fold increase in antibody titer to LPS without showing [E ． J. Ziegler et al., New. Eng. J. Med. 307: 1225-1230 (1982)]. Second, sepsis Many patients at risk of illness are immunocompromised and vaccinated Unable to provoke and / or maintain a sufficient protective response. And thirdly One or more core glycolipid antibodies The degree of cross-protection conferred by immunizing with primordial is well understood clinically. Absent.   ii) Passive immunity   To overcome some of the limitations inherent in active immunity, experimental animals Or to make an endotoxin-binding antibody that can be passively transferred to a human subject Various techniques have been used. The majority of endotoxin antibodies are (i) bacteria, LPS or Or immunization of animals or humans with its derivatives and immunization of serum or plasma Or (ii) monoclonal mouse or human antibodies by established methods And the recovery and purification of these antibodies.   The two major antibody types obtained by each method are IgM and Ig G antibody. These antibodies have only important aspects of their structure and effector function But not their antibody titers in normal and hyperimmune plasma. Much research IgM antibodies are more effective than Gram-negative bacteremia or endotoxin challenge in animals [W ． R. McCabe et al. Infect. Dis. 158: 291-300 (1988)] and humans [Id.; E. J. Ziegler et al., New. Eng. J. Med. 307: 1225-1230 (1982)] Therefore, it is suggested that it is more effective than the IgG antibody in terms of its binding activity. Only And a number of IgG preparations from immunized animal donors have been developed and used in experimental studies. It has been demonstrated to have some protective effects [D. L. Dunn et al., Surgery 96: 440-446 (1984) and S.H. J. Spier et al., Circulatory Shock 28: 235-248 (198 9)]. The advantage of IgG formulations is that IgG antibody titers increase in response to repeated immunizations This is In contrast, the IgM antibody titer is relatively constant. However, how is the immunization process? The total amount of bacterial-reactive or endotoxin-reactive antibodies in hyperimmune plasma or serum Is a small fraction of the total antibody and is highly variable from donor to donor.   In order to develop a therapeutically consistent antibody formulation, shared epitopes and specific Numerous LPS-reactive monoclonal antibodies have been made to both pitopes . Gram-negative sepsis is caused by many species, so a potential treatment Antibodies that cross-react widely have been emphasized as drugs. Two IgM Monocloners Antibody was considered the study of choice. Human origin now known as Centoxin HA-lA Antibody [N. N. H. Teng et al., Proc. Natl. Acad. Sci. USA 82: 1790-1794 (1985) ] And an antibody derived from mouse known as XOMEN-E5 [Young and Alam, US No. 4,918,163] was tested in animals and humans. Animal data show that both antibodies Binds endotoxin, neutralizes its biological activity, and suppresses gram-negative bacteremia Have the ability to Unfortunately, clinical trials in humans have shown that these trials Despite the optimism of the authors and sponsors of the trials, there are clear benefits There was no [E. J. Ziegler et al., New. Eng. J. Med. 324: 429-436 (1991); L. G reenman et al., JAMA 266: 1097-1102 (1991)]. US Food and Drug Administration Approve each antibody for the treatment of sepsis based on extensive clinical trials conducted Refused to do so.   Each antibody was identified after the symptoms of sepsis and the type of microorganism was determined. It should be noted that before it was tested in humans. After sepsis is established Anti-endotoxin antibody therapy given in It is generally considered to bring little benefit. What if these The body has an inflammatory cascade initiated by endotoxins and like TNF and IL-1 This is because the incidental triggering of the mediator cannot be turned back. Further And the high cost of each antibody (Centoxin HA-lA costs $ 3,700 per 100 mg dose) It was expected that doctors would use products that had no proven efficacy. Will limit its use [K. A. Schulman et al., JAMA 266: 3466-3471 (1991)] . Of course, these endotoxin antibodies target only Gram-negative sepsis. Therefore, there is no equivalent antibody for Gram-positive bacteria and fungi. III. Inhibition of cytokines released during sepsis   New therapies for the effects of endotoxin on the host's inflammatory response have allowed other therapies to We are heading towards blocking IL-1 and TNF function. For example, the same as IL-1 IL-1 receptor that occupies the same receptor site but does not mediate biological effects Antagonists have been identified. Blocking the IL-1 receptor by this molecule is endotoxic Mortality from elementary shock can be reduced [K. Ohlsson et al., Nature 348 : 550-552 (1990)]. IL-1 receptor antagonists appear to be well tolerated However, the dose required is extremely high (100 kg recombinant protein / kg body weight). More than mg is infused over hours to days). Required for human treatment The expected recombinant protein is 8-10g, which is very expensive (thousands of dollars) It seems to be.   TNF therapy aims to remove this mediator from the circulatory system To do. Monoclonal antibodies have some protection in experimental animals It was found that the squirrel [S. M. Opal et al. Infect. Dis. 161: 1148-1152 (199 0)], but the experiments in human patients with sepsis were not conclusive. Also in this case , These antibodies are expensive therapeutics that are given only when there are signs of sepsis. Seems like IV. Prevention   Since the treatment of ongoing sepsis poses many challenges in this way, the preventive stage Several tests were done at. These tests led to mixed results. One Hopeful research suggests hyperimmunity to core glycolipids in surgical patients at high risk of infection. It was to use plasma. Antibody protection does not reduce infection rates However, it reduced the severity of Gram-negative infections and increased patient survival [J. D. Ba umgartner et al., Lancet 2: 59-63 (1985)]. Collected from a number of normal donors, Confounding results of many studies using intravenous immunoglobulin with a wide range of antibodies Was given [J. D. Baumgartner and M.M. P. Glauser, Rev. Infect. Dis. 9: 194- 205 (1987)]. The main limitation to these studies is pooled immunoglobulin The potency of the preparations varies and seems to be relatively low [T. Cal andra et al. Infect. Dis. 158: 312-319 (1988)].   Monoclonal antibodies have also been made. These formulations will have higher potency However, its high cost and immunogenicity [S. Harkonen et al., Antimicrob. Agents Chemother ． 32: 710-716 (1988)] and abnormality Short circulation half-life (less than 24 hours) [S. Harkonen et al., Antimicrob. Agents Chem other. 32: 710-716 (1988); and C.I. J. Fisher et al., Clin. Care Med. 18: 1311- 1315 (1990)] make them unattractive prophylactic drug candidates.   Clearly there is a great need for drugs that can prevent and treat sepsis. is there. These drugs not only counteract the effects of endotoxin in Gram-negative sepsis But it must be able to protect and reduce bacteremia. These drugs It would be desirable if the agent could be administered prophylactically in a cost effective manner. Further In addition, not only Gram-negative cases, but an approach to eradicate all forms of sepsis Chi is needed.Summary of the invention   The present invention provides for the prevention and treatment of blood-borne toxin-mediated diseases, In particular, it relates to therapeutics for preventing and treating sepsis in humans and other animals. In one aspect, the invention provides high-risk patients (eg, surgical patients and other hospitalized patients). People, children with low birth weight, victims of burns and trauma, who are immunocompromised. The present invention relates to compositions and methods for preventing sepsis in humans. In other aspects The present invention contemplates the treatment of humans and animals with signs of a systemic septic reaction. are doing.   According to the invention, an antibody-antibiotic conjugate or "antibody otic" (antibody bodiotic) ”is a member of a class of compounds widely described as intravenous, intramuscular, or pod Used for internal or topical administration. Antibodies are antibiotics (antibodies For example, IgG, Ig M, IgA) covalently bound to form an antibody-antibiotic conjugate There is. Preferably the antibody is a non-specific IgG. Nonspecific means antibody population or Indicates that monospecificity within the pool is never predominant. Thus it is an antigen This is in contrast to the use of specific antibodies.   In one aspect, the present invention covalently binds non-specific immunoglobulins. Antibiotic-antibody conjugates consisting of selected antibiotics are contemplated. Immunoglobulin IgG having an Fc region and capable of binding to phagocytes by the Fc region It is preferred that   In one embodiment, the conjugate is capable of binding bacteria via antibiotics . The conjugate may be bacteriostatic, bactericidal, neither, or both.   However, the intended antibiotics are not limited to antibacterial agents. Antifungal and antiviral Agents are also contemplated. Gram-positive and Gram-negative when using antibacterial antibiotics A drug that is effective against both bacteria is contemplated.   The present invention provides not only conjugates that can bind to lipopolysaccharides of Gram-negative bacteria, but also free conjugates. Conjugates capable of binding endotoxin and neutralizing free endotoxin are also contemplated.   Preferred antibiotics include polymyxins, especially polymyxin B. it can. Polymyxin can bind free endotoxin. Known endotoxin binding Compound.   The invention also includes an antibiotic covalently linked to a non-specific immunoglobulin. For both Gram-positive and Gram-negative bacteria Intravenous therapeutic formulations are contemplated. In one embodiment of the therapeutic formulation, The biomaterial is selected from the group consisting of cephalosporins and penicillins. other In embodiments, the therapeutic formulation is (i) covalently attached to a non-specific immunoglobulin To a first conjugate comprising a first antibiotic, and (ii) a non-specific immunoglobulin It further comprises a second conjugate comprising a second antibiotic covalently bound (eg, For example, if the first antibiotic is polymyxin, the second antibiotic is Or bacitracin). In yet another aspect of the therapeutic formulation, two different Antibiotics are covalently attached to the same immunoglobulin molecule, one Lamb-positive bacteria and the other one is capable of binding to Gram-negative bacteria.   In the present invention, (a) a mammal is prepared for treatment, and (b) a protein is shared. Providing a therapeutic formulation comprising a conjugated endotoxin-binding compound, and (c) said A method of treatment which comprises administering (eg, intravenously) the formulation to a mammal is contemplated. ing. The endotoxin-binding compound may be polymyxin, the protein may be IgG It is preferably a non-specific immunoglobulin.   Treatment with antibody otic shows the effects of antibiotics alone. Expected not to exhibit toxicity and short half-life issues commonly associated with other drugs Be done. Furthermore, these conjugates promote clearance of both toxins and microbes. It is expected to have the opsonizing action of immunoglobulin.   The present invention presents signs of sepsis in mammals at risk of developing sepsis. Covalently bound to non-specific immunoglobulins before Consisting of administering a therapeutic preparation containing an antibiotic capable of binding to the bound microorganisms. It is intended for a method of treating the above mammals. In a preferred embodiment of the invention The method includes administering intravenously.   The present invention provides that the method can be applied to animals such as newborn calves and foals, humans who have undergone surgery, It is intended to be used for livestock and for trauma and burn patients. Book The method will be used to treat immunocompromised patients.   The present invention may contact gram negative and / or gram positive bacteria It may be useful in treating mammals. The therapeutic formulation used in the method of the invention is It is intended to be able to bind toxins.   The present invention further mediates the opsonization of a pathogen in a mammal infected with the pathogen. A table covalently bound to a non-specific immunoglobulin G having a mediated Fc region. Treatment of the above mammal comprising administering a therapeutic formulation containing a surface active antibiotic The method is intended. Infectious agents can be Gram-negative or Gram-positive bacteria It Surface-active antibiotics used in therapeutic formulations are polymyxins (eg, It can be polymyxin B).   One aspect of the invention resides in (a) the surface of a pathogen immobilized on a solid support. Antigen, (b) a surface-active antibiotic covalently bound to a non-specific immunoglobulin And (c) a competitor consisting of a surface antigen present in solution, Prepared by incubating the immobilized antigen with the conjugate in the presence of the competitor and washing. To remove unbound conjugate and competitors, then bind to immobilized surface antigen Diagnostic methods involving detecting bound conjugates are contemplated.   In a preferred embodiment, the above diagnostic method comprises one or more microtiter plates. Immobilizing the surface antigen in the wells of Isolation of surface antigens of this method from bacteria It is also intended to do. Surface antigens can be isolated from Gram-negative bacteria such as E. coli. And (for example, lipopolysaccharide). In addition, the competing substances used in this diagnostic method are It may consist of lipopolysaccharides from Lam-negative bacteria. In addition, the competitor is E. coli (Escherichia coli), Salmonella typhimurium, Pseudomonas aeruginosa ( Pseudomonasaeruginosa), Vibrio cholerae, Shigella flexneri (Shigella flexneri), Klebsiella pneumoniae, Salmonella enteritidis (Salmon) ella enteritiditis), Serratia marcescens, and Rhodobacter sukh Lipopolysaccharides from Gram-negative bacteria such as Rhodobacter sphaeroides It may consist of   The present invention also includes a) reacting an antibiotic with a crosslinker to form a derivatized antibiotic. , And b) reacting the derivatized antibiotic with a non-specific immunoglobulin to bind the conjugate. It is intended to be a method of synthesizing a conjugate that comprises the steps of forming. In addition, antibiotics A quality is intended to be one that binds to the surface of microorganisms. In a preferred mode Thus, antibiotics are peptides. In other preferred embodiments, such peptides are Limulus anti-lipopolysaccharide factor, and in another aspect, the peptide is It is a D-amino acid-containing peptide. Also, the peptide may bind to endotoxin. Intended. In a preferred embodiment, the antibiotic is a polymer such as polymyxin B. In xin It   In another preferred embodiment, the antibiotic used is bactericidal against Gram-negative bacteria. is there. In other embodiments, the antibiotic is bactericidal against Gram-positive bacteria. One In the above embodiment, the antibiotic is vancomycin. In one aspect, non-specific immunity The globulin is composed of the Fc region.   The present invention also includes a) reacting an antibiotic with a first bifunctional cross-linking agent to derivatize an anti-derivative. Forming a biomaterial, b) reacting the non-specific immunoglobulin with a second bifunctional cross-linking agent. To form a derivatized immunoglobulin and c) derivatized with an antibiotic Derivatization of derivatized antibiotics to form covalent bonds with immunoglobulins Synthesis of a conjugate consisting of steps for reacting with immunoglobulin to form a conjugate The method is intended.   In one embodiment of this method, the antibiotic is one that binds to the surface of a microorganism. It In a preferred embodiment, the antibiotic is such as Limulus anti-lipopolysaccharide factor (but In another preferred embodiment, which is (but not limited to) a peptide, the peptide is D-amino. It is an acid-containing peptide. In another embodiment, the peptide binds endotoxin Things. In a preferred embodiment, the antibiotic is such as polymyxin B (but (Not limited to this) polymyxin.   In one embodiment of this method, the antibiotic is bactericidal against Gram-negative bacteria. It In other embodiments, the antibiotic is bactericidal against Gram-positive bacteria. Preferred In one aspect, the antibiotic is vancomycin. In another preferred embodiment, The first and second bifunctional crosslinkers are N-succinimidyl 3- (2-pyridyldithio E) Propionate. In another embodiment of this method, the first bifunctional The crosslinker is S-acetyl mercaptosuccinic anhydride. In another aspect, The second bifunctional crosslinker is sulfosuccinimidyl 4- (N-maleimidomethyl) It is cyclohexane-1-carboxylate.   The invention also has a) i) first and second reactive sites, in any order. A cross-linking agent, the first site of which is exposed and is reactive with primary amino groups, A cross-linking agent whose second site is blocked by a cleavable group, ii) one or more Antibiotics with primary amino groups, and iii) with one or more primary amino groups A non-specific immunoglobulin, b) in any order, i) blocked Cross-linking agents with antibiotics to form derivatized antibiotics, and ii) blocked A cross-linking agent with the immunoglobulin to form a derivatized immunoglobulin And c) in any order, i) to form free derivatized antibiotics. Modified derivatized antibiotics with modified reagents and ii) free derivatized immunoglobulins Reacting the derivatized immunoglobulin blocked to form with a modifying reagent , And d) reacting the free derivatized antibiotic with the free derivatized immunoglobulin. In order to form the conjugate, the method for synthesizing the conjugate comprising each step is intended.   In a preferred embodiment of this method, the antibiotic is one that binds to the surface of the microorganism. is there. In another preferred embodiment, the antibiotic is a Limulus anti-lipopolysaccharide factor such as (But not limited to) peptides. In one embodiment, the peptide is D- It is an amino acid-containing peptide. In another embodiment, the peptide binds endotoxin Ru Things. In a preferred embodiment, the antibiotic is such as polymyxin B (but (Not limited to this) polymyxin.   In a preferred embodiment, the method of the invention comprises an antibiotic bactericidal against Gram-negative bacteria. The substance is used. In other embodiments, the antibiotic is bactericidal against Gram-positive bacteria. Is. In a preferred embodiment, the antibiotic is vancomycin. In another aspect Thus, non-specific immunoglobulins consist of the Fc region. One of this method In an aspect, the modifying reagent is a reducing agent. In a preferred embodiment, the reducing agent is dithiotray It's Thor.   The present invention further comprises a) in any order i) first and second reactive sites. A first cross-linking agent, the first moiety being reactive with a primary amino group, and A cross-linking agent whose site 2 is reactive with the maleimide group, ii) one or more primary amino groups An antibiotic having iii) a second cross-linking agent having first and second reactive sites , The first site is reactive with the primary amino group, and the second site is sulfhydryl. A crosslinker that is reactive with a drill group, and iv) a non-containing one or more primary amino groups Specific immunoglobulins are prepared, b) in any order, i) derivatized antibiotics A first cross-linking agent with an antibiotic to form, and ii) a derivatized immunoglobulin Reacting a second cross-linking agent with the immunoglobulin to form c and induce c) Reacting a conjugated antibiotic with a derivatized immunoglobulin to form a conjugate. A method of synthesizing a conjugate consisting of   In one embodiment of this method, the first crosslinker is difunctional. Preferred state The first bifunctional cross-linking agent is S-acetylmeth It is lucaptosuccinic anhydride. In another preferred embodiment, the second bridge The agent is bifunctional. In one aspect, the second bifunctional crosslinker is sulfosuccin. With imidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate is there.   The invention also includes a) reacting a non-specific immunoglobulin with a first modifying reagent to produce an acid. A modified immunoglobulin preparation, and b) oxidising the immunoglobulin preparation. Reacting with a substance and a second modifying reagent to form an antibiotic-immunoglobulin conjugate The method of synthesizing the conjugate comprising each step is intended. In a preferred embodiment, Immunoglobulins consist of the Fc region.   In one aspect of this method, the first modifying reagent is an oxidizing agent. Preferred state In like manner, the oxidant is periodate. In another aspect, the method It contains a second modifying reagent which is a base agent. In a preferred embodiment, the reducing agent is sodium borohydride. It's Rium.   The present invention also includes a) an antibiotic precursor having a limited bactericidal activity as a first cross-linking agent. To form a derivatized antibiotic precursor with increased bactericidal activity, b ) Derivatized immunoglobulin by reacting non-specific immunoglobulin with a second cross-linking agent And c) between the derivatized antibiotic precursor and the derivatized immunoglobulin Derivatized antibiotic precursor to form a covalent bond to the immunoglobulin Intended to form a conjugate by reacting with There is.   In one preferred embodiment, the antibiotic precursor is 7-aminocephalosporan. An acid and 6-aminopenicillanic acid. In another aspect, The first crosslinker is difunctional. In a preferred embodiment, the first bifunctional crosslinker is m-maleimidobenzoyl-N-hydride. Roxysulfosuccinimide ester. In another aspect, a second cross-linking agent Is bifunctional. In a preferred embodiment, the second bifunctional crosslinker is iminothiolane. Is.   The present invention further comprises a) in any order i) first and second reactive sites. A first cross-linking agent, the first moiety being reactive with a primary amino group, and A cross-linking agent whose site 2 is reactive with a sulfhydryl group, ii) one or more primary amino An antibiotic precursor having a group and limited bactericidal activity, iii) first and second A second cross-linking agent having a reactive site, the first site being reactive with a primary amino group And a second moiety is reactive with the maleimide group, and iv) 1 A non-specific immunoglobulin having one or more primary amino groups, b) any In sequence, i) a first cross-linking agent is added to the antibiotic to form a derivatized antibiotic precursor. A precursor, and ii) a second cross-linking agent to form a derivatized immunoglobulin React with immunoglobulins and c) derivatize derivatized antibiotic precursor A method for synthesizing a conjugate consisting of steps for reacting with an immunoglobulin to form a conjugate Is intended for law.   In a preferred embodiment, the antibiotic precursor is 7-aminocephalosporanic acid and And 6-aminopenicillanic acid. In another preferred embodiment , The first cross-linking agent is difunctional. In a further aspect, the first difunctional crosslinker is m -Maleimidobenzoyl-N-hydroxysulfosuccinimide ester. In another preferred embodiment, the second crosslinker is difunctional. Preferred state The second bifunctional crosslinker is iminothiolane. ItDescription of the drawings   FIG. 1A is a schematic diagram showing the design of an antibody otic of the present invention. Figure 1B FIG. 3 is a schematic diagram showing another antibody otic design of the present invention.   FIG. 2 schematically shows a means for screening modified antibiotics for antibacterial activity. Show.   FIG. 3 shows a new anti-antibody for use as a compound for the formation of conjugates with immunoglobulins. An alternative method for screening biomaterials is presented. FIG. 3A shows the maximum inhibition of bacterial growth. The means for establishing small concentrations are shown. Figure 3B shows new antibiotics for bactericidal activity. The means for screening are shown.   FIG. 4 shows a solid-phase assay for measuring the binding level of the antibodies of the present invention. Show a essay. Stage 1 shows toxins or microorganisms in the test microwells. Step Floor 2 schematically shows the binding of antibody otics. Step 3 involves the binding of secondary reagents It shows typically.   FIG. 5 shows the binding of the conjugates of the invention to LPS as measured by ELISA. You   FIG. 6 shows the binding of another conjugate of the invention to LPS as measured by ELISA. Is shown.   FIG. 7 uses free polymyxin (PMB) as measured by ELISA 3 shows inhibition of LPS binding of the conjugate of the present invention.   FIG. 8 shows the LPS of the periodate conjugate of the present invention measured by ELISA. Indicates the binding of.   FIG. 9 shows the present invention using LPS of various bacterial species measured by ELISA. Inhibition of LPS binding of the conjugate.   FIG. 10 shows the binding of the inventive conjugate to phagocytic cells in a radioactive competition assay.   Figure 11: Intravenous PM in rabbits expressed as absorbance at 410 nm 3 shows the pharmacokinetic profile of B-HIgG and HIgG.   Figure 12: Intravenous PMB-HIg in rabbits expressed as IgG concentration. 1 shows the pharmacokinetic profile of G and HIgG.   FIG. 13 shows single chain pep that is known to bind and neutralize endotoxin. Figure 3 shows the sequence of Limulus anti-lipopolysaccharide factor (LALF) from Tide. Description of the invention   The present invention is directed to the prevention and treatment of blood-borne toxin-mediated diseases, particularly in humans. The prevention and treatment of sepsis (sepsis) caused by various organisms in other animals. Therapeutics for Oki. The present invention is particularly concerned with the effects of endotoxin in vivo. Suitable for Japanese. However, the present invention can be used to treat Gram-negative and Gram-positive sepsis. It is also intended to be used. The invention is also used for the treatment of sepsis by one organism But can also be used to treat sepsis caused by multiple organisms (eg. (Eg sepsis and / or bacteremia due to Gram-negative and Gram-positive organisms). Departure Ming also has multiple antibody-antibiotic conjugates used in combination Treatment using is also contemplated. The present invention also provides an opsonizing effect (opsoni). bacteremia, viremia or Is also intended for the treatment of fungalemia.   According to the invention, soluble antibody-antibiotic conjugates, i.e. antibody otics (Antibodiotic) "intravenously, intramuscularly, subcutaneously, intracutaneously, intraperitoneally, intrapleurally, subarachnoidally , Or topically. Conjugate is at least 0.1 mg / ml, preferably at room temperature. Water soluble if it has a solubility in saline of at least 1.0 mg / ml I shall. The present invention provides a therapeutic preparation for both prophylactic and acute treatment. Intended for use in antibody otic.   The benefits of the treatment of the present invention are that soluble antibody-antibiotic conjugates are therapeutic. In the case of bacteria, (i ) Bacterial binding and opsonization, (ii) Bacterial killing (direct killing by the conjugate) And / or complement mediated And (iii) neutralization and removal of free bacterial toxins (eg Gram-negative endotoxin) And thereby prevent the onset and / or progression of septic symptoms) Is expected to be achieved.   Anti-body otic, necessary prevention with low cost and reasonable effect And the treatment is considered to be obtainable. Antibodies are fungi and It is possible to suppress ruth infection. In addition, anti-body otic is lined with bacteremia In addition, the endotoxin-mediated action can be suppressed. Long working period (eg days to weeks) Antibodies with an interval can be easily administered. Further invention Is an antibody otic that is reactive to Gram-negative organisms, as well as Gram-positive Includes antibody biotics that are reactive to living organisms, so any other known A broader protection spectrum is expected than the above method.   The present invention is also intended for use in diagnostic applications. Such diagnostic use Is a specific organism or surface structure present on the organism that is recognized by the antibiotic. Detection of LPS from its structure (ie, a receptor expressed on the cell surface that binds to antibiotics) There is a method of doing.   Descriptions of the invention include (I) design and characterization of antibody otics, (II) in Effects of antibody otics in vitro and in vivo, (III) Antibodies Uses of otic, (IV) therapeutic preparations and combinations are included. In section III, (A ) Prophylactic use in humans, (B) treatment of acute conditions in humans, and (C) veterinary The use of antibody otics for medical treatment is described. I. Design and characterization of antibody otics A. antibody   Antibodies of all types (eg IgG, Pentamers and monomeric IgM, secretory monomers IgA, IgE and IgD) are contemplated. But, It is advantageous to use a particular type of antibody. For example, Table 1 compares the properties of IgG and IgM. It is a comparison. IgM has better opsonization and complement activation benefits However, IgG has a longer half-life in vivo, which is Higher titers result from being the primary antibody generated during the next reaction. Therefore, the book The preferred antibody for binding of the invention is IgG.   Antigen-specific IgG (eg antibodies directed against bacteria) can be used but Prospecificity results in shorter half-life (and / or greater cost) of the compound You can Therefore, preferred antibodies are non-specific [C. H. J. Ford et al. , Indian J. Pediatr. 57: 29-46 (1990)].   Goers et al. (US Pat. No. 4,867,973) describes antibodies bound to antimicrobial substances. The use is described, but with an antigen-specific antibody. On the other hand, according to the present invention The conjugate uses a non-specific antibody. Goers et al. Is especially antigen-specific monochrome The binding with the null antibody is described. Monoclonal antibodies are predictive of bacteremia and sepsis It was not a step forward in prevention and treatment. These preparations are More powerful and specific than Qing, but a) extremely expensive and b) immunogenic And c) short circulation half-life (typically less than 24 hours) ).   As for cost, Centoxin, a commercially produced Is a real example, the price is about 3,7 per 100 mg dose. It's $ 00. This product has strict guidelines for acute symptoms. Even when used in line, pharmacoeconomic analysis shows that "this use is It could add $ 2.3 billion to the calculation. ”[K. A. Schulman et al., JAMA 266: 3466-3471 (1991)]. The cost of Centoxin is totally preventative. It's like you can't use it. On the other hand, the conjugate of the present invention is only a part of this number. Manufactured from costly materials (eg $ 2 per 100 mg dose) The availability of a cheap source of IgG Things.   Also, human monoclonal antibodies probably reduce the chance of immunogenicity. , The problem of short circulation half-life cannot be overcome. Patients with septic syndrome In a study using anti-lipid A human monoclonal antibodies in The half-life was about 16 hours [C. J. Fisher el al., Clin. Care Med. 18: 1311 -1315 (1990)]. In order to maintain a protective level of antibody, this drug Must be given repeatedly. The cost of such a method is also hesitant Things.   From the above, why the limitation to "non-specific immunoglobulins" is of particular importance to the present invention. It would have been made clear if it was a limitation. Non-specific IgG is easily and inexpensively obtained It does not require immunization and produces an immune response in a syngeneic setting. I don't know. Non-specific IgG does not have the problem of standardizing antigen-specific antibodies. Paid attention There is no antigen specific titer that has to be present, just add (unit dependent) The difference in potency may be left unchanged). And standardization depends on the bound ligand. Resulting in over 1000-fold increase in LPS-binding titer due to non-specific IgG binding And normalize the activity of the treatment by normalizing the bound ligand. And non-singular IgG, unlike monoclonal antibodies, has a long half-life needed for prevention. (Pooled against the 16-hour average half-life of the human monoclonal antibody described above) Polyclonal human IgG has a half-life of 21 days or more).   IgG from donors (ie humans and animals) is preferred over cell lines for cost reasons New In this regard, typically large pools of plasma Is used as the starting material. Eta is a large-scale fractionation method known in the art. There is the precipitation of nole and the precipitation of high concentration of salt [H. F. Deutsch in Methods in Im munology and Immunochemistry, (CA Williams and MW Chase, eds.) , Academic Press, New York, pp. 315-321 (1967)]. Diafiltration and ultra Isolation of immunoglobulins from Cohn Effluent III by filtration, with some complexity There is also a method [E. J. Cohn el al., J. Am. Chem. Soc. 68: 459-475 (1946). ].   This latter method is a commercially available IgG preparation of Gammim. Have been. Of course, each individual donor used to manufacture this product Tested and non-reactive in tests for exposure to or presence of pathogens Must be known to be sex. This product is intended for intravenous injection In addition, except for adjusting the pH of the solution to 4.0-4.5, protein (as a 4.5-5.5% solution) Has not been chemically modified. Isotonicity is due to the addition of maltose (9-11%) Can be   Each milliliter (ml) contains approximately 50 mg of protein, over 98% of which is The eluate portion has the electrophoretic mobility of gamma globulin. Gamma globulin More than 90% of this is monomeric. Trace amounts of IgA and IgM are included. I The distribution of gG subclasses is similar to that found in normal serum.   This commercial product exhibits a broad spectrum of opsonization and neutralizing antibody activity. . When administered intravenously, substantially 100% of the infused IgG antibody is immediately available to recipient circulation. It will be available in the circle. in The in vivo half-life is equal to or less than the 3 week half-life reported for IgG in the literature. It exceeds this. Therefore, it can be sufficiently used for the production of the antibody-antibiotic conjugate of the present invention. It can be used.   Of course, previously had anaphylaxis or a severe systemic reaction to IgG Large doses of antibody in humans should be contraindicated in certain individuals. I have to. Confirmed to be insensitive to trace amounts of other antibodies (eg IgA) You must also pay attention to what you do.   Prior to administration of the antibody-antibiotic conjugate of the present invention to humans, an antibiotic susceptibility test should be performed. Doing is good for medical practice. This covers a small amount of conjugate on the patient's arm. It can be carried out by injecting underneath. Inject the salt solution into the other arm as a control It A positive result in a hypersensitivity test is usually the formation of a swollen wormhole on the skin. Anaphylactic shock (ie General hypersensitivity reactions that occur later). For cases like this It is recommended to have adrenaline ready.   The usual dose of a commercially available intravenous immunoglobulin product is 100-200 mg / kg (2-4 ml / kg ) The body weight is administered by intravenous infusion almost once a month. Appropriate clinical response If not, or if you suspect that the levels of IgG obtained in the circulation are inadequate , This dose may be given more frequently, or 400 mg / kg (8 ml / kg) body weight May be increased up to.   The present invention is much more than that given for commercial immunoglobulin preparations. And a low typical antibody otic dose It is intended. The number of bound antibiotic molecules per immunoglobulin molecule This is especially true if it exceeds 1. The present invention is 0.1-100 mg / kg, preferably 1-20 mg Conjugate dose ranges of / kg are intended. Dosage included in this preferred range The amount of PMB included (assuming 3 molecules per IgG molecule) is 0.025-0.5 mg / kg. B. Antibiotics   Thousands of natural, synthetic, and semi-synthetic compounds Alternatively, it has been identified as having antiparasitic activity.   The first consideration in designing antibody-antibiotic conjugates is that This is how antibiotics work. The conjugate is a much larger molecule than the original antibiotic Therefore, exposed or secreted bacteria, fungi, viruses, or parasites Only antibiotics that bind to the labeled components (eg toxins) can cause the antibody carrier to Is more likely to be directed to the product. For example, penicillin antibiotics It disrupts wall synthesis and binds to surface-exposed components of certain bacteria, but Lycoside antibiotics normally bind to ribosomal subunits in the cytoplasm of cells . The former is also much better than the latter as a candidate for effective antibody-antibiotic conjugates. Of.   Antibiotics vary greatly in the type and species of organism in which they are active. For example, some antibiotics, such as polymyxin, are effective against Gram-negative bacteria Crab is effective, but Other antibiotics like Shin tend to be more effective against Gram-positive ones There is. Some things like cephalosporins, and have a broad spectrum Penicillin is relatively effective for both types. Like amphotericin Other antibiotics are primarily antifungal agents, whereas amantadine is It is active against certain influenza viruses. Anti-drugs for prevention or treatment of diseases In designing the body-antibiotic conjugate, consider the spectrum of the desired antibiotic activity. Carefully, it is active against the target pathogen and, as noted above, primarily Antibiotics that act on the exposed components of the drug substance must be selected.   As used herein, the term "pathogen" refers to an infection or disease. All organisms that are traditionally considered to be pathogens. (For example, S. aureus, S. pyogenes, S. dysenteriae, S .. flexneri Etc., or optionally a pathogen (eg, P. aeruginosa, S. ma. rcesens, S. mitis etc.).   Family of antibiotics (eg penicillins, cephalosporins, polymycins) Xins) have structural features common to all members. But this There is often a wide variety of natural and synthetic changes in the common structure, which Influences the spectrum of activity, pharmacokinetics, or other antibiotic properties. There are things to do. Within the antibiotic family, the design of antibody-antibiotic conjugates These structural differences in are important from two perspectives. First, the activity spectrum Tol can influence antibiotic choice, and second, the chemical phase between antibiotics. The difference is the antibiotics It affects the range of crosslinking chemistries that can be used to do so. For example, The variable side chain component of penicillin antibiotics is methylben in penicillin G. Although it is a dil group, the side chain group that can be changed in amoxicillin is the primary amine side. It is a phenolic group having a chain. The latter antibiotic is wider than penicillin G It has a variety of possible morphologies for crosslinking.   In Table 2, surface and / or product reactive activity against various pathogens is shown. Several families of antibiotics have been described. This is for illustration only And is not intended to limit the invention to only these compounds .   The preferred antibiotic of the present invention is polymyxin B (PMB). As mentioned above , This antibiotic binds to and neutralizes endotoxin. But used in vivo If PMB is short-lived, and the recommended therapeutic dose for systemic infection is However, the risk of nephrotoxicity is quite high.   The level of protection afforded by the present invention is determined by comparison with other known methods. It will be best understood (see Table 3). For example, a widely tested and advertised model Centroxin-HA-IA, a monoclonal antibody, binds endotoxin and binds its biological activity. It can neutralize sex. However, when compared with the IgG-PMB conjugates of the invention, Monoclonal antibodies are expensive, have low affinity, and have a short half-life. these The properties listed below still have to show clear advantages in human clinical studies. It will explain the reason why it does not exist.   Those who tried to reduce the toxicity of polymyxin B by binding to dextran There is [D. A. Handley, European Patent Application Publication No. 428486]. But deki Strand has a human half-life of only about 1 day. Immunoglobulin of the present invention A much longer half-life is obtained by using (Table 4 and Examples 24 and 25). reference). Dextran, which has no Fc receptor (FcR), also promotes opsonization. It is not known for its ability to progress or activate complement (C ').   Since it is important that antibiotics are non-toxic to host animals, the present invention Contemplate use of conjugates that are effective in the subject organism but non-toxic to the host It is something to illustrate. The non-toxicity of IgG-PMB is shown in Example 27.   As pointed out above, the present invention is responsive to Gram-positive organisms and their toxins. Anti-body otic is also intended. In one aspect, the invention Intended to use bacitracin bound to immunoglobulin Is.   In another aspect, the invention provides the use of vancomycin bound to immunoglobulin. It is intended for use.   Bacitracin is a strain of Bacillus subtilis (Tracy strain). ) Is a polypeptide produced mainly by Streptococcus pie. Logenes (Streptococcus pyogenes) and other β-hemolytic Streptococcus , Pneumococcus pneumoniae, and cross It is bactericidal for Gram-positive organisms, including certain strains of Tridium spp. Bachito Rasin blocks early steps of peptidoglycan biosynthesis and interferes with cell wall synthesis To exert its effect. Commercially available bacitracin is cheap In general, it is not well absorbed from the intestinal tract and wounds. Tan seen on systemic administration This use is usually limited to topical application because of urine, hematuria, and nitrogen retention [For example, R. Berkowand A. J. Fletcher (eds.), The Merck Manual, 16th ed ., 1992, p. 46; and G. F. Brooks et al., Jawetz, Me1nick & Adelberg's Med ical Microbiology, 19th ed., 1991, pp. See 172-173].   Systemic administration of free bacitracin causes unacceptable nephrotoxicity The benefits of bacitracin without this side effect when combined with immunoglobulins according to clarity Is obtained. However, the present invention is limited to the mechanism of action of any particular antimicrobial substance. It is not intended to be.   Vancomycin is especially suitable for Staphylococcus aureus. ureus) and Clostridium difficile Active against Gram-positive organisms. Limit the invention by the mechanism of action Although not intended, vancomycin may interfere with cell wall synthesis. It is thought that it exerts a more bactericidal action. The present invention is directed to various cross-linkers. ) And method to synthesize from vancomycin and non-specific human immunoglobulin It is intended as a conjugate. These conjugates, like the ones listed above, It suppresses bacteremia and toxin-mediated effects in Lam-positive organisms.   In one embodiment, the method first comprises vancomycin and immunoglobulin. Treated with different heterobifunctional crosslinkers, and then reacting the derivatized species with each other. The vancomycin to form non-specific immunoglobulins. Used to bind to. In a second aspect, the method comprises first Both mycin and immunoglobulin were treated with the same heterobifunctional crosslinker and then the By reacting the derivatized species with each other to form a conjugate, The use of binding syn to non-specific immunoglobulins is used. Both synthetic methods For this purpose, various crosslinker combinations were intended and tested. The following table has been tested so far Van The cross-linking compounds for reaction with comycin are listed. Some of the cross-linking agents Is insoluble in aqueous solution during its reaction with vancomycin, The exam didn't continue. However, the step of making them soluble (eg addition of solvent In addition, if the side chain of the basic vancomycin structure is further modified) It was found that various crosslinkers prove useful.   In the table, the cross-linking method, modified Bancoma reactive with immunoglobulins A group on isine (a "reactive group") or a corresponding linker on an immunoglobulin (presence) ), Solubility, and biological activity of the conjugate. Later examples are tables The representative reaction described in 1. is described. C. Conjugates and crosslinks   Numerous reagents have been developed to cross-link biological molecules [Pierce Chemica l Co., (Rockford, IL), General Catalog, pp. E-10-E-39 (1992)]. General In addition, these reagents do not react with the side chains of various amino acids in proteins or peptides. It has a responsive functional group. As shown in Table 5, various functional groups Or primary amino group, carboxyl group, hydroxyl group of other compounds or Reacts with thiol groups. In designing antibody-antibiotic conjugates, antibody and antibiotic Both reactive groups of the substance must be considered. In general, antibodies are directly bound Many reactive groups that can be used in the method (amino acids are primary amines, carboxyls, Includes xyl, thiol [after reduction]) or modifications for conjugation methods Group (glycosylated amino acids can be oxidized to aldehydes and primary amines Thiol reactive). Individual antibiotics generally It does not have so many different reactive groups and is less for binding to the antibody. Only give no choice. With the selection of antibiotics from a family of related compounds Reactive groups on the antibiotic must be taken into account in the choice of cross-linking method.   The key to modifying an antibiotic is the surface of the pathogen or its secreted production. Maintaining the ability to bind things. Modification of individual reactive groups with crosslinkers or Is an extra modification of more than one reactive group, or a large For joining to Park The more generated steric hindrance can cause loss of antibody activity. Therefore, consider the activity of the conjugate Biology of modified or cross-linked antibiotics in a simple antimicrobial assay before Conditions for preservation of antibiotic activity must be investigated by examining biological activity. Absent. Preferably, the type and concentration of crosslinking agent that preserves antibiotic activity is selected.   Different crosslinkers affect the activity of individual antibiotics and the efficiency with which they bind to antibodies. You can In designing antibody-antibiotic conjugates, the discovery of more suitable crosslinkers has been Depends on empirical analysis of conjugates prepared with varying concentrations of different crosslinkers Things.   The in vivo safety and efficacy of antibody-antibiotic conjugates depends on their activity, toxicity and And stability. The choice of crosslinker also has an impact on these aspects of conjugate performance. You can For example, in addition to affecting the activity of the conjugate given by the antibiotic Thus, the cross-linking agent used can affect the properties of the antibody. Opsonization or complement fixation The effector functions that depend on the Fc region of the antibody, such as Its position on the antibody molecule can be influenced. In addition, some crosslinkers are Causing an adverse reaction by expressing an immune response to the hapten group of There is. Furthermore, the in vivo stability of the bond formed by the cross-linking method is not It can change in an invisible form. The disulfide bond that connects the antibiotic and the antibody , Not as stable as the amide bond formed by other cross-linking agents, for example. Long term The dissociation of antibodies and antibiotics is unacceptable if prophylaxis is desired. D. Analogues   The present invention contemplates the use of antibody analogs. Antibody analogs are similar to antibodies. It is a compound that functions in a similar manner. In one embodiment, antibody fragments It is also intended to form antibody-antibiotic conjugates using (eg Fc fraction) Of. As used herein, the terms "antibody" and "immunoglobulin" refer to antibodies It includes analogs. E. New antibiotics and conjugates   Antibiotic compounds have been isolated from many different microbial, plant and animal sources, and Promising compounds continue to be discovered. Furthermore, synthetic derivatives of natural compounds and In many laboratories fully synthetic compounds such as small peptides are antibiotics Screened for activity. Use of "antibiotics" as used herein The term destroys a microorganism, inhibits its growth, or any compound that binds to it. It refers to a chemical compound (ie, "antimicrobial substance"). The term is It is not intended to be limited to the compound produced. Therefore, "antibiotic A substance is actually a microbial organism in many chemical laboratories nowadays many antibiotics But synthetically produced as well as synthetically produced Things. The polymyxin and other compounds described herein are synthetic It can be manufactured or obtained from natural sources (eg B. polymyxa). Therefore, The present invention provides for the construction of various antibody-antibiotic conjugates using antibiotics from all sources. It is intended to be total and synthetic.   Figures 2-4 are used as compounds to bind immunoglobulins. Also outlined is a method for screening new antibiotics for use. Of. The “screening method” consists of the following time-course steps.   Mode I: Antibiotics bound to cross-linker only, in liquid media and disk inhibition lawn The inhibition of the growth of the organism is examined in an assay (eg Kirby-Bauer).   Mode IIA: Antibiotics linked to immunoglobulin via cross-linking agent, solid-phase assay For binding to bacteria and bacterial toxins.   Mode IIB: Linking antibiotics to immunoglobulins without the use of cross-linking agents (eg, (Periodic acid oxidation of carbohydrate groups (CHO) of IgG), followed by bacterial analysis by solid phase assay. And binding to bacterial toxins.   Mode III: Inhibition of antibodies by inhibition of bacterial toxin bound to antibiotics Check for specificity.   Mode IV: Antibodies to inhibit growth of organisms in liquid medium Find out.   Using this method, a new antibiotic (“X”) was prepared for use in the present invention. Can be evaluated. Although not needed for research use, in a clinical setting, Developed by National Committee for Clinical Laboratory Standard (NCCLS) According to current procedures for liquid medium dilution, disc diffusion, and other methods And   For example, antibiotic X is first evaluated by Mode I. In this mode, X is combined only with the cross-linking agent "c" to form "X-c". This compound is then added to the liquid phase. Or added to solid medium. By creating only the anti-body otic part , Immunoglobulin The compatibility problem with can be avoided. Mode I is the bond chemistry of "X" It just checks the suitability. Antibiotics X unassayed to perform assay Of the same assay.   For comparison of lawn assays in Mode I, organisms were placed in Petri dishes containing agar. (Step 1, FIG. 2). A small filter paper containing a known amount of antibiotic X or X-c Place the disc on the agar surface and allow it to diffuse into the medium for 18-24 hours (step 2, Figure 2 ). After this incubation, the area of growth inhibition by X was revealed and Compare this to the region (if present) obtained by X-c (step 3, FIG. 2).   Alternatively, for Mode I, a known concentration of X or X-c in liquid medium in a test tube. Are diluted and incubated with organisms sensitive to X (Figure 3). Inn After cubating, growth was inhibited (ie, visible, as indicated by no turbidity) No growth). This value corresponds to the minimum inhibitory concentration (“MIC”) (Figure 3A). To test bacterial activity, test tubes showing bacterial growth inhibitory activity Take an aliquot and add this aliquot to the agar plate (Figure 3B). Proliferation If so, the reagent is a bacterial growth inhibitor. If no growth occurs, then Is a bactericide. The minimum bactericidal (lethal) concentration is about 100,000 organisms It is the lowest concentration of X-C or X resulting in a 99.9% reduction from the first inoculation. this The minimum bactericidal concentration (“MBC”) was determined by the method [I.S. Snyder and R.G. Finc h in Modern Pharmacology, 2d Ed. (C.R.Craig and R.E.Stitzel.eds.), Little, Brown and Company, Boston, pp. 631-640 (1986); J.E. Conte. Jr. and S.L. Barrie re ， Manual of Antibiotics and Infectious Diseases ， 6th Ed., Lea and Febig er, Philadelphia, pp. 135-152 (1988)].   Some reduction in activity is expected when X-c is compared to X. But X The more potent, the greater the reduction in X-c activity can be tolerated. As a whole, MIC and The range of 0.01 to 50 μg / ml has utility for both MBC.   When the activity of X-c is good, it is further evaluated in Mode IIA. X-c activity If poor, evaluate X in Mode IIB. Both modes IIA and IIB are covalently bonded Mode IIA uses a cross-linking agent that produces “X-c-lg” when intended for conjugation by Mode IIB uses no crosslinker and produces "X = CHO-Ig". In both cases Here, the antibody-antibiotic conjugate, or simply "antibody otic", is For example, it is assayed by a solid phase assay as shown schematically in FIG.   Toxins or organisms in solid phase assays in microwells or other suitable Can be used to coat various surfaces (step 1, Figure 4A). Anti body Otics are then added for binding studies (step 2, FIG. 4A). Standard cleaning A process is used to prevent non-specific binding. The antibody portion of the conjugate is then the second reagent (Has an enzyme reporter group such as horseradish peroxidase Goat anti-human IgG antibody) (step 3, see FIG. 4A). Then for the enzyme Of the appropriate substrate (not shown) to produce a colorimetric signal.   If the toxin was used in a solid phase assay, compare X-c-Ig binding to that of X-CHO-Ig be able to. When using organisms, the binding It should be noted that it is not mediated by Ig Fc receptors. Unbound Ig Can be used as a control for this purpose.   To avoid coupling by metamorphosis or other artificial factors, the Mode IIA or Should be evaluated in Mode II I for those conjugates that showed activity in Mode II B. Absent. As shown in Figure 4B, this simply added free antibiotic and allowed for binding. It is only to show that they compete specifically.   The next part of the evaluation is to bring the antibody-antibiotic conjugates to growth inhibition and / or bactericidal activity. Including testing (Mode IV). This is the same as the assay shown in Figure 2. And the difference here is not the complete antibiotic (X-c) but the complete conjugate X-c-Ig ( Or X-CHO-Ig) is evaluated.   Both X-c-Ig and X-CHO-Ig show good toxin binding in mode II, but It may exhibit poor antibacterial activity in do IV. Nevertheless combined If the specificity of is confirmed in Mode III, these compounds are candidates for diagnostic agents Is. Alternatively, they simply bind free toxin in vivo and thereby It can be used to reduce the load of endotoxin.   By carefully considering the consequences of each of these steps, any antibiotic The substance can be analyzed for its potential use in the form of antibody otics. After these in vitro studies, antibiotics were tested for in vivo attenuated toxicity and drug Can be evaluated for dynamics. "Antibody" and "immunoglobulin" are antibodies It is meant to include analogs. F. Antibiotic precursors and conjugates   As defined herein, "antibiotic precursor" refers to limited in vitro and Reactant used in the synthesis of semi-synthetic antibiotics with in vivo antimicrobial activity . Therefore, such compounds are not typically used as antimicrobial agents. Yes. However, the present invention provides a "latent" for such compounds by the methods described herein. Intended to activate "resident" antimicrobial capacity in binding to immunoglobulins To do.   In one embodiment, the method comprises the step of using a cross-linking agent as the first step. Derivatizing the biogenic precursors reveals their antimicrobial potential. This These derivatized antibiotic precursors are themselves comparable to free antibiotic precursors. Have an enhanced antimicrobial effect. Second step, derivatized antibiotic It forms a conjugate between the substance precursor and human immunoglobulin. This latter stage , The advantages demonstrated by the conjugates described above (increased half-life, decreased nephrotoxicity, etc. ) Is formed. II. Effects of antibody otics in vitro and in vivo A. In vitro reactivity of Fc region   In the previous section, the main problem is that the antibiotic part of the conjugate is the same as the original antibiotic. Or was it a similar reactivity? However, immune globuli It has to be emphasized that N is not just an inactive carrier. antibody The Fc part of Escherichia coli removes pathogens by two mechanisms different from the effect of antibiotics. Can mediate. First, after binding of the antibody to the antigen, the Fc region activates the traditional complement pathway. It is known to be able to sexualize and ultimately result in the dissolution of organisms. First 2. Due to the binding of the conjugate to bacteria, the Fc region of phagocytes (eg macrophages) Recognition of the organism leads to uptake of organisms or opsonization, and / or killer Cell lysis is guided [LE. Hood et al., Immunology, 2d Ed., The Benj amin / Cummings Publishing Company, Inc., Menlo Park, pp. Visit 339-340 (1984) See).   The present invention provides antibody-antibiotic conjugates having the ability to bind Fc receptors on phagocytes. Is intended. In the competitive binding of the antibody-antibiotic conjugate of the invention Binding to such cells should be substantially the same as for normal IgG Is preferred.   Although the present invention does not activate complement systemically, it binds complement and causes pathogenesis. It is intended for antibody-antibiotic conjugates that facilitate body killing. Furthermore, Fc territory Conjugates that bind to phagocytes via the tract and facilitate pathogen clearance are contemplated. That is , Antibody-antibiotic conjugates in patients treated, opsonization and sepsis Of pathogens of infectious diseases (opsonophagocytosis, opsonophagocytosi s) is intended to be mediated or enhanced. B. Effects of conjugates in vivo   The form in which the conjugate will be used in vivo (for acute conditions, preventative, etc.) ), But the conjugate lies behind a range of overall potency of host immune mediators. It These immune mediators, of course, look like endogenous antibodies to bacteria and their toxins. Humoral immune mediators are included.   In this regard, several studies have linked human humoral immune status to Gram-negative infections. It reports an unexpected relationship between them. Is Pseudomonas aeruginosa a help from sepsis? Titers of total IgG and core antigen-specific anti-LSP levels in patients Significantly higher than that of patients who both died [M. Pollack et al., J. Clin. Invest. 72: 1874-1881 (1983)]. Similarly, I for the patient's infected organism There was a correlation between gG titers and the frequency of shock and death [S.H. Zinner and W.R. McCabe, J. Infect. Dis. 133: 37-45 (1976).]   These studies show that due to weakened humoral immune defenses, Gram-negative sepsis and endotoxicity It has been shown that this may be the case for patients on the verge of primaemia. For this reason books The invention, in one aspect, comprises the immunological condition of the host prior to the administration of the antibodyotic. It is intended to measure the condition. This measurement aligns with total IgG and IgM levels. Screen potentially dangerous groups for endotoxin core antigen-specific IgG and IgM levels Can be performed by training [B.J.Stoll et al., Serodiagnosis an d Immunotherapy 1: 21-31 (198 7)]. Elderly, newborn or premature infant born full moon [W. Marget el al., I nfection 11: 84-86 (1983)], patients with malignant tumor [C. Stoll el al., Infe ction 13: 115-119 (1985)], those who are going to undergo abdominal surgery, long-term catheter Patients wearing or wearing artificial respiration, as well as burns and other Screening is considered to be particularly important for patients with trauma.   If immune conditions are poor (eg low total IgG levels and low levels of antibacterial antibodies). Bell), the effect of antibody-antibiotic conjugates is expected to be the most dramatic. Inn Use of conjugates aids in intrinsic antibacterial defense when the main immune status is good Will be.   For optimal in vivo treatment, the conjugate itself should It must be effective, non-toxic, and non-immunogenic. Therefore, the conjugate of the present invention Are Gram-positive and Gram-negative organisms normally associated with sepsis (eg E. coli, K. pn eumoniae, P. aeruginosa, S. pyogenes, S. aureus, S. epidermidis etc.) Intended to be effective. That these conjugates are non-toxic to the host animal Intended. Like other chemotherapies, the conjugate is effective against infectious organisms It is necessary to do no harm. In addition, infection with subsequent administration of the conjugate To enhance the host's response to the organism and prevent symptoms such as blood disorders, The coalescence itself must be non-immunogenic. This feature makes the host's immune system You can focus on fighting infected organisms, rather than attacking the conjugate for therapeutic purposes. These conjugates may be administered multiple times to the same animal (ie, potentially pathogenic) Of the host) to the conjugate itself, as It is important not to produce antibodies. The production of such antibodies is Cause rapid disappearance of conjugates or cause severe and potentially life-threatening hypersensitivity reactions. Easy to strain.   Non-immunogenic or weakly immunogenic due to high concentration of amino acids in D-configuration Conjugates are also contemplated. Synthetic peptides consisting entirely of D-amino acids are generally immune-reactive. Cannot be generated [M. Se1a. in Advances in Immunology. Vol. Five, (F. Dixon and J. Humphrey, eds,), pp. 29-129 (1966)]. Therefore, the whole Binding of synthetic antimicrobial substances consisting of D-amino acids to antibodies is advantageous in the present invention Will. III. Uses of antibiotics A. Prophylactic use in humans   Diagnosis of sepsis is difficult. First, the progression of sepsis is a constant poison in the circulation It does not require the release of elementary substances nor the presence of organisms in the circulation. Therefore, sepsis Many of the patients who die from the disease have never had bacteremia [R.C. Bone, Ann ． Intern. Med. 115: 457-469 (1991)]. Second, even if bacteria are detected, This detection is time consuming and often too long for practical use.   For these and other reasons, the present invention prior to the onset of symptoms (and thus prophylactically) It is intended for the use of antibody otics in humans. In particular The present invention provides a prophylactic agent for patients at high risk of infection as well as sepsis. It is intended to use antibody otics.   Patients who are at high risk include those who have undergone surgery (especially Elderly patients), children who gave birth with low weight, patients with burns and trauma. Trauma patients are particularly difficult to diagnose because of the variety of invasive processes they undergo. In addition, trauma patients often use intravenous tubes, mechanical ventilators, Foley catheters, etc. Often connected to a dog's equipment. Depending on the degree of trauma and available veins Often, trying to change an IV tube is not possible [ E.S. Caplan and N. Hoyt, Am. J. Med. 70: 638-640 (1981)].   Most patients with multiple traumas have fever due to the stress of the trauma itself. , The white blood cell count increases. Therefore, conventional methods of indicating infection indicate progressive infection. Sometimes it is not shown.   For this reason, current clinical practice indicates that treatment with antibiotics may be relevant for certain indications. I'm going for the shortest time possible. In general, the average course of infections described in the literature The excess is 7-10 days. Prophylactic antibiotics are recommended for open fractures, abdominal penetration injury and respiratory mucus. Used only in the three cases of face-penetration injury with membrane damage. this In these cases, antibiotics are only used for 3-5 days depending on the injury .   In contrast, the present invention prophylactically treats all trauma patients with antibody otics. It is intended to be treated. The reduced toxicity of the conjugate and its circulation Due to the longer dwell time, the present invention allows anti-body injury immediately after admission to trauma patients. It is intended to administer tics. In fact, antibody otic At the first moment when clinical treatment became available (for example, ambulance treatment) It can be successfully administered.   Unlike the short (ie 3-7 days) protection period obtained with intact antibiotics , By using the antibody-antibiotic conjugates of the present invention over the entire dangerous period The trauma patient is protected.   Burn patients have many of the same problems as diagnosis and treatment of infections. Burn This is relevant in acute cases, since the degree of Becomes a problem.   Sepsis appears in blood cultures of burn patients about 4 days after sepsis Reported [M. Meek el al., J. Burn Care Rehab. 12: 564-568 (1991)] . Therefore, treatment with the conjugates of the invention may be used to prevent burn trauma as a means of preventing septic reactions. Is particularly suitable immediately after. In addition, in severe cases, anti-body Consideration should be given to preventing traumatic sepsis by topical administration of ku.   Importantly, burn patients are exposed to both Gram-negative and Gram-positive organisms. Is Rukoto. Burn patients have bactericidal activity against both Gram-positive and Gram-negative organisms. They are particularly suitable candidates for their therapeutic preparation. It is responsive to both groups of organisms A single antibiotic (eg cephalosporin or broad spectrum Antibiotics such as penicillin), and (i) nonspecific immunoglobulins. A first conjugate comprising a bound first antibiotic, and (ii) a non-specific immunoglobulin A second conjugate comprising a second antibiotic covalently bound to brin (eg, the first antibiotic The biological substance is polymyxin and the second antibiotic is bacitracin). Includes therapeutic "mixture" preparations. Alternatively, use two different antibiotics in the same immunoglobin. It can be covalently attached to the brin molecule.   The use of blood cultures, etc. is also useful in diagnosing neonatal sepsis. Has been shown to be unreliable. In fact, culture in clinical practice is at risk Little or no impact on antibiotic treatment policy decisions for active children Seems not to do [T.J. Zuerlein et al., Clin. Ped. 29: 445-447 (1990)] . Therefore, there are great advantages in applying the conjugates of the invention (ie, antibiotics). The substance can be used without fear of toxicity and its longer circulation half-life does not necessarily mean Can be treated with antibiotics without extending the hospital stay).   Also, patients who have undergone surgery can successfully use the conjugates of the invention. It represents a dangerous group. Prophylactic use of antibiotics in current practice Is limited to very narrow areas (eg, selective colorectal procedures, cholecystectomy) , Hysterectomy, and caesarean section) [R.L. Nichols in Decision Making in Surgical Sepsis, B.C. Decker, Inc., Philadelphia, pp. 20-21 (1991)]. Intravenous administration of 1-2 grams of broad spectrum antibiotics during induction of anesthesia Can be Additional doses can be given during the dilatative procedure or after surgery However, prevention for more than 24 hours is not instructed. 24-hour antibiotic prophylaxis It is considered sufficient to control pollution. Antibiotics for more than 24 hours Continuing protection is costly and requires the use of short serum and tissue half-life antibiotics. Especially when Most importantly, continued antibiotic prophylaxis And the excessive risk of the emergence of drug-toxic and resistant strains.   In contrast, as demonstrated in rabbits in the examples herein, Due to the longer serum half-life of the conjugate, multiple injections Prolonged protection against sepsis is given without spending money. further, Since the immunoglobulin distribution is primarily in the intravascular compartment, the use of the conjugates of the invention is Use reduces the risk of rupture of the body compartment system. Therefore, the conjugate of the present invention is free (For example, in more surgical procedure types). B. Treatment of acute symptoms in humans   As pointed out above, the present invention provides a therapeutic preparation for the acute treatment of antibody otic. It is also intended for use in goods. In this case, the treatment would be delayed after the infection was detected. And / or administering an antibody-antibiotic conjugate after suspected sepsis.   Evidence of Gram-negative infection is (1) nuclei above 38 ° C or below 35 ° C. Heart temperature, (2) 12x10⁹Higher than / L or 3x10⁹Peripheral blood leukocyte count lower than / L (chemotherapy (Not by law), or at least 20% immature morphology, (3) 48 o'clock Growth of Gram-negative organisms from blood cultures removed within Areas where rum-negative infections are documented or suspected.   Current medical practice admits that there is no specific medical treatment for sepsis [R.L. Greenman el al., JAMA 266: 1097-1102 (1991)]. Systemic septic reaction Is arterial hypotension (maximum blood pressure less than 90 mmHg or sharp decrease of 30 mmHg), metabolism Acidosis (base deficiency> 5 m Eq / L), low systemic vascular resistance (systemic vascular resistance <800 dies /S.cm^Five), Tachypnea (breathing rate> 20 / min or breathing when mechanically breathing) 10 L / min) or otherwise unexplained renal dysfunction (urine output <30 ml / h) or low lung dysfunction Characterized by at least one.   Antibodies of the present invention are used prior to systemic infection where possible It must be emphasized that is ideal. For example, the conjugate is It can be administered as soon as bacteremia or fungalemia is detected. Similarly, Coalescence is when there is clear evidence of infection at a particular site (eg, trauma, sinusitis). , Meningitis, respiratory system, gastrointestinal system, urinary system infection, etc.).   Primary bacteremia typically occurs in some patients without other apparent sites of infection. Is defined as two or more blood cultures of the same bacterial organism that have arisen. Sinusitis has become Purulent nasal discharge, radiographic image of sinusitis, or purulent substance aspirated from sinus Is diagnosed for patients with at least two of:   The lower respiratory tract is a common site of infection. Pneumonia in an intubated patient has fever, Diagnosed in patients with leukocytosis and numerous Gram stains of polymorphonuclear leukocytes It Pneumonia is a new infiltrate that cannot be eliminated by intensive physical therapy Can also be diagnosed in patients with (this last criterion excludes pulmonary diastolic dysfunction) Useful for). C. Veterinary treatment   Sepsis (septicemia and sepsis) is not limited to humans. Gram Yin Infection with sexually transmitted bacteria causes significant morbidity and mortality in newborn animals such as calves. The cause is [D.D. Morris et al., Am. J. Vet. Res. 47: 2554-2565 (1986)] . Interestingly, the body Humoral immune status is also associated with susceptibility to sepsis, which is primarily This is due to passive transmission from milk. For this reason, the present invention is in one aspect And is intended to measure the immune status of animals prior to administration of antibody otic is there. This measurement screened newborn calves for total circulating serum immunoglobulins. Can be performed (eg, by ELISA).   Conjugates preventively if immune status is compromised (eg low total IgG levels) Should be used for. Conjugate use is high if the animal's immune status is good Gram-negative bacterial sepsis remains prevalent in newborn calves despite having antibody levels It may be necessary for the acute treatment of blood.   The present invention also contemplates treatment of other animals. 10 days after birth in heavy distress In foals within 30 years is the most serious problem [A.M. Hoffman et al., J. Ve t. Int. Med. 6: 89-95 (1992)]. Symptoms that often indicate the risk of sepsis are weakness and metabolism. Disability and dehydration. In one aspect, the invention has such an index. Or for the prophylactic treatment of foals within 10 days of age at risk of infection It is intended to use body otics.   Positive blood cultures are found in less than half of the cases, but these positive It is very unlikely that animals found to survive. Therefore, the present invention was found by culturing. Of any animal with signs of sepsis, whether or not Intended to use antibody otic for acute treatment . IV. Therapeutic preparations and combinations   The present invention contemplates the use of a soluble antibody body therapeutic composition. To do. The invention is also intended to be limited by the particular properties of said therapeutic preparation. Not of. For example, such compositions may be physiologically acceptable liquids, gels, or Or solid carriers, diluents, auxiliaries and excipients. In addition, Antibodies together with other therapeutic agents, including unbound immunoglobulins. May be used for.   As mentioned above, these therapeutic preparations are useful for veterinary medicine in mammals, such as farm animals. Similar to other therapeutic agents for clinical and human clinical use Can be administered by the method. Generally, the dose required for therapeutic effect is the dosage form, administration method , As well as the particular needs of the individual host.   As for the administration method, the antibody otic is intravenous, intramuscular, or arachnoid. It can be used for sub-or topical (including topical ocular) administration. Of such administration The formulation for is an effective amount of antibody otic in sterile water or saline. Including.   On the other hand, the formulations are binders, fillers, carriers, preservatives, stabilizers, emulsifiers, buffers. , Commonly used additives such as excipients, eg pharmaceutical grade mannitol , Lactose, starch, magnesium stearate, sodium saccharin, cell It may include sucrose, magnesium carbonate and the like. These compositions are typically Contains 1% -95%, preferably 2% -70% active ingredient.   The composition is preferably manufactured as an injectable liquid solution or suspension, Solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.   The antibody otic of the present invention is a compatible physiologically acceptable diluent or Often mixed with excipients. Suitable diluents and excipients are, for example, water, physiological Saline, dextrose, glycerol, or a combination thereof. Further In addition, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, Stabilizers or pH buffers may be included.   If you need repeated doses, give a free antibiotic It may be beneficial to first remove all anti-hapten antibodies. After this anti Body otic can be administered. Experiment   The following examples serve to illustrate certain preferred aspects and forms of the invention. Yes, and should not be construed as limiting the scope.   In the description of the following experiments, the following abbreviations are used: eq (equal amount); M (molar concentration Degree); μM (micromolar concentration); N (normal); mol (mol); mmol (mmol) Μmol (micromol); nmol (nanomol); gm (gram); mg (milligram) ); Μg (microgram); ng (nanogram); L (liter); ml (milliliter) Torr); μl (microliter); cm (centimeter); mm (millimeter) ); Μm (micrometer); nm (nanometer); ℃ (Celsius); AUFS ( Absorption units full scale); hr (time); sec ( Second); min (minute); IV (intravenous); Ig (immunoglobulin), FCS (fetal calf serum) , CFU (colony forming unit); ELISA (enzyme-linked immunosorbent assay); Aldrich (Aldrich Ch emical Co., Milwaukee. WI); Amicon (Beverly.MA); Baxter (Deerfield, I) L); BBL (Becton Dickinson Microbiology Systems. Cockeysville. MD); Bio -Rad (Richmond, CA); Corning (Corning, Inc., Corning, NY); Falcon (Lin coln Park, NJ); Lee (Lee Laboratories, Grayson, Georgia); Harlan Sprag ue-Dawley (Harlan Sprague-Dawley, Indianapolis, IN); ICN (ICN Biomedica ls, Costa Mesa, CA); Mallinckrodt (Mallinckrodt. St. Louise, MO); Pharm acia (Pharmacia, Inc., Piscataway, NJ); Pierce (Pierce Chemical Co., Ro ckford, IL.); Prochem (Prochem. Inc., Rockford, IL); Scientific Produ cts or S / P (Scientific Products. McGraw Park, IL); S & S (Schleicher & Schuell, Inc., Keene, NH); Sigma (Sigma Chemical Co., St. Louis, MO.) Spectrum (Spectrum, Houston, TX); Whatman (Whatman, Inc., Clifton, NJ ).   In some of the examples below, purification of the product from the reactants was performed at various times. Column chromatography. Use standard terms understood by those of ordinary skill in the art. Used to describe the purification of For example, the "eluent" is bound to the column matrix. The desired combined product (if present) can be dissociated and the column matrix It is a chemical solution that passes through the column and contains the "eluate". Dissociated product (if present Is released from the column matrix, and the “eluate” is obtained by elution with the “eluent”. It will be.Example 1 Binding of Antibiotics to Human IgG Using a Carbodiimide Crosslinker   This example describes an attempt to attach an antibiotic to a carrier (in this case an antibody). Things. In this regard, K. Hanasawa et al. On the chemistry of carbodiimide Have described the binding of PMB to fixed fibers [Surg. Gyn. & Ob. 168: 323- 331 (1989)]. In this example, polymyxin B (PMB) was bound to human IgG. The ability of the carbodiimide crosslinker to do so was investigated.   1-Ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) It is known to crosslink proteins and peptides between mines and carboxylic acids . In this example, (a) EDC-mediated cross-linking of PMB and IgG, and (b) LPS. An enzyme-linked immunosorbent assay (ELISA) of the conjugate that binds to is used. a) EDC-mediated cross-linking of PMB to IgG   In all examples, including this one, exogenous pyrogens (eg LPS) Inhibit the binding reaction, absorb PMB or conjugate, or block the activity of PMB conjugate. Locks glassware, solutions, and other materials and reagents Measures were taken to exclude it.   Human IgG (Sigma) and PMB (Sigma) each containing a pyrogen at a concentration of 8 mg / ml. MES (2- [N-morpholino] ethanesulfonic acid) buffer solution (0.1 MMES, 0.09M  It was dissolved in NaCl, pH 4.7). Mix 0.5 ml of each solution and add 0 of the mixture. .15 ml of 0.4M EDC, 0.2M EDC, 0.1M EDC (Pierce) in MES buffer or control Incubated with 0.15 ml of the solution for 2 hours at room temperature. 2.7 ml TBS (50 mM Tris-HCl , 150mM NaCl, pH7.2) The reaction was stopped more. Do this 5 times separately against 1500 ml of PBS four times Exchanged and dialyzed for 36 hours at 4 ° C (dialysis tube molecular weight cutoff is 12-14,000 , Scientific Products). Samples containing 0.2 mg / ml human IgG were stored at 4 ° C. b) Enzyme-linked immunoassay of EDC-PMB conjugate bound to LPS   To determine if the binding of PMB facilitated the binding of IgG to LPS, A simple indirect binding assay was performed. 96-well microtiter plate (Fa lcon) in each well with E. coli in PBS. 1 of 2.0 μg / ml solution of coli 0111: B4 LPS (Sigma) 00 μl was coated, but for control wells PBS without LSP was added. 4 ° C After overnight incubation at room temperature, the coating solution was decanted and all Wells were washed 3 times with PBS. 5 mg / ml bovine serum albumin (BSA, Sigma) The non-specific binding sites were blotted for 2 hours at room temperature by adding 100 μl PBS containing I clicked. After removing the block solution by decantation, remove the odor in (a) above. A sample of the conjugate prepared by the above procedure was diluted to an initial concentration of 10 μg / ml IgG in PBS containing 1 mg / ml BSA. And then serially diluted 5-fold. Commercially produced rabbit anti-E. coli 0111 : B4 antiserum (Lee Laboratories, lot M25082) positive control antiserum first 1: 100 Diluted to. Incubate two 100 μl of each sample for 2 hours at room temperature and play BBS-Tween 20 (0.1M boric acid, 0.025M Na borate, 1.0M NaCl, 0.1% Tween 20, pH Washed 3 times with 8.3) and twice with PBS-Tween20 (0.1% Tween20 (v / v)) And finally washed twice with PBS.   Incubated with human antibody conjugate to detect bound antibody Wells of goat anti-human IgG (whole molecule) -alkaline phosphatase conjugate (Sigma) 1 Incubate with 100 μl of 500 dilution and incubate with rabbit serum. Plated wells with goat anti-rabbit IgG (all molecules) -alkaline phosphatase conjugate ( Sigma) and incubated with 100 μl of a 1: 500 dilution for 2 hours at room temperature. First 2 Discard the antibody solution and wash the plate with BBS-Tween 20 and PBS-Tween 20 as above. Purified, then 50 mM Na₂CO₃， 10mMMgCl₂, Washed twice at pH 9.5. After 45 minutes at room temperature Absorbance at 410 nm in each well, using the diluent control wells as blanks for Dyn Measured on atech MR700 plate reader. Tables 6 and 7 show rabbit control serum and And results for ECD-conjugates.   The results in Table 6 show that the positive control sera were specific for LSP-coated wells as expected. It is shown that they are bound in various forms. With these data, the design of ELISA is LSP It is confirmed that the bound antibody can be detected.   The results in Table 7 seem to indicate that IgG-PMB bound to LSP by ECD-crosslinking. Is. However, between 5 and 1 μg / ml the titration of conjugate drops relatively suddenly. To confirm that the binding observed was specific, binding was blocked by PMB. Must be antigen-dependent, not possible .   The above ELISA was repeated, but in this case the highest binding by ELISA The concentration of EDC-conjugate (10 μg / ml) was fixed and polymyxin B was 5 times continuous. Incubation with dilutions (starting at 10 mg / ml) and LPS-binding activity was measured. In addition, binding was tested in control wells containing no antigen. The results are shown in Tables 8 and 9 Shown in. Binding of the conjugate was inhibited only at the highest PBS concentration tested and binding Observed binding was observed as the body showed significant binding to wells containing no antigen. Clearly, most of the cases are not specific. This is PMB and I It may indicate an inappropriate type or number of linkages between gGs, and Since IgG that has not been treated with a cross-linking agent shows almost no binding to LSP, the The bridge indicates that nonspecific binding is generated.   Apparently, the carbodiimide chemistry does not work at a practical level. In fact It is clear that the interaction of cross-linking agents with antibiotics is rather complex. PMB and P We must consider that there are three types of reactions possible: MB, IgG and IgG, and IgG and PMB. Yes. Only the last reaction is useful. Example 2 Binding of Antibiotics to Human IgG Using Disuccinimide Ester   Various cross-linking agents and chemistries have been tried in an attempt to overcome the difficulties seen with EDC conjugates. The effect was tested. Talmadge and Siebert test hydroxysuccinimide ester PMB binding via drugs is described [J. Chrom. 476: 175-185 (1989)]. this According to the method, this example spans peptides and proteins via their amine groups. A homobifunctional cross-linking agent that bridges, bis- (N-hydroxysuccine suberate) The ability of imide ester) (DSS) to bind PMB to IgG was examined. Implementation Examples are (a) DSS-mediated cross-linking of PMB and IgG, and (b) binding that binds to LPS. Including body ELISA. a) DSS-mediated cross-linking of PMB to IgG   Prepare pyrogen-free PBS in pyrogen-free water (Baxter) and Stock solutions of gG (40mg / ml) and PMB (40mg / ml) in pyrogen-free PBS Dissolved. Prepare a 60 mM stock solution of DSS in 100% dimethyl sulfoxide (DMSO). Made. When this solution was diluted to 6.0 mM DSS in PBS, some precipitation was observed. It was Stock solution of human IgG and PMB containing 20mg / ml IgG and 20mg / ml PMB in PBS Was prepared. A 2-fold serial dilution of DDS stock solution (0.15 ml) was added to a fixed volume (0.15 ml). ) IgG / PMB stock solution to prepare 5 different conjugates It was The five DSS concentrations obtained were 3.0 mM, 1.5 mM, 0.75 mM, 0.375 mM and 0.0 mM DSS. there were. After 1 hour incubation at room temperature, add 2.7 ml TBS The reaction was stopped more. A mixture of the five is described in Example 1 for the EDC conjugate. Dialyzed against PBS as listed. The dialyzed conjugate obtained was 1 mg / ml IgG It had a final concentration of and was stored at 4 ° C. b) ELISA of DSS bound to LSP   The ELISA was performed using an initial concentration of 10 μg / ml DSS conjugate and the same control rabbit anti-E. coli 0111 The same procedure as in Example 1 (b) was performed using: B4 antiserum. First join up The results of the sey are shown in Table 10.   The results show a low level of binding that correlates with the concentration of DSS used. The specificity of this binding is then determined by its ability to inhibit PMB binding and its dependence on antigen Tested by examining sex. The assay was performed on EDC conjugates in Example 1 (b). The procedure was the same as described above. The results are shown in Tables 11 and 12.   These results indicate that the DSS conjugate binds somewhat nonspecifically . The pattern of PMB inhibition showed that the highest concentrations showed no inhibition of binding, with intermediate PMB concentrations It is unusual in that it obviously interferes.   These results show that some level of specific binding exceeds a significant amount of nonspecific binding. Is showing. Control rabbit sera at 1: 500 and 1: 12,500 dilutions bound 1 each .766 and 0.380, which are virtually all antigen-dependent. Relatively low here The total level is Hydroxysuccinimide ester reagents like DSS crosslink for PMB and IgG It shows that it is not very effective as a drug. This is the amine-ami used It may also be due to the chemical action or the nature of the DSS reagent. We are DSS PB Attention was paid to the degree of insolubility in S. Perhaps the more water-soluble form of DSS It will perform well. Furthermore, for both EDC and DSS conjugates, PMB is exposed to a large excess of crosslinker, which binds to PMB's LPS The ability could be hindered. Example 3 Two-step binding of PMB to IgG using water-soluble analogues of EDC and DSS   In the two preceding examples, the cross-linker was present in molar excess over IgG and the antibody And both antibiotics were mixed at the same time. In this example, IgG is first cross-linked. Modified with agent to remove crosslinker, then PMB was added to the coupling reaction. This one The method should improve the binding activity of PMB and reduce the non-specific binding of IgG. U To obtain a more water-soluble amine-amine coupling reagent, BS, a soluble analogue³(Pierce) was used. This embodiment uses (a) EDC IgG-PMB two-step binding; (b) BS³-Stage binding of IgG-PMB using And (c) ELISA of conjugates that bind LPS.   a)Two-step binding of IgG-PMB using EDC   0.75 ml of a 4 mg / ml concentration of IgG solution dissolved in MES buffer is described in Example 1. And 0.45 EDC solution 0.75 ml dissolved in MES buffer at room temperature for 2 hours Mixed for a while. Inject into a sterile 10 ml pipette with pyrogen-free MES buffer Pass 1.5 ml of reaction mixture through an equilibrated Sephadex G-10 (Pharmacia) column. The unreacted crosslinking agent was removed by. Collect the flow-through fractions and dilute each fraction 1:40 OD₂₈₀Was measured to determine the IgG content. Contains 2.37 mg of IgG per ml The peak fraction that was divided into two fractions. Add 1.5mg of PMB to one side and dissolve , Nothing was added to the other (control). TB after overnight incubation at room temperature Stop the reaction with S to bring the final IgG concentration to 0.2 mg / ml. I adjusted. Both samples were dialyzed as described in Example 1 (a) and stored at 4 ° C. It was   b)2-step binding to PMB of IgG with BS ³   0.75 ml of 20 mg / ml IgG solution was added to 6.0 mM BS.³Mixed with 0.75 ml of solution. Each melt The solution was prepared by dissolving in PBS, and the mixture was incubated at room temperature for 1 hour. Unopposed The appropriate crosslinker was removed as described in Example 3 (a) above and the peak IgG fraction was removed. Identified and pooled. Make two equal fractions with an IgG concentration of 8.35 mg / ml , 7.5 mg of PMB was added to one and dissolved, nothing was added to the other. One at room temperature After overnight incubation, the reaction was stopped with TBS and the conjugate was dialyzed and reconstituted. The final IgG concentration was adjusted to 1.0 mg / ml.   c)ELISA of conjugates that bind LPS   This LPS binding assay was performed without a BBS-Tween20 wash and also with a PBS-T Example (1 except that the concentration of Tween 20 in washing ween 20 was lowered to 0.05% (v / v) ). The results are shown in Tables 13 and 14.   The 0.2M EDC IgG-PMB conjugate showed high levels of binding but contained LPS This is partially nonspecific, as evidenced by binding to control wells It was due to the combination. Further evidence of non-specific binding caused by EDC cross-linking However, the PMB-free conjugate (this is somewhat comparable to the presence or absence of antigen) (Showing binding with wells at enemy levels).   BS³The conjugate showed no specific binding to LPS at the concentrations tested. . However, they also show no significant non-specific binding and do not induce non-specific binding of IgG. That this crosslinker may not be as problematic as EDC in terms of causing Indicated.   BS³The background binding of the conjugate was low, so the concentration of this conjugate was And the concentration of LPS applied to the well is 10 times (2 μg LPS / wafer). And the ELISA was performed again. This increased the sensitivity of the assay. The results shown in Table 15 are BS³Conjugate increases LPS binding activity above background Indicates that it has.   At the same time, the results for the two-step conjugates described in this example show that EDC is acceptable. Is a water-soluble analogue of DSS, which results in unspecific levels of nonspecific binding BS³Has a modest level of specific binding in the form of this two-step conjugate and is non-specific It shows that the physical combination hardly occurs. BS³Obtained by using To determine whether it is possible to achieve high levels of LPS binding activity of Another two-step conjugation method with the above cross-linking agent was tested. Example 4 PMB and IgG using amine-sulfhydryl coupling chemistry by SMCC Three-stage binding   In the previous example, the highly specific binding between the IgG-PMB conjugate and LPS was examined. As T.T. Kitagawa et al. [J. Assoc. Anal. Chem. (1985)] in a three-step method similar to that of sulfosuccinimidyl 4- (N-male). Imidomethyl) cyclohexane-1-carboxylate [sulfo-SMCC] Was used to test another crosslinking method. In this example, (a) P using sulfo-SMCC was used. ELI of a conjugate that binds MB and reduced IgG, and (b) LPS SA was included.   a)Three-step binding of PMB IgG   In the first step of this method, the Ig was treated by treatment with 2-mercaptoethanol. A reactive thiol group was created in G. In this method, first, 4.0 mg of IgG Pyrogen free 0.1M NaPO_Four It was dissolved in 0.45 ml (pH 6.0). Next, 5 mM EDTA, 0.1 NaPO_Four, Dissolved in pH 6.0 0 Add 50 μl of 1 M 2-mercaptoethanol and incubate at 37 ° C for 1.5 hours It was Sample 0.5 ml with 0.1 M NaPO_Four, 5mM EDTA, 5ml equilibrated with pH 6.0 Sephadex G -10 column to separate free 2-mercaptoethanol and contain IgG Fractions were identified and pooled.   The second step of this method involves the preparation of PMB activated by maleimide. did. It is soluble in 50 mM sodium borate buffer pH 7.6 (pyrogen free) A solution of 0.16 mg / ml of PMB and 1.5 ml of PMB solution was dissolved in the same borate buffer solution. Included a mixture of 1.5 ml of 46 mg / ml sulfo-SMCC (Pierce) solution (for each reaction The final concentration was 0.053 mM). This "SMCC activated" PMB at 30 ° C 6 Incubated for 0 minutes.   In the third step of this method, 0.65 ml of reduced IgG was activated by SMCC. Incubation with 0.65 ml of PMB.   The concentrations of these two reactants were 0.0265 mM PMB and 0.013 mM IgG (molar The ratio was 2: 1). After incubating for 20 hours at 4 ℃, 0.1M 2-mercapto 8.7 μl of a fresh solution of ethanol was added and incubated at room temperature for 20 minutes. Equal The IgG concentration was adjusted to 1.0 mg / ml with PBS. Conjugate sample to 2 x 800 ml P By dialysis against BS for 20 hours, Was purified by gel filtration using a Sephadex G-10 column equilibrated with PBS. A control reduced human IgG fraction was prepared from the reduced IgG pool and three preparations were prepared at 4 ° C. Saved in.   b)ELISA of conjugates that bind LPS   LPS binding assay was performed by applying 2 μg of LPS per well, BBS -No Tween 20 washes and Tween 2 in PBS-Tween 20 washes Same as the method described in Example 1 (b) except that the 0 concentration was reduced to 0.05%. Met. Blocking solution and sample dilutions are pyrogen-free PBS and And low endotoxin BSA (Sigma). The results are shown in Table 16.   Although SMCC IgG-PMB showed slightly higher binding to LPS than the control. , The overall binding level was that of the positive control rabbit anti-E. Coli 0111: B4 antiserum. (1.097 at 1: 25,000 dilution). Reduced IgG used for cross-linking Since it may have only a few thiol groups that can be formed, higher concentration of activity PMB may promote the reaction more effectively.Example 5 Binding of IgG to antibiotics without bifunctional crosslinker   In all of the previous examples, the antibiotic polymyxin was covalently linked to IgG. A free bifunctional crosslinker was used in an attempt. The resulting configuration is Comparable to LPS binding activity of immune serum Could not yield a conjugate with LPS binding activity. In the absence of antigen Since binding was observed in, probably one active PMB molecule was added to each IgG molecule. There may have been conjugates that had less than individual pieces. Antibiotics without the use of bifunctional crosslinkers Periodate of the carbohydrate group of IgG to test the means of binding substances to IgG PMB using acid oxidation (DA Handley, European Patent Application Publication No. 428486) Amine-reactive alkanes that have the potential to react with and be reduced to achieve stable covalent bonds. The dehydration group was prepared.   In this example, (a) the period of IgG in a sodium acetate buffer solution having a pH of 4.0 was used. Binding of polymyxin B with IgG that has been subjected to oxidative oxidation and (b) periodate oxidation Included.   a) Periodate oxidation of IgG in sodium acetate buffer at pH 4.0 is 5 m g human IgG was dissolved in 1 ml of water, and this solution was added to 200 μl of sodium acetate pH4.0 ( H₂0.3 g of sodium acetate and 960 μl of glacial acetic acid dissolved in 100 ml of O) and 200 μl of 0. 2M NaIO_FourWas achieved by mixing with [J. W. Goding,Monoclonal Antibod ies: Principles and Practice , Academic Press, New York, p. 84 (1986) strange Law]. After leaving it at room temperature for 15 minutes in the dark, use the P-10 column and 50mM Na.₂CO₃, Using pH 9.5 The periodate solution was removed by gel filtration.   b) For the binding of periodate-oxidized IgG and PMB, 10 mg of PMB was added to the above (a ) Was added to IgG prepared in step 1) and incubated at room temperature for 1 hour, then 100 μl of NaBH₃ Add CN (4mg / ml), incubate at room temperature for another hour, then in PBS overnight 4 It was carried out by dialysis at ° C.   Examination of LPS binding activity (data not shown) showed that the prepared conjugate was inactive. It was shown to be sex. These results show that periodate of IgG without cross-linking agent Suggests that oxidation is an ineffective means of covalently linking antibiotics and antibodies ing.Example 6 Derivatization of antibiotics with cross-linking agents: preservation of antibiotic activity   Significant implications for one-step or multi-step methods for conjugating antibiotics to antibodies The question is whether the method of attachment reduces or inactivates antibiotic function. To determine the optimal crosslinker concentration for derivatization of PMB in a multi-step binding method Therefore, the effect of crosslinker concentration on antibiotic activity was confirmed (see Mode IA above). reference). In this example, (a) modification of PMB with SPDP and content of free crosslinker were performed. Release, and (b) assay of antimicrobial activity of derivatized PMB was included.   a) Modification of PMB with SPDP and separation of free cross-linking agent PMB was mixed in three different molar ratios (2: 1, 3: 1 and 4: 1) It was 2.1mg, 3.15mg or 4.2mg SPDP dissolved in dimethyl sulfoxide (Pierce) dissolved in 0.5 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0 5 mg Of PMB and incubated for 30 minutes at room temperature with occasional agitation. Then P Chromatography on a 15 ml Swift desalting column equilibrated with BS-EDTA Free crosslinker was removed from each sample by. Peak image containing derivatized PMB The minutes were collected and pooled.   b) An assay for the antibacterial activity of derivatized PMB is based on the disk inhibition assay (see Figure 2). )) Was carried out. Trypticase Soy Agar (TSA; BBL) Plated on and made a confluent layer of bacteria. Next, 1/4 inch blank paper A disc (BBL) was applied to the surface of the layer and 20 μl of each test solution was applied. Three After an overnight incubation at 7 ° C, the inhibition zone surrounding the disc was observed. this Results (data not shown) show that SPDP vs. PMB is either 2: 1 or 3: 1. PMB derivatized as a ratio is active, compared to a molar ratio of 4: 1 The ratio derivatized antibiotic is shown to be inactive. Therefore, SP Derivatization of PMB with DP resulted in a ratio of SPDP to MB of 3: 1 or Must be smaller than this.Example 7 Binding of SPDP-PMB to IgG   In Example 6, the concentration of SPDP crosslinker that preserves the antibiotic activity of polymyxin B was determined. Once determined, the derivatized antibiotic was treated with the Traut's reagent using the sulfhydryl group (- SH) A conjugate between SPDP-PMB and IgG by reacting with the introduced IgG Was prepared.   This example uses (a) derivatization of PMB with SPDP and (b) Traut reagent. Derivatization of IgG, (c) Traut-IgG binding to SPDP-PMB, And (d) evaluation of the LPS binding activity of the conjugate was included.   a) Derivatization of PMB with SPDP was carried out using 50 μl of dimethyl-sulphoxy. 1ml of 7μmol SPDP (2.1mg) Of 10 mM PMB dissolved in 50 mM sodium borate, 300 mM NaCl, pH 9.0, It was carried out by incubating at room temperature for 30 minutes using a rotary shaker. Trial Charge 20mM NaPO_Four, Equilibrated with 150mM NaCl, 1mM EDTA, pH7.2 (PBS-EDTA) 15ml It was applied to a Swift desalting column (Pierce) to remove unbound crosslinker. Pee Fractions were pooled and stored at 4 ° C.   b) Derivatization of IgG with Traut's reagent was performed in a 5-fold molar excess (concentration 0.2 mg / ml Stock solution (100 μl) of Traut reagent (pierce) 1 ml of 50 mM triethanolamine, 0.1 Add 5 mg IgG dissolved in 5M NaCl, 1 mM EDTA, pH 8.0, and store under nitrogen for 45 minutes. Incubation was carried out at a warm temperature. P-1 equilibrated with excess Traut reagent in PBS-EDTA It was removed by gel filtration using a 0 column. The peak fractions were combined.   c) The binding between Traut-IgG and SPDP-PMB is 3.5 mg Traut-IgG. 18 hours after adding IgG and 2 mg of SPDP-PMB (77 times molar excess of PMB) It was carried out by incubating at room temperature. P-10 color equilibrated with PBS-EDTA Separation of the conjugate from free SPDP-PMB by gel filtration using a membrane (50 ml), Peak fractions containing IgG were pooled, pooled and stored at 4 ° C.   d) Evaluation of the LPS binding activity of the conjugates was performed by measuring the LPS binding ability of each conjugate in (c). It was performed by evaluation in an ELISA assay (see Figure 4). This result is The Traut IgG-PMB conjugate was shown to have limited binding activity (Data not shown).Example 8 Binding of SPDP-PMB to SPDP-IgG   In Example 7, Traut's reagent did not yield a conjugate with retained antibiotic activity. Since it was confirmed, the derivatized antibiotic was treated with SPDP to activate the amino group. (NH₂) Is reacted with IgG converted to a sulfhydryl group (-SH) to give SPDP- The conjugate was prepared between PMB and IgG.   In this example, (a) derivatization of PMB with SPDP and (b) SPDP were used. Derivatization of IgG, (c) binding of SPDP-IgG and SPDP-PMB, and (D) Included evaluation of LPS binding activity of the conjugate.   a) Derivatization of PMB with SPDP was performed as described in Example 7. did.   b) Derivatization of IgG with SPDP was carried out by adding 20 μl of 20 mM SPDP to 1 ml of Add 10 mg IgG dissolved in 50 mM sodium borate, 300 mM NaCl, pH 9.0 and shake. It was carried out by incubating at room temperature for 30 minutes. Free cross-linking agent, 100 mM vinegar 15 ml Swift desalting column equilibrated with sodium acidate, 100 mM sodium chloride, pH 4.5 It was removed by chromatography using. Collect the peak fractions and use Centriprep-3 It was concentrated using a 0 concentrator (Amicon). To this sample, add 250 μl of 100 mM sodium acetate. Add 7.7 mg of dithiothreitol dissolved in 100 mM sodium chloride, pH 4.5. And incubated for 30 minutes at room temperature. 15 ml equilibrated with PBS-EDTA Reapply the sample to the Swift desalting column at₂₈₀Peak image with values Collect the minutes, pool, Concentrated using a Centriprep-30 concentrator (Amicon).   c) The binding between SPDP-IgG and SPDP-PMB is a combination of the following reaction products. Add a sprinkle:       -5 mg SPDP-IgG and 2 mg SPDP-PMB        (43 times molar excess of PMB)       -2 mg SPDP-IgG and 2 mg SPDP-PMB        (107 times molar excess of PMB) It was carried out by incubating at room temperature for 18 hours. Equilibrated with PBS-EDTA By gel filtration using a P-10 column (50 ml), the conjugates were separated into free SPDP- Separated from PMB. Fractions containing PMB-IgG conjugates were pooled, pooled, Stored at 4 ° C.   d) Evaluation of the LPS binding activity of the conjugates was performed by measuring the LPS binding ability of each conjugate in (c). These conjugates were evaluated in an ELISA assay and Traut binding prepared in Example 7 was performed. It was carried out by comparing with the body. The result (see FIG. 5) is SPDP-Ig. Both G-PMB conjugates have significant activity, namely Traut IgG-PMB binding It has been shown to have significantly higher activity than that of the body.Example 9 Binding of PMB and IgG using long chain SPDP cross-linking agent   SPDP is an effective agent for derivatization and cross-linking of IgG and PMB It was demonstrated that a longer spacer arm was added between IgG and PMB. To determine whether it enhances the activity of the conjugate. Luho-LC-SPDP) was tested. In this example, (a) sulfo-LC-SPD was used. P Derivatization of PMB used, (b) Induction of IgG with sulfo-LC-SPDP Incorporation, (c) Binding of derivatized IgG to derivatized PMB, and (d) ELISA Evaluation of the conjugate activity according to   a) Derivatization of PMB with Sulfo-LC-SPDP was performed at a concentration of 9 mg / ml. 35 μl of e-LC-SPDP solution was added to 1 ml of 50 mM sodium borate, 300 mM NaCl, pH9. By adding 10 mg PMB dissolved in 0.0 and incubating at room temperature for 30 minutes Carried out. Gel filtration using a 1.5 x 35 cm P-2 column equilibrated with PBS-EDTA Free crosslinker was removed by filtration. Combine peak fractions containing derivatized PMB And stored at 4 ° C.   b) Derivatization of IgG with Sulfo-LC-SPDP yielded 0.3 mg of sulfo-LC-SPDP. LC-SPDP dissolved in 50 mM sodium borate, 300 mM NaCl, pH 9.0 10 mg Performed by incubating for 30 minutes on a rotary shaker in addition to IgG did. 5 ml S equilibrated with 100 mM sodium acetate, 100 mM sodium chloride, pH 4.5 Derivatized IgG was separated from free crosslinker using a wift desalting column (Pierce) and Oak fractions were collected and pooled. Next, add 250m of the same sodium acetate buffer to this sample. Add 7.7 mg of dithiothreitol dissolved in μl to reduce and incubate at room temperature for 30 minutes. I had a cube. Gel using 10 ml P-10 column equilibrated with PBS-EDTA Excess reducing agent was removed by filtration. Peak fractions were collected and pooled.   c) Derivatized IgG and derivatized PMB are bound to each other by adding 2.5 mg IgG to 2.5 mg PM. B (107-fold molar excess of PMB), and 3.5 mg IgG to 1.4 mg PMB ( 43 times molar excess of PMB) In addition, it was carried out by incubating at room temperature for 18 hours. PBS-EDT The IgG-PMB conjugate was retained using a 50 ml P-10 gel filtration column equilibrated with A. Separated from the reaction mixture.   d) The sulfo-LC-SPDP conjugate was evaluated for the activity of the conjugate by ELISA. It was shown to have no greater activity than the short SPDP molecule (see Figure 6).Example 10 Inhibition of specific binding between antibodies and LPS by free antibiotics   To confirm whether the antibody-antibiotic conjugate binding observed in Figure 5 is specific , Blocked conjugate binding using free antibiotics (see Mode III above). Please refer). In this example, (a) a mixture of antibody otic and free antibiotics is used. And (b) the conjugate and LPS in the presence of different concentrations of free antibiotic. A degree of binding assay was included.   a) Antibodyotic and free antibiotic mixture contains 1 mg / ml BSA 1:12 of SPDP IgG-PMB conjugate dissolved in PBS-Tween 20 (0.05%) 5 dilutions (32 μg / ml) and polymyxin at a concentration of 0-20 μg / ml dissolved in the same buffer Was added by adding an equal amount. Then 0-2 μg PMB and 3.2 μg of conjugate 200 .mu.l of this mixture containing .alpha. Were assayed for binding activity.   b) the degree of binding between the conjugate and LPS in the presence of different concentrations of free antibiotic. The assay was carried out in the same manner as in Example 1, except that 200 μl of antibodies / free antibiotic mixture was added. 2 μg E. coli 01 as described 11: B4 LPS coated and blocked 96-well microtiter plate clot It was carried out by adding to L. Wells were washed and goat anti-human Ig-alkaline Add a phosphatase and use a MicroELISA reader exactly as in Example 1. Binding was assayed quantitatively.   The results are shown in Fig. 7. These results show that the free polymyxin is IgG-PMB and LP. It is shown to competitively block the binding of S. Obviously, anti body Is specifically bound to LPS (ie, via the PMB part that becomes the conjugate). do it).   The inhibition curve was examined by antibody 16 μg / ml solution (l.1 x 10^-7M) concentration is 40 ng / ml (2.6 x 1 0^-8M) PMB is blocked in binding to LPS by 50%, A slight degree of prominent PMB binding is shown. If there is one PMB molecule for each PMB If you do (PMB concentration for IgG 1.1 x 10^-7Equal to), equimolar concentration It would be expected that some degree of free PMB would block binding by 50%. This anti Since that l / 4 concentration of free PMB is needed to block body otic, Conclude that there is at least one PMB molecule for every four IgG molecules. You can In fact, SPDP-modified PMB has 4-fold lower antibiotic activity than free PMB. The actual degree of binding between IgG and PMB is higher than that calculated above since Probably at least 4 times higher (ie, likely for each IgG molecule At least one PMB is bound).Example 11 Binding of IgG in NaPO ₄ using periodate oxidation   In Example 5, a procedure for coupling an antibiotic to IgG without the use of a bifunctional crosslinker. A step (ie periodate oxidation of the carbohydrate groups of IgG) was attempted. This is one Part contains periodate oxidation of IgG in pH 4.0 sodium acetate buffer, It was not possible to produce a conjugate with different activity. This failure depends on the reaction conditions It may have been different, so different reaction conditions were explored. In this example, (a) Periodate oxidation of IgG in phosphate buffer, and (b) polymyxin B and period Includes binding of iodate-oxidized IgG.     a) Periodate oxidation of IgG in phosphate buffer was performed using 10 mg of human IgG. 1 ml of 50 mM NaPO_Four， Dissolved in pH7.2, 0.011g sodium metaperiodate (final Concentration of 50 mM). After leaving at room temperature for 30 minutes, 50 mM NaP O_Four, Periodic by gel filtration using a 10 ml P-10 gel filtration column equilibrated at pH 7.2. The acid was removed. The peak fractions containing antibody were pooled and concentrated to 1.5 ml.     b) For the binding of periodate-oxidized IgG and PMB, use 10 mg of PMB in (a) above. Add 5 mg or 3 mg of prepared IgG and incubate at 4 ° C overnight with gentle shaking. Incubate and then 20 mM NaPO_Four, 0.1 mg / ml NaBH dissolved in pH 6.5. Room with CN It was carried out by reducing at warm temperature for 3 hours. For gel filtration using a 10 ml P-10 column The IgG-PMB conjugate was separated from the remaining reaction products.   Examination of LPS binding activity (see Figure 8) confirmed that the prepared conjugate was active. Indicated. This is in contrast to the conjugate prepared in Example 5 Target.Example 12 Antibacterial activity of IgG-PMB conjugate   Since it was confirmed which conjugate of IgG-PMB has LPS binding activity, The biological activity of the body was examined (see discussion of Mode IV above).   Since polymyxin has direct antibiotic activity, the bound polymyxin also It was also considered to be active. Whether the conjugate has some antimicrobial activity To confirm, IgG-PMB bound by SPDP (107x PMB) IgG-PMB conjugate (PMB) via molar excess, Example 8) and periodic acid : IgG is 3: 1 ratio, minimum inhibitory concentration (MIC) and minimum for Example 11) The bactericidal concentration (MBC) was measured. This example includes (a) preparation of E. coli inoculum, (b) MIC measurements and (c) MBC measurements were included.   a) To prepare the E. coli inoculum, first incubate E. coli HB101 overnight on TSA agar. It started by culturing at ° C. Bacteria count 1.2x1 in sterile saline 0⁸Suspend at a concentration of 1 / ml and then trypticase-Soy Broth (TSB; BBL) 5x10^FiveIndividual/ Diluted to ml. This concentration can be confirmed by dilution plating. I accepted.   b) Measurement of MIC for each conjugate and native polymyxin B control is 5x10 degrees^Five0.5 ml of each inoculum / ml was added to 0.5 ml of the 2-fold dilution series of each conjugate. 12 x 75 mm, sterile It was performed by incubating overnight at 37 ° C. in a culture test tube. MIC is acceptable Defined as the lowest concentration of conjugate or PMB that resulted in complete inhibition of visual growth .   For the PMB control, the MIC was found to be 0.03 lμg / ml, while the SP The MIC was found to be 0.25 mg / ml for the DP conjugate. 3: 1 (PM B: IgG) Periodate conjugate was found to have a MIC of 0.031 mg / ml. This is about 1000 times higher than that for native PMB, and SPDP IgG- 8 times lower than the number for PMB conjugate. Therefore, the IgG-PMB conjugate is Both retain antibacterial activity, and the periodate conjugate has the highest activity. Show. The difference between the numbers of PMB and IgG-PMB is that if PMB activity is complete during binding. , And if one PMB molecule binds to each IgG molecule, Ig Ig in that the MIC will increase 100-fold due to the size of G It partially reflects the larger size of G (about 100 times PMB). Observed The 1000-fold change that the PMB activity was reduced by binding and / or Suggest that not all IgG molecules are bound. However , When small surface-active antibiotics bind to much larger proteins, It is surprising that bacterial growth can be prevented.   c) The MBC of each conjugate was measured by allowing it to grow on TSA agar overnight at 37 ° C. Was performed by plating serial dilutions of the mixture in (b) above that did not show It was MBC is a PMB conjugate that causes over 99.9% killing of viable bacteria in the primary inoculum. Was defined as the lowest concentration of For PMB, MBC should be 0.031 μg / ml Is decided For SPDP IgG-PMB, 0.5 mg / ml, for periodate conjugate It was 0.031 mg / ml. Ig that suppresses bacterial growth and kills contacting bacteria The ability of G-PMB conjugates indicates that these compounds are effective in preventing or treating bacteremia. Suggesting that it can be.Example 13 Effect of IgG-PMB conjugates on complement and LPS activation of complement   Both immunoglobulins and LPS have the potential to interact with complement. L Interaction of PS with complement exacerbates the inflammatory response to endotoxemia or bacteremia. You can In this example, IgG-PMB binding interferes with complement activation by LPS. The physical ability was tested. In addition, immunoglobulins can also trigger adverse complement reactions. Since it can be [S. Barandun et al., Vox Sang. 7: 157-174 (1962)], the conjugate alone The ability to activate complement was also confirmed. This example shows that (a) activation of complement Measurement of sufficient LPS concentration, and (b) LPS-induced complement using IgG-PMB Blocking activation.   a) The measurement of LPS concentration sufficient to activate complement involves measuring different concentrations of LPS. In addition to a standard amount of complement source (CH50 reference standard; Sigma), calculate the amount of complement consumed. Performed by determining by titration with sensitized sheep red blood cells (SRBC) It was [A. Chohen et al. Immunol. 146: 4234-4241 (1991)].   40 μl of reference standard, 80 μg, 8 μg, 0.8 μg, 0.0 μg large intestine Add 40 μl of a solution containing bacterial LPS or GVB + 2 buffer (Sigma), mix and mix 3 Incubated at 7 ° C for 30 minutes. Then add 5 μl of each mixture or blank control. Or add 10 μl aliquots to CompQuick CH50 tubes (Sigma) and repeat repeatedly. Mix well and incubate at room temperature for 60 minutes with occasional mixing. Next The tube was centrifuged at 600xg for 10 minutes at 4 ° C and any hemolysis present in the supernatant was removed by a control blank at 415 nm. It was measured against the lysate of the black solution. CH of each mixture₅₀The value is calculated as Was:   The results are shown in Table 17.   These results indicate that preincubation of the complement source with LPS It is consumed and complement becomes unavailable to act on SRBC in the second step of the assay. Which indicates that. The effect of LPS was concentration dependent.   b) Blocking LPS-induced complement activation with IgG-PMB is described in Example 7. The SPDP-bound IgG-PMB prepared as described above was mixed with LPS and then It was performed by examining the effect of pretreated LPS on complement activation. 1.5 μg E. coli 026: B6 LPS to 7.5 μg IgG-PMB or 15 μl pair Illumination buffer was added and incubated at 37 ° C for 60 minutes. 30 μl of complement (above (a ) Or GVB + 2 buffer to each sample, and add 60 ° C. at 37 ° C. Incubated for minutes. Each mixture Add 20 μl of compound to CompQuick CH50 tube (Sigma), mix and mix for 60 minutes at room temperature Incubated. Centrifuge the tube as described in (a) above to remove hemolysis at 415 nm. Was quantified by. The results are shown in Table 18.   These results show that pre-incubation of IgG-PMB with LPS It is shown that the effect of LPS on complement activation is impaired. Furthermore, Ig The G-PMB conjugate alone has no effect on complement activation. This is , IgG cross-links with PMB to the extent that it exerts a detrimental effect on the spontaneous complement reaction Suggest that it did not disturb the structure of IgG. The ability of IgG-PMB conjugates to block the LPS effect and its apparent safety is Conjugates have both prophylactic and therapeutic value against bacteremia and endotoxemia Suggests thatExample 14 Improved IgG-PMB conjugate   Example 7 Method (SPDP) and Example 11 Method (Periodic Acid Oxidation / Shishing) To improve the activity of IgG-PMB conjugate prepared by It was Both groups of conjugates have much higher levels of conjugates than conjugates prepared using other chemistries. Since it showed LPS binding, by increasing the degree of PMB substitution for IgG, And even higher levels of binding could be achieved. Achieve greater substitution The two mechanisms that were adopted to achieve this were the reactants (IgG and PMB) at the binding step. ) Increasing concentration and using more highly derivatized SPDP-PMB Was to do. In this example, (a) a novel periodate IgG-PMB conjugate Preparation, (b) preparation of a novel SPDP IgG-PMB conjugate, (c) conjugation with LPS ELISA assay of combined conjugates, (d) MIC and MBC determination of conjugates , And (e) measurement of degree of binding by amino acid analysis.   a) Preparation of the novel periodate IgG-PMB conjugate was performed with 1 ml of 50 mM NaPO 4._Four， PH 30 mg IgG dissolved in 7.2 with 10.7 mg sodium periodate (Sigma) It was carried out by oxidizing for 30 minutes at room temperature. 1 ml of reaction mixture was added to 50 mM NaPO_Four， PH Applied to a 15 ml Swift desalting column equilibrated with 7.2 and the peak IgG fraction was Ig Pooled to a G concentration of 7.1 mg / ml. This Ig mix containing 0.0476 μmol IgG To 1 ml of the product, 20 mg of PMB (14.44 μmol) was added and incubated at 4 ° C. overnight. . For 1.0N HCI The reaction mixture to pH 6.5 and 10 μl of 10 mg / ml NaBH₃Add CN solution, 4 at room temperature Incubated for hours. The conjugate was then chromatographed using a 10 ml P-10 column. Stored at 4 ° C on a fee.   b) The preparation of the novel SPDP IgG-PMB conjugate is first described in Example 6. As a derivative of PMB with a molar ratio of SPDP: PMB of 2: 1 or 3: 1 Was carried out. For each reaction, 15 μl of 20 mM S dissolved in DMSO Incubate for 30 minutes at room temperature with PDP solution with intermittent shaking. Derivatized 5 mg of IgG dissolved in 0.5 ml with. Equilibrate with acetate buffer The derivatized IgG was purified using a purified 15 ml Swift desalting column and the peak fractions were pooled. And concentrated on a Centriprep-30 concentrator (Amicon). Dissolve in 1.8 ml acetate buffer To 5 mg of IgG, 7.7 mg of dithiothreitol dissolved in 250 μl of acetate buffer Was added and incubated at room temperature for 30 minutes. Then equilibrate with PBS-EDTA Each sample was purified using a separate 15 ml Swift desalting column. Derivatized with SPDP For each sample containing about 5 mg of IgG, a SPDP: PMB molar ratio of 2: 1 or 5 mg of PMB derivatized 3: 1 was added and incubated at room temperature for 18 hours . Next, for gel filtration using a P-10 column (50 ml) equilibrated with PBS-EDTA. Each conjugate was separated from free SPDP-PMB and the peak fractions were collected, pooled and Stored at 4 ° C.   c) ELISA analysis of conjugates that bind LPS shows E. coli 0111: B4 L Performed as described in Example 1 using PS (Sigma). Created in (a) above Of the periodate conjugate and the two SPDP conjugates prepared in (b) above Dilution, LPS-coated and uncoated 96-well microtiter Binding to the wells of the plate is shown in Table 19 as the average of duplicate samples.   These results show that SPDP: PMB is a 3: 1 conjugate (hereinafter, "3: 1 SP "DP: PMB conjugate") has the highest specific LPS binding activity. Is shown. The binding activity of SPDP: PMB is 2: at a concentration of 0.8 to 4.0 μg / ml. The binding was about 2-4 times that of the conjugate of 1 and the periodic acid conjugate.   d) For the determination of the conjugates MIC and MBC, E. coli HB101 was used as a susceptibility test strain. Was carried out as described in Example 12. The results are shown in Table 20.   Compared with these measurements of the conjugate tested in Example 12, the novel periodate The acid conjugate is 4 times more potent and the 3: 1 SPDP: PMB conjugate is 2 times more potent. Surprisingly , Periodate conjugate had lower LPS binding activity in ELISA than 3: 1 SPDP: PMB conjugate. , But shows stronger antibacterial activity. Probably PMB with SPDP and The modification of IgG compared to the binding of native PMB and periodate-treated IgG It may improve the binding efficiency but reduce the antibiotic activity.   e) The degree of binding by amino acid analysis is 2: 1 SPDP Ig-PMB, 3: 1 SPDP Ig-P Amino acid pairs of MB and periodate Ig-PMB conjugates The results were compared to native human IgG and free polymyxin B control samples. It was carried out by feeding. A new structure that makes up 6 of the 10 amino acid residues of PMB The major amino acid diaminobutyric acid (DAB) was the main component detected and quantified .   A total of 5 samples were analyzed. They are as follows: 1. Free PMB (5Oμl H₂25 pmol in O) 2. Periodate IgG-PMB (600 pmol in 100 μl PBS) 3. SPDP3: 1 Ig-PMB (600 pmol in 100 μl PBS) 4. SPDP2: 1 Ig-PMB (600 pmol in 100 μl PBS) 5. Human IgG (600 pmol in 100 μl PBS)   Transfer each sample to a glass hydrolysis tube rinsed three times with 100 μl of pure water. And then concentrated to dryness in a vacuum centrifuge. 500 μl of distilled 6N HCl in each sample tube, 10 μl of 2-mercaptoethanol and 10 μl of 50% phenol aqueous solution were added. The sample tube was then purged with nitrogen gas and capped. Heat the sample at 110 ° C for 22 hours and add the sample. It was decomposed with water and concentrated to dryness again. PMB sample with 500 μl of 0.2 N sodium citrate buffer The buffer solution was suspended in pH 2.2, and the other four samples were suspended in 250 μl of this buffer. Enough After mixing, the sample solution was passed through a nylon membrane syringe filter with a pore size of 0.2 μm. .   Each hydrolyzate filtered using a Beckman Instruments 6300 amino acid analyzer 20 μl of solution was analyzed. The analyzer is a Beckman 10 cm cation exchange HPLC column, Beckman Sodium buffer system, 60-minute analytical procedure, and measurements at 570 nm and 440 nm wavelengths Equipped with a Beckman ninhydrin reagent detector using the measured absorbance. Detector Sensitivity is 1.0 AUFS for PMB samples and 0.5 AUFS for the other four samples Set.   Gilson HPLC Sy for all data collection and peak integration calculations stem Controller 712 v. 1.1 Using software package (Middleton, WI) It was carried out. The identification of sample peaks and amino acid concentrations are Mino Acid Standard Mix (Beckman Standard, Lot # A108039) and (S)-(+) 2,4 -Using diaminobutyric acid dihydrochloride (Aldrich Chemical, lot # 07301 CY) Determined in comparison with the analysis. The results of amino acid analysis are shown in Table 21.   The number is the total number of amino acids expected in the measured percentage of each amino acid. Approximate for each sample determined by multiplying (eg, 1320 for human IgG) Represents the calculated amino acid composition. The number of moles of PMB per mole of IgG is the detected feature. Calculated by dividing the number of existing DAB residues by 6 (number of DAB residues / PMB) .   The results show that the 3: 1 SPDP conjugate has the highest degree of binding (on average, PM per IgG molecule). B3.7 molecule). This is because this conjugate was measured by ELISA. Consistent with having the highest defined LPS binding activity (see (b) above) ing. The 3: 1 SPDP conjugate contains, on average, twice as many PMB molecules as the 2: 1 SPDP conjugate, This explains the 2-fold greater activity of the 3: 1 SPDP conjugate in the LPS binding ELISA. Will reveal. Periodate IgG-PMB is also well bound and this binding The coalesced showed the highest antibacterial activity. Providing antibacterial activity with greater periodate binding In contrast, SPDP binding appears to lead to the highest LPS binding activity. Will be This is due to steric differences in the way PMB binds to IgG and / or Or may reflect the different effects of the two binding chemistries on PMB activity. Absent.Example 15 Use of IgG-PMB conjugates as diagnostics: Cross-reactivity of IgG-PMB with different Gram-negative LPS antigens   The IgG-PMB conjugate showed binding to E. coli 0111: B4 LPS, but this species One of many Gram-negative pathogens that can cause toxemia and bacteremia IgG-PMB in a diagnostic format using competitive ELISA It was interesting to see if the conjugate could detect LPS of other species. Book Examples are (a) application of E. coli 0111: B4 LPS to microtiter wells, (b) Incubation of IgG-PMB conjugate with several different concentrations of LPS, And (c) Binding assay of E. coli 0111: B4 LPS with conjugate in the presence of competitor Was included.   a) E. coli 0111: B into the wells of a 96-well microtiter ELISA plate Application of 4 LPS was performed as described in Example 1 (1 mM EDTA and 2 μg). Apply 100 μl per well of non-pyrogenic PBS containing LPS and mix at 4 ° C. Incubated overnight). Wells were washed with PBS-0.05% Tween 20, 10 mg / ml endotoxin Blocked for 90 minutes at 37 ° C with PBS containing elemental BSA.   b) IgG-PMB conjugate, Escherichia coli 0111: B4 (control standard), S. typhimurium Fungus (Salmonella typhimurium), Pseudomonas aeruginosa, Vibrio cholerae (Vibrio cholerae), Shigella flexneri, and Klebsiella pneumoniae ( Klebsiella pneumoniae), Salmonella enteritidis, Serratia marcescens (Serrati) a marcescens) and Rhodobacter sphaeroide s) (all obtained from Sigma; tad Rhodobacter is List Biologicals Laborator Incubation with different concentrations of LPS purified from y, Campbell, CA) The IgG-PMB conjugate prepared by setting the SPDP: PMB molar ratio to 3: 1. 250 μl of a 7.2 μg / ml solution of the combination (see Example 13) in PBS-containing 1 mg / ml BSA. Performed by adding 250 μl of 0.05% Tween 20 and incubating at 37 ° C . 100 μl of these mixtures was added per well.   c) Binding assay of E. coli 0111: B4 LPS with the conjugate in the presence of competitor Was measured by incubating 100 μl of the mixture from (b) above at 37 ° C for 1 hour. Decided The plate is then washed and the wells are PBS-0.05% Twe containing 1 mg / ml BSA. Alkaline phosphatase-conjugated goat anti-human IgG diluted 1: 500 with en 20 (Sigma ) At 37 ° C. for 1 hour, washed again, and p-nitrophenyl Incubate in luphosphate for 30 minutes, 410 as described in Example 1. Absorbance was read in nm. The results are shown in Fig. 9. The result is four different eyes (or der) gram-negative bacteria, LPS antigens derived from a total of 9 bacteria are IgG- Established to effectively compete for binding of PMB to E. coli 0111: B4 LPS I testify. These results show that LPS from various bacterial species can be detected and quantified. IgG-PMB conjugates can be used for Suggest that it may be therapeutically effective against a wide range of Gram-negative bacteria and endotoxins ing.Example 16 Neutralization of in vivo effect of endotoxin by IgG-PMB   Endotoxin (LPS) can trigger lethal reactions in vivo. IgG-PM To confirm whether the B-conjugate can neutralize the lethal effects of endotoxin, Kock's well-characterized and accepted model mouse was used [C. Gal anos et al., Proc. Natl. Acad. Sci. USA 76: 5939-5943 (1979)]. In this example, (A) Determination of the minimum lethal dose of endotoxin in galactosamine-sensitized mice, and (b ) Included neutralization of endotoxin lethality by premixing with IgG-PMB.   a) The minimum lethal dose of endotoxin in galactosamine-sensitized mice is different. 20 mg D-galactosamine dissolved in 400 μl PBS at a dose of E. coli 0111: B4 LPS -HCI was administered to co-administered C57B1 / 6 mice. the latter Specific hepatotoxic agent increases the susceptibility of laboratory animals to endotoxin several thousand fold. It is a sex acting agent [C. Galanos et al., Proc. Natl. Acad. Sci. USA 76: 5939-5943 (1 979)]. To perform this measurement, E. coli 0111: B4 LPS (List Bis) dissolved in PBS was used. 1-500 ng of 20-mg D-galactosamine (ologicals Laboratory, Campbell, CA) (Sigma) and injected intraperitoneally. 10-25 ng of endotoxin is mostly or It was found to be usually sufficient to kill all mice within 24 hours. Endotoxin The lethal variability may be related to the different ages of the mice used. This amount of LPS was used for neutralization experiments as 10 ng was the minimum effective lethal dose.   b) Neutralization of lethality of endotoxin by premixing with IgG-PMB was performed in E. coli 0111: B 4 LPS 50 ng, periodate conjugated IgG-PMB 5 mg (as described in Example 14) 5 mg) or unconjugated control human IgG (Sigma) and PBS Incubate with 100 mg of dissolved D-galactosamine, 1 part of each mixture Was carried out by intraperitoneal injection into C57BL / 6 mice. The results are shown in Table 22. . Survival was assessed after 24 hours.   Since the number of animals used in this experiment was small, repeat the experiment using the following animals. did. That is, a) treatment with endotoxin, D-galactosamine and normal human IgG. 12 control mice, and b) endotoxin, D-galactosamine and excess. Iodate oxidized Ig There were 12 mice in the experimental group that received G-PMB. One mouse for each component The doses were the same as above and the experiment was repeated exactly as above. Table of results 23.   The results of these two experiments show that IgG-PMB exerts a lethal effect on endotoxin in vivo. It has been demonstrated to neutralize, and the IgG-PMB conjugate is due to Gram-negative bacteria. It suggests that it may be useful in the prevention or treatment of sepsis.Example 17 Prevention of endotoxin lethality by prophylactic administration of IgG-PMB conjugate   In the previous example, the conjugate or control IgG was mixed with endotoxin and the mixture was added to D -IgG-PMB conjugate by administration to mice with galactosamine The ability to neutralize the lethality of endotoxin in vivo was investigated. The result is that the above conjugate is It was shown to neutralize endotoxin. The ability of this conjugate to neutralize endotoxin lethality A more thorough test is to use a different route at a different time than the administration of endotoxin. Is to administer the conjugate. Furthermore, to prove its preventive value, A lower dose of conjugate was used. This example demonstrates that IgG-PMB or control IgG Belly of lethal dose of endotoxin and D-galactosamine after intravenous injection and 1 hour later Intracavitary administration was included.   20 CS7BL / 6 mice each weighing 20 g were dissolved in 100 μl of PBS 200 μg (5 animals) or 400 μg (8 animals) IgG-PMB conjugate (in the case of Example 14) (Periodic acid conjugate) prepared in the same manner as above) or 400 μg of control human IgG (7 animals) Was administered via the tail vein. After 90 minutes, each mouse had 10 ng dissolved in 200 μl PBS. E. coli 011l: B4 LPS endotoxin and 20 mg D-galactosamine were administered intraperitoneally It was After 24 hours, the number of surviving mice in each group was recorded. The results are shown in Table 24.   This result indicates that a 10-20 mg / kg dose of IgG- administered intravenously PMB was administered from a subsequent lethal challenge of intraperitoneally administered endotoxin in test animals. Is sufficient to protect the. These findings show that IgG-PMB That prophylactic administration of the conjugate will prevent endotoxin mediated effects, And this conjugate neutralizes endotoxin outside the vascular compartment It suggests that you can.Example 18 Preservation of IgG effector function in IgG-PMB conjugates: Fc receptor binding   One of the functions of IgG is to perform opsonization, which is a microbe and toxin produced by phagocytes. , The removal of antigens, etc. This property of IgG (which is Fc region promoted) was prepared with SPDP or periodic acid To confirm whether the IgG conjugate remains intact, IgG-PM The binding of B to human monocytes / macrophages was tested by a competition assay. This Essay is a hybrid recombinant antibody fragment fused to a cell surface viral receptor. Similar to the assay employed to test Fc receptor binding activity [D.J. ． Capon et al., Nature 337: 525-531 (1989); Traunecker et al., Nature 339: 68-70. (1989)]. This example illustrates (a) a control conjugate of PMB and human albumin (non-F c receptor-binding human protein-PMB conjugate), and (b) human U93 Of an IgG-PMB conjugate that binds to the Fc receptor of the 7 monocyte / macrophage cell line The assay was included.   a) Specific properties of IgG-PMB conjugates can be determined by To compare with that of the MB conjugate, the SPDP chemistry of Example 7 was used to Bumin was coupled to PMB (albumin is not glycosylated; Periodate chemistry of 5 was not applicable to albumin). Albumin and PMB Coupling with was carried out in three steps in the same manner as described in Example 7. First stage The floor is at room temperature with 2.1 mg SPDP dissolved in 50 μl dimethylsulfoxide. PMB dissolved in 50 mM sodium borate, 300 mM NaCl, pH 9.0 for 30 minutes It contained 10 mg of derivatization. Described in Example 7 using a 15 ml Swift desalting column. Free crosslinker was removed as described above.   10 mg human serum albumin with 1.2 mg SPDP (dissolved in 25 μl DMSO) Derivatize and dissolve in 1 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0, room temperature And mixed for 30 minutes. Using a 15 ml Swift desalting column equilibrated with PBS-EDTA, pH 7.5 Peak containing SPDP-albumin by removing free crosslinking agent by gel filtration Fractions were collected, pooled and concentrated on a Centriprep-30 concentrator. Use 10 μl of 10 N NaOH The pH of the sample was raised to 8.0, and 15.4 mg of the solution was dissolved in 200 μl of pyrogen-free water. It was reduced with dithiothreitol for 30 minutes at room temperature. Reduced derivatized albu The min was purified by gel filtration using a 15 ml desalting column and a Centriprep-30 concentrator. Concentrated.   By mixing the two solutions prepared above and incubating overnight at room temperature The reduced derivatized albumin was coupled to SPDP-PMB. 50 ml P-10 The conjugate was separated from SPDP-PMB by gel filtration using a column.   b) IgG binding to the Fc receptor of the human U937 monocyte / macrophage cell line -A PMB conjugate as described by Capon et al. [Nature 337: 525-531 (1989)]. Assayed in a similar manner. First, 1x10^-81 x 10 from M^-12M's¹²⁵I-IgG 2x10 in 0.5 ml PBS containing 2 mg / ml BSA and 0.1% sodium azide^FiveOf By incubating with U937 cells,¹²⁵I-labeled human IgG [¹²⁵ I-IgG stock solution concentration is 16 μg / ml = 1.07 × 10^-7M] (New England Nuclear, Boston , MA) binding saturation curve was prepared. Incubate the cell suspension at 37 ° C for 60 minutes. , Centrifuged at 1500 × g for 3 minutes and washed 3 times with incubation buffer. Next, the Bioscan “Quick Count” benchtop radioisotope counter (Bioscan (Washington D.C.) was used to measure the radioactivity of the cell pellet. Bond¹²⁵ I-Ig concentration is 1 x 10^-8It turned out to be saturated with M. Therefore, this concentration Used in the competition assay described below.   Due to competition experiments,¹²⁵I-IgG (1x10^-8M) with 2 mg / ml BSA and 2 x 10 in 0.5 ml PBS containing 0.1% sodium azide^FiveU937 cells, And different concentrations of unlabeled competing protein [ie human IgG, IgG- Prepared with PMB (SPDP), IgG-PMB (periodic acid), and (a) above. Human albumin-PMB]. Incubate the cells Radioactive, washed and bound¹²⁵The amount of I-IgG was quantified as described above. Human race 12,029 cpm of labeled IgG bound to cells in the absence of combined protein did. The results of this competition assay are plotted in FIG. Human IgG and And both In the IgG-PMB conjugate, these three concentrations are 10^-8M and 10^-6Competing equally in M In that respect, it is clear that it has similar binding properties to U937 cells. . This result indicates that periodate with SPDP and PMB or with the carbohydrate side chain Modification of IgG by acid oxidation does not impair the ability of IgG to bind to Fc receptors Is shown. This is because the conjugate is mediated by the phagocyte antigen / microorganism Fc receptor mediated It suggests that it can promote the psoninating action. As expected, Hitaru Bumin-PMB conjugate is 10^-6Showed no competitive binding activity at concentrations up to (Data not shown). Therefore, this conjugate promotes opsonization You cannot do it.Example 19 Preparation of antibody-antibiotic conjugate having activity against Gram-positive bacteria: IgG-bashi Thracin   Gram-positive bacteria are responsible for about 1/3 of cases of sepsis. Against these microbes It would be desirable to have an IgG-antibiotic conjugate having the activity to activate. this For purposes of IgG and bacitracin, two surface-active Gram-positive antibiotics And a conjugate was made with vancomycin. In this example, (a) IgG Periodate activation and (b) binding to bacitracin and vancomycin Included.   a) Periodate activation of IgG was performed using 30 mg of human IgG and 1 ml of 50 mM NaPO._Four , Example 5 using 50 mM sodium periodate dissolved in pH 7.2 for 30 minutes at room temperature Implement as described in (b) did. Activated IgG was purified using a 15 ml Swift desalting column (Pierce) and The fractions were pooled.   b) Binding with bacitracin and vancomycin yielded 18.6 mg bacitracin 7.1 mg of activated IgG and 19.7 mg of vancomycin to 7.1 mg of activated IgG Performed by adding and each solution was incubated at 4 ° C. overnight. Then in Centrifuge these mixtures to remove the precipitate formed during cubation. More clarified. The reaction mixtures were adjusted to pH 6.5 with 1.0 N HCl and μl NaCNBH₃The solution (10 mg / ml) was added and incubated at room temperature for 4 hours. next The conjugate using a 15 ml Swift desalting column equilibrated with PBS, PBS-EDTA, pH 7.2 did.Example 20 Antibacterial activity of IgG-antibiotic conjugate against gram-positive bacteria   To confirm whether the conjugate prepared in Example 19 has antibacterial activity, Epidermal Staphylococcus obtained from Dr. Edward Balish of the School of Medicine and Microbiology, University of Sukhumin Fungus (Staphylococcus epidermidis)MIC and MBC of these conjugates against Was assayed. This strain is Gram-positive, DNase-negative, Mannitol salt-negative , Coagulase negative, and novobiocin sensitive. In this example, (a) table Preparation of Staphylococcus epidermidis inoculum, and (b) free and conjugated antibiotics Measurements of MIC and MBC of material were included.   a) Preparation of S. epidermidis inoculum by plating the bacteria on TSA overnight at 37 ° C. , 5x10 bacteria^FiveSuspended in TSB at 1 / ml It was carried out by   b) Measurement of MIC and MBC of free and bound antibiotics is 0.5 ml S. epidermidis inoculum of 0.3125-10 μg / ml free antibiotic or 12.5-250 It was carried out by mixing with 0.5 ml of solution containing μg / ml of each conjugate. MI C is defined as "minimum concentration of compound that inhibits visible growth" and MBC is "inoculum Kills more than 99.9% of the bacteria initially present in the solution (solutions that show no visible growth) Plated and measured; see Example 10) Concentration ". The results are shown in Table 25. Shown in. Example 21 Susceptibility to Gram-negative sepsis and endotoxemia with antibody-antibiotic conjugates Treatment of humans   As mentioned earlier, studies have been conducted on human humoral immune status and Gram-negative infections. It suggests a causal relationship between susceptibility. The present invention is most in need of antibiotics To determine the subpopulation to Intended for screening humans with poor immune status. In this example, (a) Patient total IgG and IgM level assay, (b) patient endotoxin core antigen feature Allogeneic IgG and IgM level assay, (c) Patient immunoglobulin levels And healthy controls, and (d) total immunoglobulin or core antigen-specific immunity Immunoglobulins and / or to patients with significant defects in epiglobulin levels Involved administration of immunoglobulin-antibiotic conjugates.   a) Patient total IgG and IgM level assay is based on Beckman automated immunization Stoll et al. By nephelometry using a scientific system (Beckman Instrument, Brea, CA). , Serodiagnosis and Immunotherapy 1: 21-31 (1987). Be implemented.   b) Endotoxin core antigen specific IgG and IgM level assay is ELISA It is carried out by. Purified E. coli J by diluting plasma or serum and different sample dilutions 5 Endotoxin and Salmonella minnesota R595 Endotoxin The binding level with and was quantified and compared with a known standard of purified anti-endotoxin antibody (B. J. Stoll et al., Serodiagnosis and Immunotherapy 1: 21-31 (1987); Po llack et al. Clin. Invest. 72: 1874-1881 (1983)].   c) Comparison of patient immunoglobulin levels with healthy controls is based on patient vs. control groups. Total IgG and IgM levels (as mg / ml sample), and Endotoxin core antigen-specific IgG and IgM levels in the two groups (1 ml sample) Per μg). All IgG is normal control 80% or less of the level, and / or endotoxin core antigen-specific IgG and IgM Patients with blood glucose below 60% of normal control levels Considered to be at risk of Lam-negative infection and endotoxemia.   d) a significant deficiency in total immunoglobulin or core antigen-specific immunoglobulin levels. Immunoglobulin and / or immunoglobulin-antibiotic binding to patients with depression Administration of the combination provides normal or near normal general and antigen-specific humoral protection. Implemented to recover. Intravenous to restore normal IgG levels Immunoglobulin (Sandoz Forschungsinstitut, Vienna, Austria; Hyland Thera Available from peutics, Duarte, CA; or Cutter Laboratories, Berkeley, CA ) 3% solution twice a day, immunoglobulin level within the normal range of 10% Administer until rising.   The IgG-PMB conjugate of the present invention is an antibody in which all antibody molecules can bind to endotoxin. As it consists of a molecular population, it is necessary to restore or increase the level of antigen-specific antibody. It is sufficient if there are far fewer IgG-PMB conjugates than total IgG. 1 per kg body weight A single intravenous dose of ~ 20 mg IgG-PMB conjugate was used as an endotoxin-specific antibody. Given to restore levels to 100% or above normal levels.Example 22 Gram-negative sepsis, endotoxemia and gut with a mixture of antibody-antibiotic conjugates Treatment of humans susceptible to rum-positive sepsis   Because of the causal relationship between human humoral immune status and susceptibility to infection, Gram Antibody levels against Gram-positive bacteria as well as against sex and endotoxin Need to recover. This is against both these classes of bacteria and endotoxins Multiple with activity This is achieved by administering a mixture of antibody-antibiotic conjugates of. This embodiment Includes (a) identification of humans at risk of infection, and (b) antigen specificity. And a mixture of antibody-antibiotic conjugates to restore total immunoglobulin levels and And, if necessary, included administration of pooled whole human immunoglobulin.   a) Identification of humans at risk of infection is by the means specified in Example 21. Therefore, it is carried out.   b) Antibodies-antibiotics for the restoration of antigen-specific as well as total immunoglobulin levels Of a mixture of cytoplasmic conjugates and, if necessary, pooled total human immunoglobulin IgG-PMB (1 to 20 mg / kg) was intravenously administered once and IgG-bacitra was administered. Gram-negative and gram-positive with a single intravenous injection of syn-conjugate (1-20mg / kg) It is carried out by increasing the level of each reactive antibody. If the whole immune Intravenous immunoglobulin (Sand oz Forschungsinstitut, Vienna, Austria; Hyland Therapeutics, Duarte, CA ; Or available from Cutter Laboratories, Berkeley, CA) for 1 day Twice until immunoglobulin level rises from normal to within 10% ItExample 23 Long-term prevention of endotoxin lethality using IgG-PMB conjugate   The long-term prophylactic effect of IgG-PMB conjugate was examined by D-galactosamine-sensitized model mouse. Was used [C. Galanos et al., Proc. Natl. Acad. Sci. 76: 5939-5943 (19 79); V. Lehmann et al. Exp. Med. 165: 657-663 (1987); and M.A. Freudenberg and C.I. Galanos, Infect. I mmun. 56: 1352-1357 (1988)]. Human IgG-PMB conjugate dissolved in PBS ( 1 mg of each of 10 male C57Bl / 6 mice (prepared as described in Example 14) Was injected intravenously. Ten control mice had unbound human I dissolved in PBS. 1 mg of gG was also injected intravenously. Bound IgG and control IgG solutions were both The concentration was also 8 mg / ml, so the injection volume was 125 μl per mouse.   The in vivo experiments described in Examples 16 and 17 show that 1 hour of endotoxin challenge When administered intravenously before, a very small amount of IgG-PMB conjugate of about 25 μg was completely removed. It has shown that it can provide various protections. Here we have a defense win after 24 hours. I checked the window.   Twenty-four hours after administration of PMB-bound IgG or control IgG, mice in both groups were E. coli 0111: B4 endotoxin (# 201; List Biological Laboratories) , Campbell, CA) was administered by intraperitoneal injection. Ie endotoxin Sonicated 1 mg / ml stock solution of Branson 2000 using a Branson 2000 water bath sonicator , 100 times diluted with PBS to prepare a working solution of 10 ng / μl. 240 mg D-galactosa Mine hydrochloride (# G-1639; Sigma Chemical Co., St. Louis, MO) was added to two silicone 0.1m as carrier protein by measuring in Reacti vial (Pierce) It was dissolved in 2.4 ml PBS containing g / ml bovine serum albumin (BSA). 12 μl 1 Add 0 ng / μl endotoxin solution (l20 ng) to each vial and mix the solution for 15 minutes at room temperature. It was Each vial contains 10 ng of intoxication per injection Sufficient solution to make 12 injections of 200 μl consisting of the enzyme and 20 mg of galactosamine. Contained. Each mouse in both groups received 200 μl of the above mixture intraperitoneally. Mau Su was given food and water ad libitum and was observed for 24 hours, ending at death. Guess the probability To determine the result, use the Fisher's exact method itself and Analyzed [F. Mosteller et al.Probabilty With Statistical Applications， Addi Son Wesley, Reading, MA (1970)]; Significant protection was defined by comparing the experimental group with the control group. It was defined as p value <0.05 when solid.   The results of this prophylactic study are summarized in Table 26.   These results indicate that IgG-PMB conjugates can be administered intravenously as a prophylactic , And significant protection from endotoxin lethality for at least 24 hours after administration of the conjugate. It shows that you can get control.Example 24 Determination of relative half-life of PMB-HIgG conjugate and HIgG in rabbit   This example gives some indication of the half-life of HIgG in rabbits when bound to PMB. Describe the experiment to confirm whether the effect of. Male new zealand white Using rabbits (10 lb, 12 months old) The pharmacokinetic study was carried out. On day 0 of the experiment, two rabbits each 150m 3 mg of PM dissolved in 10 mM phosphate buffer (pH 7.1) containing M sodium chloride The B-HIgG conjugate was injected once intravenously. At the same time, two rabbits in the control group had the same strain. 3 mg of HIgG dissolved in the buffer solution was injected intravenously. Both test and control samples Upon inspection, it was found to be pyrogen-free. 1 hour after the first injection And 5 hours later and 1st, 2nd, 3rd, 4th, 7th, 10th after the first injection. The rabbits were bled on days 14 and 14. Serum samples were collected for the presence of HIgG Stored at -70 ° C until tested.   Sandwich ELISA to detect the presence of HIgG in rabbit serum samples A was developed. Add 50 mM carbonate to each well of the microtiter plate (Corning). 100μ containing 25μg / ml goat anti-human IgG (Sigma) dissolved in buffer pH 9.5 l of solution was applied. After incubating overnight at 4 ° C, remove the coating solution and All wells were washed 3 times with PBS-Tween-20 (0.05% Tween-20 dissolved in PBS). Ten By adding PBS containing mg / ml BSA (Sigma), the remaining antigen-binding site Blocked at 37 ° C for 1 hour.   Test serum samples stored at -70 ° C were thawed immediately before the assay and contained 0.1% BSA. It was diluted 1:10 with PBS-Tween-20. All samples in two aliquots And added to the wells of a microtiter plate (200 μl / well). Test serum Normal rabbit serum diluted 1:10 with the same diluent used for the sample was added per well. Negative control wells were prepared by adding 200 μl. As a positive control Then, dilute normal HIgG to 20 μg / ml with the same diluent, and then use 8 steps of serial 1: 4 dilution. The dilution was carried out to 0.00031 μg / ml. Create a standard curve using the corresponding OD values, From there the corresponding HIgG levels of the test serum samples were determined. Plate at 37 ° C for 1 Incubated for an hour and washed 3 times with PBS-Tween-20. Dissolved in PBS-Tween-20 Alkaline phosphatase-conjugated goat anti-human IgG diluted 1: 500 in 0.1% BSA (Sigm a) was added to the wells and incubated at 37 ° C for 1 hour. Well using PBS-T After washing 4 times with 50 mM Na₂CO₃, PH 9.5 and 1 mM MgCl₂Dissolved in 1mg / mlp -100 μl of nitrophenyl phosphate (Sigma) was added to all wells. The The rate was shielded from light and allowed to develop for 20-30 minutes at room temperature. Dynatech MR 700 Microphone Absorbance at 410 nm was measured using a Loplate reader.   A blank containing only the conjugate and substrate by averaging the absorbance of two wells The well absorbance was subtracted to correct for background. Absorbance vs. H A standard curve of IgG log concentration was plotted. The absorbance of the test sample is the straight line of the standard curve Quantified from a portion.   Of PMB-HIgG (R3 and R4) and HIgG (R1 and R2) Serum clearance curves are shown in Figures 11 and 12. In Figure 11, the HIgG concentration The corresponding absorbance at 410 nm is plotted against days. Figure 12 Is a graph showing the IgG concentration as the number of μg per ml of serum over time. Figure From both 11 and 12, the serum half-life of PMB-HIgG was unbound. Apparently, it is similar to that of IgG. Half of human IgG in human Since the reduction period is about 21 days, Studies suggest that the conjugate may have a long half-life. Therefore, In the example of the present invention, the present inventors found that the serum was still active in rabbit serum 2 weeks after the administration of the conjugate. It proved that sexual conjugates could be detected.Example 25 Detection of LPS binding of PMB-HIgG conjugate after 2 weeks in rabbit circulation   This example shows that the PMB-HIgGLPS binding activity was 2 weeks after circulation in rabbits. It describes an experiment to see if it still exists. This study , Male New Zealand White Rabbit (4.5 kg (10 lb), 12 months old) went. Two rabbits each containing 50 mM sodium chloride containing 50 m 3 mg of PMB-HIgG conjugate in M phosphate buffer was administered intravenously once on day 0. I gave it. At the same time, 2 mg of HIgG in the same buffer was intravenously injected into two control rabbits. Shot Testing of both test and control samples showed no pyrogens . Rabbits were bled 1 and 5 hours after the first injection, then 1, after the first injection, Blood was collected on days 2, 3, 4, 7, 10 and 14. Serum samples were collected and PMB-H Stored at -70 ° C until tested for IgG conjugate activity.   Activity of PMB-HIgG conjugates from rabbit serum (ie, ability to bind LPS A simple indirect binding assay was used to detect a. 96-well microta Each well of an Iter plate (Corning) was prepared from E. coli O11: B4 (Sigma). Coated with 100 μl of 20 μg / ml solution of LPS in PBS. Control wells are Coated with PBS only (no LPS). Coat after overnight incubation at 4 ° C The solution was removed and all wells were washed 3 times with PBS-Tween20. Remaining anti PBS containing 10 mg / ml BSA (Sigma, for tissue culture) as the original binding site By the addition of 3 Blocked at 7 ° C for 1 hour. Remove this blocking solution and wash with PBS-Tween Test rabbit serum samples diluted 1:10 with 20 were added. P as a positive control The MB-HIgG conjugate was also diluted in 10% normal rabbit serum and added to the wells. each Samples were incubated in duplicate for 1 hour at 37 ° C and the plates were incubated with PBS- Washed with Tween 20 three times.   The wells were plated with goat anti-human IgG-alkaline to detect bound antibody. Reactive phosphatase-labeled antibody (Sigma) at 1: 500 dilution with 100 μl of 1: 500 dilution Incubated for hours. After removing this secondary antibody solution, the wells were PBS- Washed with Tween 20 four times. 50 mM Na₂CO₃And 10 mM MgCl₂In 1 mg / ml substrate [p-nitrophenyl phosphate (Sigma)] in each well Was added to. Color development after incubation at room temperature for 15-20 minutes with Dynatech MR7 It was measured at 410 nm using a 00 microplate reader.   The results of this LPS binding assay are shown in Table 27. Rabbit serum collected on day 14 The conjugates from S. al. Bound to the LPS coated wells. This is because this conjugate is in the rabbit It is shown to be still active after 2 weeks of circulation. Example 26 PMB-IgG conjugate does not elicit an immune response when administered intravenously to rabbits   This example demonstrates whether anti-PMB antibodies are elicited in rabbits by conjugate administration. This is to explain an experiment for confirming whether or not. 3 mg P for each of two rabbits The MB-HIgG conjugate was administered intravenously on day 0. Additional injections in these rabbits (Booster) was given at the second, fourth and seventh weeks. Two rabbits as a control Each received 3 mg of single HIgG at the same planned date and time as the experimental group. Conclusion Blood was collected from all rabbits every 2 weeks after administration of either combined or single IgG. It was Serum was collected and stored at -70 ° C until tested for anti-PMB antibody.   A simple indirect binding assay was developed to detect anti-PMB antibodies in rabbit serum. Fired. Each well of 96-well microtiter plate (Corning) PMB in toxin PBS ( Sigma) was coated with 100 μl of a 200 μg / ml solution. Control wells are PBS Only (no PMB). After overnight incubation at 4 ° C, the coating solution was After removal, all wells were washed 3 times with endotoxin-free PBS-Tween 20. Remaining PB containing 10 mg / ml BSA (Sigma, for tissue culture) as an antigen-binding site Blocked by the addition of S for 1 hour at 37 ° C. Remove this blocking solution 1:10, 1: 100, 1: 1000 and 1: 1000 with 2% normal rabbit serum A test rabbit serum sample diluted to 0 dilution was added. Positive control antiserum (Niwato Re-anti-PMB immunoglobulin, Ophidian Phamaceutic, Madison, Wisconsin als Inc.) was also diluted as the test rabbit serum sample. Each sample in 2 series at 37 ℃ And incubated for 1 hour. After this incubation, plate the plates Washed 3 times with BS-Tween 20.   Wells incubated with rabbit serum to detect bound antibody were 1:50 of goat anti-rabbit IgG-alkaline phosphatase labeled antibody (Sigma) Incubated with 100 μl of 0 dilution and incubated with chicken antibody The goat anti-chicken IgG (complete molecule) -alkaline phosphatase conjugate (Sigma) incubated with 100 μl of 1: 500 dilution for 1 hour at 37 ° C. . After removing this secondary antibody solution, the wells were washed 4 times with PBS-Tween 20. 50 mM Na₂CO₃, 10 mM MgCl₂1 mg / ml p-nitrof in Phenyl phosphate (Sigma) was added to each well. 15-20 minutes at room temperature Color development after incubation is performed by Dynatech MR700 Micro Play It measured at 410 nm using the reader.   The results in Table 28 show that the positive control antibody bound PMB as expected. . This confirms that the designed ELISA can detect PMB binding antibody. Things. Results in Table 29 (A for each sample 2 series)₄₁₀Shown as reading) Shows that none of the rabbit serum samples bound to PMB, anti-PMB It shows the absence of antibody. These results show that when PMB is administered intravenously Proven not to be immunogenic even on heterologous protein carriers with repeated injections I have revealed.   The lack of immunogenicity of this peptide is related to its D-amino acid content. Can be said to be. Because these residues will not be recognized by the immune system Because. Example 27 IgG PMB is not toxic   To investigate the safety of using the IgG-PMB conjugate, a toxicity test was conducted. . Relatively high in female SD rats (Harlan Sprague-Dawley) weighing 250-300 g The conjugate was administered intravenously twice at a low dose (about 16 mg / kg). Then this test Serum samples taken at various times during the period and signs of abnormalities in major organs I analyzed.   The study consisted of 5 groups of 3 rats per group . After acclimatization for 7 days, the rats of groups 1, 2 and 3 were conjugated with PMB. 4 mg of rat IgG was injected intravenously on day 0 and again on day 2 (to rat IgG See (E) below for PMB conjugate formation in Were injected with 4 mg of unconjugated rat IgG on day 0 (IgG control); The rats of group 5 served as normal controls (the conjugate was also normal rat IgG Did not inject). Blood was collected from these rats by cardiac puncture and is shown in Table 30. Killed to see the organ abnormalities shown.   Immediately after cardiac puncture, blood smears (2 slides / rat) were prepared and leukocyte percentage For staining, it was stained with Diff-Quik (Baxter Healthcare, McGaw Park, IL). The rest Rino blood (2-7 ml) was coagulated at 2-8 ° C. The clot is 2000 rpm Precipitated by centrifugation for 10 minutes at 37 ° C, collect serum, and perform blood chemistry analysis (SMA C12) and CH_{50 EZ}Measuring Complement Activation by the Complement Assay (Myr, Florida) M. Diamedix Corporation) in two aliquots and frozen at -70 ° C. Cut the organs of interest (kidney, liver and spleen) shown in Table 30 from each rat. And formalin phosphate addition until immediately sectioned for histopathology slides Fixed in buffer (50 mM sodium phosphate, 10% formaldehyde).   A. Biochemical serum analysis for liver and kidney function   All rat serum samples (18 total: groups 1, 2, 4 and 5 to 3; And groups 3 to 6) DuPont DimensionAR (Wilmington, Delaware) DuPont Co.) for 12 test items (SMAC12): Glucose Glucose, blood urea nitrogen (BUM), creatinine, uric acid, calcium, albumin , Total protein, cholesterol, total bilirubin, alkaline phosphatase, Spartate transferase (SGOT / AST), and lactate dehydrogena (LDH). The average value for each group was calculated (Table 31) and the experimental group (1 2) and 3) compared to the control group (4 and 5), is there any significant difference? saw. These experimental results are used to analyze the experimental data of 20 female SD rats. Normal for each assay issued by The range was also compared (data provided by Harlan Sprague-Dawley).   Standard laboratory tests for liver disease include serum levels of bilirubin, AST, Alkaline phosphatase, LDH, albumin, and some non-standard Glucose measurements are included. Renal function includes urea, creatinine, and calciu It can be evaluated by measuring plasma levels in the serum [J.F. Zilva, P.R ． Pannall, Clinical Chemistry in Diagnosis and Treatment, Yearbook Medic al Publishers, Chicago, IL (1984)]. Lactate dehydrogenase discussed below Except for the values (LDH), Table 31 shows the values for experimental groups 1, 2 and 3. It shows no significant difference compared to control groups 4 and 5. Furthermore, all Is the value within the normal range for each assay in this series of rats? Or something close to it.   Values for LDH vary considerably between groups, with most values in rat blood. Beyond the normal range for Qing LDH (data from Harlan Sprague-Dawley) There is. Lactate dehydrogenase is found in the liver, heart, skeletal muscle, brain, kidneys and red blood cells. Found in high concentration. High levels of certain isozymes indicate liver or myocardial damage However, in this test, the control rats also showed high LDH values, which It is suggested that the high LDH value is unrelated to the IgG-PMB conjugate. In vitro (when a blood sample was drawn, or when serum was difficult to separate from blood cells) Haemolytic reactions that may have occurred (if they did not) also raise serum LDH levels. Let (J.F. Zilva and P.R. Pannall, Clinical Chemistry in Diagnosis and Treatment, supra), which explains the high value in this study. May be.   Day 5 (3 days after second injection), Day 7 (5 days after second injection) and 14 The measured value of the serum sample taken on the day (12 days after the second injection) was IgG-P. It is also worth noting that it does not show a significant difference that can be attributed to the administration of MB conjugate. To do. In other words, on days 5, 7 and 14 for all serum components measured Day values are close to or within their respective normal range ( (Except LDH), so that this conjugate has any acute changes in the condition of these test rats. It shows little or no significant aging that would be expected if It   B. White blood cell percentage   Blood smears from each rat were examined under oil immersion (1000x) to determine leukocyte percentage. I put it out. At least 100 white blood cells on each slide Counted above, each cell type (lymphocyte, monocyte, neutrophil, eosinophil, and basophil) -Centage was calculated to give the leukocyte percentage for each rat. Each group Take the average of the percentages for rats within and from groups 1, 2 and 3 Comparing the percentages with the percentages from groups 4 and 5, there was something significant in the leukocyte population. I checked if there was a difference. In addition, examine the morphology of red blood cells on each slide to A rough estimate of the number of platelets to be performed was made. The results of the differentiated leukocyte count are summarized in Table 32. It   Table 32 shows significant differences in the percentage of each cell type between groups. There is no. These percentages are for rats obtained from Harlan Sprague-Dawley. Somewhat different from the normal reference value of (neutrophils about 5-10% more than expected) 5-10% less), which is seen in both normal controls and experimental groups, This finding suggests that it is independent of IgG-PMB conjugate administration. Red Blood cell morphology appeared normal and platelets were abundant in all slides examined.   C. Organ histopathology   The organ of interest is excised and fixed in formaldehyde phosphate buffer. It was Sections are prepared as described below and stained with hematoxylin and eosin. Colored.   Kidney: Full length central longitudinal section through the center   Liver: Transverse section through liver lobules   Spleen: horizontal axis section   These slides were examined for organ abnormalities and found to be normal .   D. Analysis of serum complement activity   Immunoglobulins and immunoglobulin complexes have the ability to activate the complement system ing. This type of complement activation mediated by IgG-PMB conjugates It will exacerbate the inflammatory response to toxemia or bacteremia. In addition, normal complement function Inhibition would impair the complement-mediated host defense mechanism. In this example, Ig The in vivo effect of G-PMB conjugates on serum complement activity was investigated.   Rat serum samples are treated with EZ complement CH₅₀Assay (Diamedix C, Miami, FL orp.) for total hemolytic complement activity (CH₅₀) To I analyzed it. To confirm the effect of this conjugate on complement activity, untreated controls CH obtained from rat₅₀Values from IgG and IgG-PMB conjugate treated rats CH obtained₅₀Compared with the value.   Referring to Table 33 above, CH between any of the groups tested₅₀There is no significant difference in the value Yes. Given that IgG-PMB conjugate-mediated complement activation occurs in vivo, this For the treated animals compared to untreated control rats (group 5) Of conjugate-treated rats (groups 1, 2 and 3) due to exhaustion of complement components of₅₀value Would have been reflected as a decrease in Similarly, inhibition of normal complement function is not associated with untreated controls. Of the combined treatment group when compared to the group CH₅₀It would be indicated by a decrease in the value. Unconjugated IgG also activates serum complement activity It was found to have no effect on (group 4). Therefore, these results are It is shown that intravenous administration of IgG-PMB conjugate has no side effects on serum complement activity. It is something.   E. FIG. NaPO_FourRat IgG using periodate oxidation of IgG in Formation of the conjugate   PMB was conjugated to rat IgG using periodate oxidation of IgG . This is as follows: a) Periodate oxidation of IgG in phosphate buffer, followed by Included conjugate formation of PMB to periodate oxidized IgG.   a) Periodate oxidation of IgG in phosphate buffer was 25 mg rat IgG (Sigma) with 1 ml of 50 mM NaPO_FourDissolve in (pH 7.2) buffer, 10. By adding 7 mg of sodium metaperiodate (final concentration 50 mM) went. Incubate for 30 minutes at room temperature with gentle swirling every 5 minutes And then 50 mM NaPO_Four(PH7.2) 15ml Swift equilibrated with buffer Periodate was removed by gel filtration on a desalting column (Pierce). A_{28 0} The peak fractions containing the highest amount of antibody, followed by absorbance, were pooled.   b) For the formation of a conjugate between periodate-oxidized IgG and PMB, add 75 mg PMB to acid. 20 mM NaPO was added to the purified IgG and gently shaken overnight at 4 ° C._Four(PH 6.5) in 0.1 mg / ml NaBH₃By reducing with CN at room temperature for 2-3 hours. I went. 50 mM phosphate (pH 7.5) containing 150 mM NaCl (PB For gel filtration on a 15 ml Swift desalting column equilibrated with S) The PMB-IgG was separated from the rest of this reaction product.   The activity of PMB-rat IgG conjugate was determined by the LPS binding assay described above. It was measured. The results in Table 34 show that the PMB-rat IgG conjugate has excellent LPS binding activity. It shows that it had sex. Example 28 Demonstration of opsonophagocytosis of IgG-PMB conjugate   Opsonin IgG class antibody enhances phagocytic clearance of extracellular bacteria Mediate important immune effector functions by [Raff et al., J. Infect. Dis. 16 3: 346-354 (1991)]. In this way, opsonin IgG is directed against bacterial pathogens. Play an important role in the host defense mechanism that protects [Rozenberg-Arska Et al., J. Med. Microbiol. 22: 143-149 (1991)]. The purpose of this example is to Whether the IgG component of the PMB conjugate retains this important effector function Was to find out. This is an E. coli microbiota using an IgG-PMB conjugate. Pretreatment of the product with the phagocytic uptake by the human monocyte cell line U937 (opsonin phagocytosis) It was performed by evaluating whether to enhance the effect). Opsonine phagocytosis The essay is the potential of immunoglobulin preparations used to treat bacterial infections. It provides a useful tool by which therapeutic efficacy can be evaluated [Hill et al., Am. J. Med. 61-66 (1984)]. This example illustrates (a) opsonization of IgG-PMB conjugates. Assay for potency and (b) Determination of minimum effective concentration of IgG-PMB conjugate Included.   A. Assay for opsonophagocytosis of IgG-PMB conjugate   The opsonophagocytic capacity of IgG-PMB conjugates was determined by modifying the published method. It was measured using the essay procedure [Gemmell et al., J. Clin. Invest. 67: 1249-1256 (198 1) and Bohnsack et al., J. Immunol. 143 (10): 3338-3342 (1989)]. E. coli strain H B101 Were grown on TSA (BBL) at 37 ° C. for about 20 hours. Then this microorganism 1 x 10 in PBS (pH 7.2)⁸It was suspended at a concentration of CFU / ml. This suspension Place multiple 1.0 ml aliquots of the solution in separate microseparation tubes and Was centrifuged at 4 ° C. and about 14,000 × g for 5 minutes. Then, the resulting precipitate Resuspend each in 1.0 ml volume of one of the following opsonin or control solutions. I let you.   1. MIC (0.062 mg / ml) IgG-P for E. coli HB101 MB conjugate (prepared by periodate oxidation as described in Example 14 (a)) ) (MIC was measured as described in Example 12).   2. 2 × MBC (0.25 mg / ml) IgG-for E. coli HB101 PMB conjugate (same as above) (MIB was measured as described in Example 12) ).   3.0.062 mg / ml IgG control (unconjugated product) (for # 1 above) All controls; this was the same IgG used to generate this conjugate ).   4.0.25 mg / ml IgG control (unconjugated product) (for # 2 above) Control).   5. PBS control (no IgG or conjugate), ie PBS (pH 7.2) only .   Incubate these 5 suspensions at 37 ° C for 60 minutes with periodic mixing It was opsonized. After opsonization, separate these suspensions as above. Each of the obtained precipitates was separated with 0.5 R of R supplemented with 10% FCS. PMI1640 medium (This was referred to as "medium" in the rest of this example). Five For each separate polypropylene culture tube (S / P), prepare 1x in medium 10⁶1.0 ml of U937 cell suspension containing U937 cells / ml was added. In addition, in each culture tube, of the opsonized E. coli suspension prepared above 0.1 ml of one was added. 1.0 ml U937 cell suspension and 0.1 ml P A sixth control group containing BS (PBS control) was also prepared. at this point, 1 x 10 for each culture tube⁶U937 cells, and 2 × 10⁷Large intestine containing E. coli microorganisms The ratio of bacteria to U937 cells was 20: 1. Then these 6 cultures Incubate the tube at 37 ° C with constant shaking for 60 minutes to allow phagocytosis. Let After the incubation, place the culture tubes on ice for a few minutes for further feeding. I stopped using it. Then, centrifuge these 6 culture tubes at 4 ° C. and 500 × g for 10 minutes. Released. The resulting precipitate is cold PBS (while centrifuging as in the previous step) 3 The cells were washed twice to remove extracellular E. coli microorganisms. Each of these final precipitates was Resuspend in 2 ml cold PBS and add a 40 μl volume of these suspensions to a microscope slide. Smears were prepared by applying to glass. Air dry these smears It was fixed by immersing it in 100% methanol for 5 seconds and air-drying again.   Sowter-McGee staining method that differentially stains intracellular bacteria and peripheral cytoplasm of host cells Rev. [Sowter and McGee, J. Clin.Pathol. 29: 433-437 (1976)]. These smears were stained. Hydrate slides by soaking them in water for about 60 seconds. And then in methyl green-pyrronine (MGP) solution (Sigma) for 5 minutes. I put it in. Wash slides in water for 15-20 seconds, then light green post-stain (H₂At O Immersion in diluted 0.25% Sigma Light Green SF Yellow) for 3-5 seconds. water After rinsing for 15-20 seconds in water, soak the slides in 100% ethanol for 5 seconds. Then, it was immersed in xylene for 5 seconds. Allow the slides to air dry and then cover glass. I put it.   Evaluation of opsonophagocytosis was performed blinded by light microscopy. To each experimental group About 100 or more U937 cells are randomly counted and 1 or 2 or more cells are obtained. The percentage of cells containing the internal E. coli microorganism was given.   The results of this study are shown in Table 35.   MICs for E. coli microorganisms and IgG-PMB at concentrations equal to 2 × MBC Treatment of the microorganisms with the conjugate results in the microbial growth of over 50% of U937 cells. The thing was taken in by phagocytosis. The unbound form of the same IgG with comparable concentrations of the microorganism Incorporation resulted in very little or no uptake. IgG-PMB binding If neither coalesced nor IgG is present, no phagocytic uptake of E. coli microorganisms occurs. Yes (PBS control group). These results show that IgG-PMB conjugate IgG Part retains opsonin effector function, and IgG-PMB binding It has been demonstrated that coalescence enhances the phagocytic clearance of bacterial microorganisms.   B. Determination of the minimum effective concentration of IgG-PMB conjugate   The minimum concentration of IgG-PMB required to mediate opsonophagocytosis is defined as M To test this conjugate at IC and sub-MIC concentrations (low MIC) More measured. As an additional control, a corresponding series of albumin-PMB (Alb -PMB) conjugate solutions were also tested at concentrations comparable to IgG-PMB conjugates. Next The conjugates and control solutions of Example 1 were tested for opsonophagocytosis in (a) of this Example. The assay was performed according to the procedure described.   1. MIC (0.062 mg / ml) IgG-P for E. coli HB101 MB conjugate (the same as used in (a) of this example).   2. 1/2 MIC (0.031 mg / ml) IgG-for E. coli HB101 PMB conjugate (same as above).   3. 1/4 MIC (0.0155 mg / ml) IgG for E. coli HB101 -PMB conjugate (same as above).   4. E. coli HB101 1/8 MIC (7.75 μg / ml) IgG-P MB conjugate (same as above).   5.062 mg / ml Alb-PMB conjugate (described in Example 18 (a)). Prepared as above) (this group served as a control for # 1 above) ).   6.031 mg / ml A1b-PMB conjugate (same as above) The loop served as a control for # 2 above).   7.0155 mg / ml Alb-PMB conjugate (same as above) (this The group served as a control for # 3 above).   8.7.75 μg / ml Alb-PMB conjugate (same as above) Boop served as a control for # 4 above).   9.0.062 mg / ml IgG control (unconjugated product).   Ten. PBS control (no IgG or conjugate), ie PBS (pH 7.2) only .   The results of this study are shown in Table 36.   E. coli using concentrations of MIC or below for IgG-PMB conjugates Treatment of microorganisms with U937 cells at all IgG-PMB concentrations tested The amount of phagocytic uptake of the microorganism was increased. Corresponding series of Alb-PMB bindings The coalesces did not show significant levels of opsonophagocytosis at the concentrations tested and were unbound. So was the somatic IgG control. These results show that the IgG-PMB conjugate is significant. Various levels of opsonophagocytosis at clinically relevant low MIC concentrations, And this conjugate is opsonophagocytosis In order to be able to mediate both the functional PMB and IgG moieties of this conjugate at the same time. Prove that you are required.Example 29 Antibacterial activity of IgG-PMB conjugate against clinically relevant bacterial strains   Bacteria known to be human pathogens (see Example 29 below) The MIC and MBC values for the strain were compared to IgG-PMB conjugate and native PMB control. I measured it. This example includes (a) preparation of the conjugate, (b) preparation of the bacterial inoculum, and (C) MIC and MBC measurements were included.   A. Preparation of conjugate   To more efficiently remove free (unconjugated) PMB from the final conjugate sample Periodate as described in Example 14 (a), except for the following changes. IgG-PMB conjugates were prepared by oxidation. Final conjugate solution was 1.0% PBS containing 0.1% Tween 20 and then adjusted to contain Was chromatographed on a P-10 column using as the eluent. Pore volume Concentrate the substance in the (void volume) and then use column chromatography as described here. It was further purified by chromatography.   B. Preparation of bacterial inoculum   The microorganisms were grown to the following test microorganisms as described in Example 12 (a). An isolated inoculum was prepared by: E. coli strain EC5; Pseudomonas aeruginosa ) Strain ATCC27312; Pseudomonas aeruginosa strain Strong; and Pseudomonas aeruginosa strain 3.   C. Measurement of MIC and MBC   IgG-PMB conjugates and native PMB control MICs and MBCs were tested in Example 12. Measured for each of the test organisms as described in (b) and 12 (c) .   The results of MIC measurement are shown in Table 37.   The results of MBC measurement are shown in Table 38.   This bacteriostatic and bactericidal activity of IgG-PMB conjugates against pathogenic bacterial strains Have demonstrated that the compounds may be effective in the prevention and / or treatment of bacteremia.Example 30 Prophylactic administration of IgG-PMB conjugate protects rats against colibacillemia   Gram-negative bacteremia and endotoxic shock can trigger lethal reactions in vivo. Real However, irreversible Gram-negative bacteremia is a major cause of infection and death in hospitals. [S.M. Wolf, N. Eng. J. Med. 307: 1267-1268 (1982)]. Especially large Enterobacteriaceae species are hard to overlook despite the potential use of powerful antibiotics Continued to be associated with high mortality. E. coli strains with K1 type capsules Up to 24% of blood culture isolates [G.W. Count and M. Tur ck, J. Clin. Microbiol. 5: 490 (1977)] as a causative agent. [L.D. Sarff et al., Lancet 1: 1099 (1975)]. It is the nosocomial gram in adults Negative bacteremia [MP Weinstein et al., Rev. Infect. Dis. 5: 35-53 (1983)] and children Pyelonephritis [G.H. McCracken et al., Hosp. Pract. 9:57 (1974)] most frequent It is a common cause. In addition to K1 type, human blood E. coli isolate also contains O antigen serotype And O18 lipopolysaccharide is most often associated with bacteremia [A.S. Cross et al. J. Infect. Dis. 149: 184-193 (1984)]. E. coli serotype O18: K1 is D ． Rowley, Br. J. Exp. Pathol. 35: 528-538 (1954) or H.P. Smith, J. Gen. Mi crobiol. 136: 377-383 (1990) are highly pathogenic human pathogens as defined by any of Proliferates in vivo from a small inoculum, bypasses host defenses and causes extra-intestinal infection .   In vivo efficacy against bacteremia caused by pathogenic bacteria such as Escherichia coli O18: K1 To test IgG-PMB conjugates for force, D.E. Schiff et al., Infect. I mmun. 61: 975-980 (1993), the established animal infection model was used. this The model includes many criteria that are important in assessing the toxicity, efficacy and safety of immunotherapeutic agents. And some of these criteria are A.S. Cross et al., Infect. Immun. 61: 2741-2747 (1993). Among other things, this model is An infection model rather than a Dell alternative to using high doses of non-pathogenic strains Rats were boosted with a low dose of pathogenic bacteria. Medium due to experimental evidence In contrast to the venom model, the model of infection more accurately reflects the course of human sepsis. And are shown. Infection models include a) virulence factors (ie specific K antigens or Human sepsis with an invasive phenotype with a planar LPS antigen (smooth LPSantigen) Bacteria causing disease are used [I. Orskov and F. Orskov, J. Hyg. Camp. 95: 551-575 ( 1985)]; b) mimics the normal progression of sepsis from the lesion to confluence; A level of circulating bacteria is produced that is consistent with bacteremia on the floor [D.E. Diezman et al. Pedia tr. 85: 128-131 (1974)]; and d) endotoxin levels similar to those experienced clinically. Induce physiological physiological cytokine responses such as triggering TNF movement and triggering TNF motility [A.S. Cross et al., Infect. Immun. 61: 2741-2747 (1993) Have been used].   In this example, prophylactic treatment of neonatal rats with IgG-PMB resulted in E. coli O1. 8: Tested for protection against bacteremia and death caused by K1. This Example of (a) determination of lethal dose of E. coli O18: K1 in neonatal rats; And (b) Include in vivo protection against E. coli O18: K1 using IgG-PMB. Included.   A. Measurement of lethal dose of E. coli O18: K1 in newborn rats   Five-day-old pathogen-free SD rats (Charles, Wilmington, MA) River Laboratories) were subcutaneously inoculated with different doses of E. coli O18: K1. K.S ． E. coli strains obtained from Kim Children's Hospital (Los Angeles, CA) Name C5) was isolated from the cerebrospinal fluid of the child. Brain Heart Infusion (BHI ) Diluting an overnight culture of E. coli C5 in medium 1:40 with fresh medium to Approximately 1 × 10 until several growth stages⁸OD of 0.25 corresponding to bacteria / ml₆₂₀Up to I let you. By centrifuging these cells with sterile saline (0.9% NaCl) And washed twice and diluted with saline to different cell densities. Add each dilution to BHI agar Plates were plated and incubated at 37 ° C to give the actual cell number.   Rats were inoculated subcutaneously with approximately 370-2620 bacteria in 100 μl of sterile saline. It was A typical lethal infection with E. coli O18: K1 causes bacteremia within 18 hours. , Some rats died within 24-72 hours after infection. About 300 bacteria are LD₅₀ Approximately 50% of newborns die, while most or all rats die It has been found that 1500 bacteria are usually sufficient to be used. 150 Since 0 to 2500 bacteria correspond to the minimum effective lethal dose, E. coli at this concentration was It was used for an in vivo bacteremia protection test using gG-PMB.   B. In v against E. coli O18: K1 (C5) using IgG-PMB conjugate ivo protection   To confirm whether administration of IgG-PMB conjugate can protect in vivo In addition, a large lethal dose was obtained after pretreatment of rat neonates with IgG-PMB or control IgG. Enterobacter C5 was administered. IgG used as carrier in conjugate and as control Is a human bone shown to be non-reactive with E. coli C5 by ELISA It was a myeloma protein. Using this myeloma IgG as a carrier, PMB binding As a result, the reactivity between the Escherichia coli and the IgG-PMB conjugate is exclusively PMB. It was confirmed that it was due to the bond between the and lipopolysaccharide.   To 11 to 12 4 to 5 day-old SD rats (body weight: 10 g) in each group, 100 30 μg, 100 μg or 300 μg IgG-PM in μl endotoxin-free PBS B-conjugate or control IgG was administered ip. PBS (combined) for untreated groups (Body diluent) only. After 2 hours, about 2560 of PBS in 100 μl E. coli C5 bacteria were subcutaneously administered to each rat from the back of the head. 24 hours after infection, live Survival, survival with lesions and mortality were recorded. The results are shown in Table 39. The lesion was very hemorrhagic and was at or near the inoculation site. Have lesions All newborns were found to be bacteremia 24-48 after the wound appeared Died in time. For bacteremia, blood was collected from the tail vein and treated with BHI broth. Dilute 20-fold and add 20 μl aliquots (5% sheep red blood cells) to blood agar plates. Detected by plating on the plate.   These results indicate that 30 μg IgG-PMB conjugate / newborn, or 300 μg / After a dose of kg was given to all animals against the development of E. coli lesions or mortality It is shown to be sufficient to provide full protection for 24 hours. In contrast, untreated Protected by 30 or 100 μg IgG / newborn dose compared to loop Not. Newborns treated with the highest dose of IgG (300 μg) were protected (not It may be a non-specific phenomenon.   These findings indicate that IgG-PMB conjugate administered prophylactically causes sepsis caused by Escherichia coli. It proves that the progress of can be stopped. This will prevent the death of the newborn And that lesion lesions could be prevented. These results show that this conjugate is Intended to be able to control infection by being distributed throughout the body even in the area outside the vascular compartment. I taste. Example 31 Derivatization of 7-aminocephalosporanic acid with sulfo-MBS   This example also illustrates the attachment of a heterobifunctional crosslinker to an antibiotic precursor. Of. This example illustrates 7-A, an antibiotic precursor that does not show significant antibacterial properties. Mincephalosporanic acid, m-maleimidobenzoyl-N-hydroxysulfo Derivatization with succinimide ester (sulfo-MBS) is outlined.   To derivatize 7-aminocephalosporanic acid, 2.9 mg of 7-amido was used. Nocephalosporanic acid (Sigma) was added to 1.0 ml of 50 mM phosphate buffer (pH 6. 65) and dissolved in 10.7 μl of 1. 0N NaOH was added during mixing to bring the pH back to 6.65. Then 4.6 mg sulfo-MBS (Pierce) was added and dissolved with mixing. This mixture Was incubated for 4.5 hours with stirring at ambient temperature. 10 mg ethanol Incubation was continued for an additional 17 minutes with the addition of luamine.   This reaction mixture (0.5 ml) was applied to a 1.5 × 20 cm column of Bio-Rad P2 resin. Applied The eluent is 50 mM phosphate buffer (pH 6.65), which is 0.5 Run at ml / min. 280n eluate (ie, liquid collected at the bottom of the column) Followed for absorbance at m. Fractions were collected in 0.5 ml increments. 2 main sucks A peak appeared. One was 42 minutes in the center and the other was 47.5 minutes (that Aminocephalosporanic acid and reaction products, respectively). Paired with the leading edge of its first peak The corresponding fractions were pooled (2.0 ml, 3.42A).₂₈₀). 1.4 μl of 100% β -Mercaptoethanol was added to this mixture and then sterile Whatman 0.45 Filtered using a Micron Puradisc. This putative S-hydroxyethylthio- The MIC of maleimidobenzoyl-N-aminocephalosporanic acid ester is yellow. Staphylococcus aureus, 3.7A for 7-aminocephalosporanic acid₂₈₀Compared to 0.86A₂₈₀Was measured.   A control was also tested for the above derivatized precursor. Sulfo-MBS, β-mercap To the concentrations used above for ethanol and ethanolamine for S. aureus The activity was evaluated. These compounds were inactive.Example 32 Derivatization of 6-aminopenicillanic acid with sulfo-MBS   This example is directed to different antibiotic precursors of the heterobifunctional crosslinker of Example 31. To explain the combination of. This example shows an antibiotic that does not show significant antibacterial properties. Outline derivatization of the precursor 6-aminopenicillanic acid with sulfo-MBS It   To derivatize 6-aminopenicillanic acid, 12.1 mg of 6-aminopenicillanic acid was used. Disilanic acid (ICN) was added to 2.5 ml of 50 mM phosphate buffer (pH 6.65). Dissolved. This solution is continuously mixed with a stirrer and a magnetic stirrer, and H was followed. Sulfo-MBS (24.1 mg, Prochem, Inc.) was added and 1. The pH was adjusted to 6.85 with 0N sodium hydroxide solution. Ambient temperature of this mixture And incubated for 2.5 hours.   Whatman LRP-2 was equilibrated with 10% methanol water in this reaction mixture. Applied to a 1.5 x 20 cm column of resin (C18 reverse phase). 10% of this column Develop with methanol at 1.0 ml / min for 5 minutes, then 10-90% methanol water The gradient was developed for 30 minutes. Include the final peak material with absorption at 280 nm. Collect the eluate (eluted at 26 minutes) and reduce it using a Labconco Centravap concentrator. It was concentrated to dryness under pressure. This derivatized aminopenicillanic acid was added to 1.0 ml of 50 mM Dissolved in phosphate buffer + 1.0 mM EDTA (pH 6.65). This induction The MIC of the immobilized aminopenicillanic acid is Measured to be 8 μg / ml compared to 250 μg / ml for disilane acid. It was   Purified human IgG (40 mg, Sigma) was added to 2.5 ml of 50 mM triethanolamine. Min, dissolved in 1.0 mM EDTA (pH 8.0) and magnetically stirred. And continuously stirred with a stirrer. 13 mg / ml iminothiolane in water (Trau 100 t of Reagent, Prochem) was added. pH tracking 1.0N sodium hydroxide Adjusted to 8.0 with Lithium. This mixture was incubated for 2 hours at ambient temperature . Then, this mixed solution was added to Spectra / Gel ACA202 (Spectra) 2.5 × 20c. m column. This column is 50mM sodium phosphate buffer, 1.0m Elution with MEDTA (pH 6.5) at 2.0 ml / min. Absorption at 280 nm Was tracked. Collect material in pore volume containing iminothiolane IgG and pool I did. The concentration of iminothiolane-modified IgG was 5.0 mg / ml.   This derivatized aminopenicillanic acid (0.5 ml, 9.0 mg / ml) was added to 1.75 Mix with ml of iminothiolane IgG and mix for 10 minutes at ambient temperature with ink And then incubated overnight at 2-8 ° C. Bring this mixture to ambient temperature It returned and incubated, stirring. After 20 minutes, 146 μl of 10 mM N -Ethylmaleimide (Pierce) was added and the incubation continued for 4 hours. Pass this reaction mixture through Uniflo-Plus filter (S & S) to Spectra / Gel ACA It was applied to a 2.5 × 20 cm column of 202 (Spectrum). This column is 50m Elution with M sodium phosphate buffer (pH 6.5) at 2.0 ml / min. 280n The absorption at m was followed. MBS aminopenicillanic acid: pores containing IgG Material in volume is collected, pooled and placed in a Centricon concentrator. Concentrate using Amicon and pass through Uniflo-Plus filter (S & S) . This MBS aminopenicillanic acid: IgG is 0.65 against Staphylococcus aureus. It was found to be inactive at mg / ml.   A control was also tested for the above derivatized precursor. Sulfo-MBS, β-mercap To the concentrations used above for ethanol and ethanolamine for S. aureus The activity was evaluated. These compounds were inactive.Example 33 Derivatization of amoxicillin with sulfo-SMCC   This example demonstrates the sulfo-S of amoxicillin, an antibiotic showing significant antibacterial properties. Figure 3 outlines derivatization with MCC. To derivatize amoxicillin , 23 mg of amoxicillin trihydrate (ICN) with 2.5 ml of 50 mM phosphoric acid Added in buffer (pH 6.65). This suspension is stirred with a magnetic stirrer. The pH was monitored by continuous mixing with a roller. Sulfo-SMCC (23 mg, Proc hem, Inc.) and adjust the pH to 7.0 with 1.0 N sodium hydroxide (periodic) Readjusted to 7.0). This mixture was incubated for 4 hours at ambient temperature (This reaction mixture was initially cloudy due to suspended amoxicillin, but with time Became transparent).   This reaction mixture was equilibrated with 10% methanol water, Whatman LRP-2 tree. Applied to a 1.5 x 20 cm column of fat (C18 reverse phase). This column is 10% 5 minutes at 1.0 ml / min with Tanol After opening, it was developed with a primary gradient of 10-90% methanol water for 30 minutes. 280 Collect the eluate containing the material of the final peak absorbing at nm (eluted at 27.5 minutes). Therefore, it was concentrated to dryness under reduced pressure using a Labconco Centravap concentrator. This derivatization Amoxicillin was added to 1.0 ml of 50 mM phosphate buffer + 1.0 mM EDTA (pH 6). .65). The MIC of this derivatized amoxicillin is 8 μg against fungi, compared to 250 μg / ml for native amoxicillin / Ml.Example 34 Derivatization of cefadroxil with sulfo-SMCC   In the following example, cefadroki, an antibiotic active against Gram-positive bacteria, is used. Syl is used as a cross-linking agent 4- (N-maleimidomethyl) cyclohexane-1-carvone. Reaction with sulfosuccinimidyl acid (sulfo-SMCC).   Cefadroxil (Sigma) in 50 mM phosphate buffer (pH 6.65) 3. It was dissolved at 0 mg / ml and sulfo-SMCC was dissolved at 2.6 mg / ml. Incubate for 1 hour 55 minutes at ambient temperature with agitation, then add ethanolamine Was added at 3.4 mg / ml and the incubation was continued for another 42 minutes.   This reaction mixture (0.5 ml) was mixed with 1 of Sephadex G10 resin (Pharmacia). Applied to a 0.5 x 13 cm column. The eluent is 50 mM phosphate buffer (pH 6.65). ), Which was run at 0.5 ml / min. Absorbance of eluate at 280 nm Tracked about. Fractions of 1.0 ml each were collected. Two main absorption peaks appeared . on the other hand Had 22 minutes in the center and 36 minutes in the other (reaction product and cefadr Kishiru). Fractions corresponding to the leading edge of the first peak were pooled (3.0 ml, 3.5A₂₈₀). 300 μl of 100 mM β-mercaptoethanol was added to this mixture And then filtered using a sterile Whatman 0.45 micron Puradisc. This putative S-hydroxyethylthiomaleimidomethylcyclohexanecarbo The MIC of xy-N-cefadroxyl ester is 0.028A About Natural Cefadroxil₂₈₀1.6A compared to other MIC₂₈₀ Was measured. Therefore, this derivatized cefadroxil is relatively inactive there were.Example 35 Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB, and Sulfo-S Derivatization of vancomycin with MPB   In the following example, vancomycin is an antibiotic active against Gram-positive bacteria. Was reacted with four different heterobifunctional crosslinkers.   Vancomycin in phosphate buffer was reacted with each of the following compounds: 6- [ 3- (2-Pyridyldithio) propionamide] hexanoic acid sulfosuccinimine Zil (sulfo-LC-SPDP), m-maleimidobenzoyl-N-hydroxy Sulfosuccinimide ester (sulfo-MBS), (4-iodoacetyl) a Sulfosuccinimidyl minobenzoate (sulfo-SIAB), and 4- (p-ma) Reimidophenyl) sulfosuccinimidyl butyrate (sulfo-SMPB). All 4 types Crosslinkers react with primary amino groups on the vancomycin molecule to form amide bonds To do. Vancomycin derivatized with sulfo-LC-SPDP is suitable It has a sulfhydryl group that can react with maleimide on derivatized IgG when exposed to conditions. To do. Vancomycin derivatized with sulfo-MBS is a reduced IgG or Having a maleimide group capable of reacting with any of the sulfhydryl groups of derivatized IgG , The reaction is due to the addition of the maleimide to the carbon-carbon double bond. Finally, Vancomycin derivatized with Rufo-SIAB is a reduced IgG or derivative Having an iodo group capable of reacting with any of the sulfhydryl groups of derivatized IgG The reaction is due to nucleophilic substitution with the iodo group. Sulfo-MBS, Sulfo-SIAB, Su Ruho-SMPB or sulfo -SMCC (Pierce) in 50 mM sodium phosphate buffer (pH 7.15) Solution of 10 mM vancomycin (ICN) at a concentration of 20 mM with mixing Dissolved in. The mixtures were incubated at ambient temperature with agitation. 4 No further traces of precipitates and reaction products formed in the mixture of all species .Example 36 Iminothiolane and 4- (N-maleimidomethyl) cyclohexane-1-carbo Conjugate formation of vancomycin to IgG with sulfosuccinimidyl phosphate   Soluble derivatized vancomycin suitable for further conjugate formation to immunoglobulins Since the results of the previous examples to obtain the above were unsatisfactory, iminothiolane (Traut reagent) was used to derivatize vancomycin to obtain 4- (N-maleimido isomer). Cyl) cyclohexane-1-carboxylate sulfosuccinimidyl (sulfo-SM Another cross-linking method for derivatizing IgG with CC) was investigated. This reaction is The process proceeds in three steps outlined in. First, derivatization of vancomycin with Traut reagent To do. Second, non-specific immunoglobulins are derivatized with sulfo-SMCC. First 3, these derivatized vancomycin and derivatized IgG were reacted with each other and bound. Let the body form.   a) Reaction of vancomycin with iminothiolane   33.4 mg of vancomycin (ICN) 2.0 ml of freshly degassed 50 Dissolved in mM triethanolamine, 1.0 mM EDTA (pH 8.0) buffer Let Dissolve 34.5 mg of Traut reagent (Pierce) and add N₂Gas that bahia Blow into the bottle I plugged it up. Incubate for 1 hour and 15 minutes while shaking this vial I got it.   This mixed solution (0.5 ml) was added to Sephadex G10 (Pharmacia) 1.5 × 1. Applied to a 3 cm column. The eluent was freshly degassed 50 mM phosphoric acid, 1.0 It was mMEDTA (pH 6.65) buffer, which was run at 0.5 ml / min. 1 Fractions of 0.0 ml were collected and the first peak fraction (column stomatal dose) was pooled. (3.0 ml).   Pool from G10 column with 4 ml of Bio-Rad Affi-Gel 501 organomercury resin , 1.0 cm diameter column. This 501 resin is 50 mM sodium acetate (PH 5.0) (acetic acid buffer) 25 ml, 4.0 mM acetic acid secondary water in acetic acid buffer Preliminarily washed with 25 ml of silver and 25 ml of acetate buffer, 50 mM phosphoric acid, 1.0 mM It was equilibrated with EDTA, 0.5% Tween 20 (pH 6.65). This Was applied at 0.2 ml / min to obtain 50 mM phosphoric acid, 1.0 mM EDTA, The column was washed with 0.5% Tween 20 (pH 6.65). 13 minutes later The speed was increased to 1.0 ml / min. Continue to wash for a total of 85 minutes and use natural non-thiolane Vancomycin chloride was removed. 50 mM phosphoric acid, 1.0 mM EDTA, 0.5% Tze Freshly prepared 10 mM β-mercaptoethanone in corn 20 (pH 6.65) With Affi-Gel 501 resin at 1.0 ml / min. Was eluted. The first 5 fractions, each 1.0 ml, from the baseline Is also significantly larger A₂₈₀It was pooled because it had (0.116A₂₈₀Or 29 μg / ml, 5.0 ml). About this putative iminothiolane vancomycin All MICs were determined to be 2.4 μg / ml for S. aureus. Banco The MIC for mycin is 1-2 μg / ml.   The derivatized vancomycin was concentrated with a Labconco Centravap and 1.0 ml of water was added. And the material was added to Sephadex G10 (Pharmacia) 1.5 × 13. applied to a cm column. The column was washed with 50 mM sodium phosphate buffer, 1.0 Eluted with mM EDTA, 0.5% Tween 20 (pH 6.65). Medium pore volume Material was collected and pooled.   b) Reaction of IgG with sulfo-SMCC   Generally, 15 moles of sulfo-SMCC were used per mole of IgG. Than this IgG precipitated with a high molar ratio of cross-linking agent. 40 mg human IgG (Sigma, Talog # I4506, Lot # 063H-88751) in 2 ml of 50 mM phosphorus. 1.6 mg of sulfo-SMCC (Proche) was dissolved in acid buffer (pH 7.1). m, catalog # CL207, lot # 03092) was added with mixing. This Of the mixture was incubated at room temperature for 45 minutes and then loaded onto an ACA202 gel filtration column. Excess crosslinker was removed by chromatography. 2 ml sample, 50 mM 2.5 × 20c equilibrated with MES, 0.5% Tween 20 (pH 6.5) buffer m ACA202 gel filtration column (Spectrum) and eluted with the same buffer. The first peak corresponding to this activated IgG was collected and the absorbance at 280 nm was collected. Tracked.   c) Derivatized vancomycin and derivatized IgG conjugate formation   To finally conjugate derivatized vancomycin and derivatized IgG To 2.95 ml of 4.4 mg / ml SMCC: I gG (from step b above) was added to 14 ml of 0.74 mg / ml iminothiola Vancomycin (from step a above). Ambient temperature of this solution Incubated at 200 rpm on a rotary shaker. 100 minutes after 1 hour and 5 minutes Add μl of 3.5 mg / ml 2-mercaptoethanol and incubate for another 25 minutes. Incubation was continued. Using Amicon Centricon 30, add about 2 parts of this solution. Concentrated to ml.   This sample is a 2.5 × 20 cm sample of Spectra Gel ACA202 resin (Spectrum). A ram was loaded and eluted with PBS + 0.1% Tween 20 at 0.1 ml / min. 1 Fractions of 0.5 ml were collected. Fractions in pore volume were pooled and sterile filtered. This result Coalescence activity was measured by the standard MIC test against S. aureus. this The conjugate was found to be inactive at 1.1 mg / ml against S. aureus It was.Example 37 S-Acetylmercaptosuccinic anhydride (SAMSA) and 4- (N-maleimide) Methyl) cyclohexane-1-carboxylate sulfosuccinimidyl (sulfo-S Conjugate formation of vancomycin to IgG in MCC)   Of the previous example in which an attempt was made to obtain a vancomycin-IgG conjugate having antibacterial activity. Since the result was unsatisfactory, S-acetylmercaptosuccinic anhydride (SA Vancomycin was derivatized with MSA) to give 4- (N-maleimidomethyl) Cyclohexane-1-carboxylic acid sulfosuccinimidyl (sulfo-SMCC) To Another cross-linking method was used to derivatize IgG using. This reaction is outlined below It progresses in 5 steps. First, vancomycin is derivatized with SAMSA. First 2. Unreacted both derivatized vancomycin and free unreacted vancomycin. Separate from the crosslinker. Third, derivatized vancomycin is free of unreacted van Separate from comomycin. Fourth, non-specific immunoglobulin with sulfo-SMCC Is derivatized. Fifth, purified derivatized vancomycin and derivatized IgG are To form a conjugate.   a) Reaction of vancomycin with SAMSA   20.4 mg of vancomycin (Sigma, Catalog # V2002, Lot # 43 H1090) [14 μmol] was dissolved in 200 μl of water. 1.2 ml of satiety Add sodium succinate slowly with stirring and place the mixture on ice. And cooled to 4 ° C. The mixture was slightly cloudy. Add 200μ to this mixture 1 dimethyl sulfoxide (DMSO, Mallinckrodt, Catalog # 5507, 121.8 mg of SAMSA (Sigma, mosquitoes) dissolved in solution # 5507KLDL). Talog # A1251, Lot 3 # 120H5017) was added. track pH It was The initial pH was 8.1 and was 6.8 after the addition of SAMSA. At a constant speed Incubate the reaction mixture at 4 ° C for 1 hour with stirring and then at room temperature. Incubate for 1 hour.   b) Separation of excess crosslinking agent   This mixture was used to remove excess crosslinker from modified and unmodified vancomycin. G-10 color equilibrated with 50 mM sodium phosphate buffer (pH 7.1) Mu (2.5 x 20 cm, Pharmacia). First containing both modified and unmodified vancomycin Peaks were collected and stored at 4 ° C.   c) unmodified bar by affinity chromatography on an organic mercuric column. Purification of modified vancomycin from ncomycin   Manufacturer of 5 ml Affi-Gel 501 organic mercuric agarose column (Bio-Rad) Was prepared according to the manufacturer's instructions. This column is loaded with 50 mM phosphate buffer (pH 7 Equilibration in step 1). SAMSA modified vancomycin is a protected sulfhydride Contains a ryl group. Deprotect it with hydroxylamine hydrochloride before applying it to the column. Protected. Hydroxylamine hydrochloride was added to the modified vancomycin solution to a final concentration of 0.2M. And the mixture was incubated at room temperature for 5 minutes. Af this sample Apply to a fi-Gel 501 column at a flow rate of 0.5 ml / min, then apply this column to 10 mM2- (N-morpholino) ethanesulfonic acid, 1 mM EDTA, 0.5% Tze Baseline A with buffer 20 (pH 6.5)₂₈₀Was washed until Bound modified vancomycin containing 20 mM 2-mercaptoethanol Elute with the same wash buffer. The activity of this modified vancomycin is It was determined to be 2.6 μl / ml by MIC test against cocci.   d) Reaction of IgG with sulfo-SMCC   The reaction was carried out as described in Example 35 step b (above).   e) Formation of conjugate between derivatized vancomycin and derivatized IgG   Modified vancomycin is a buffer containing 20 mM 2-mercaptoethanol. It was in the liquid. This 2-mercaptoethanol Benzoylated Dialysis Tubing (Sigma, Catalog # D7884, Lot # 43 H7085) and removed by dialysis. 750 μg vancomycin (about 0 0.5 μmol) and 7.5 mg (0.05 μmol) maleimide-activated IgG were used. This mixture was incubated at room temperature for 1 hour. 30 moles per mole of IgG 2-Mercaptoethylamine was added and the mixture was incubated at room temperature for 20 minutes. To block unreacted maleimide sites. Gel filtration chroma 2-mercaptoethylamine and unreacted vancomycin by topography The conjugate was purified from. This sample was added to 0.01 M containing 0.1% Tween 20. ACA202 gel filtration column (equalized with phosphate buffered saline (pH 7.2)) 2.5 × 20 cm, Spectrum). Track the absorbance at 280 nm It was The first peak containing the vancomycin-IgG conjugate was collected. This bond Body activity was measured by the MIC test against S. aureus. The yellow of this combination The MIC against Staphylococcus aureus was found to be 0.438 mg / ml. This example shows vancomycin derivatized with SAMSA and sulfo-SM. Conjugate formation with IgG derivatized with CC results in active conjugate Has proved. This conjugate was tested against S. aureus and found to be effective It turned out to be an antibacterial agent.Example 38 Conjugate form of Limulus anti-lipopolysaccharide factor to IgG by periodate oxidation of IgG Success   This example demonstrates the periodate oxidation of human immunoglobulin to human IgG. 2 illustrates the formation of a conjugate of the Limulus anti-lipopolysaccharide factor (LALF). L ALF is a single-chain peptide known to bind to and neutralize endotoxin. is there. H.S. See Waver et al., Infection and Immunity 60: 2506 (1992). . The sequence of this peptide is shown in FIG. LALF conjugate to this IgG After formation was complete, the conjugate was transformed into E. coli 0111: B4 lipopolysaccharide (LPS) and It was tested for binding to both E. coli HB101. LALF binding to LPS The binding properties of body and PMB conjugate were also compared.   a) Preparation of LALF-IgG conjugate   Follow inhibition of LPS-induced cytolytic activation using spectrophotometric LAL assay Purification was carried out. Briefly, L. polypemus (L. polyp transformed cells from hemus) under non-endotoxin conditions and lysed by addition of distilled water And centrifuged at 5,000 xg for 30 minutes. Extract the precipitate with 3M urea It was The extract was filtered through a 30,000 Da cutoff membrane to obtain a 8,000 Da cutoff. Concentrated by toff membrane. The debris was mixed with 3M urea-10 mM ammonium acetate. Apply to a cation exchange column (CM Sepharose) equilibrated at (pH 5.5) And eluted stepwise with 0.15, 0.25, 0.5M NaCl. Its 0.5M The NaCl peak was equilibrated with water-0.2% trifluoroacetic acid on a C-4 reverse phase column. (Vydac, Hesperia, CA). Set this column to 0. Step with 25, 35 and 50% isopropanol containing 2% trifluoroacetic acid Eluted. 50% isopro The propanol peak was freeze-dried and returned to the state immediately before use. This final product has a high reverse phase Higher than 95% pure by high performance liquid chromatography and SDS-PAGE It turned out to be degree.   In this case, purified LALF was obtained from a member of Cape Cod. Lyophilized LALF Dissolved at 15 mg / ml in 50 mM sodium acetate (pH 5.0). this MIC of native LALF for E. coli HB101 is 16 μg / ml I understood.   10 mg of purified human IgG (Sigma) was added to 1.0 ml of 50 mM sodium phosphate ( It was dissolved in pH 7.1). 10.7 mg of sodium metaperiodate was added to this I It was added to the GG solution and dissolved with mixing. Rotate this mixture at ambient temperature Incubated for 30 minutes at 180 rpm on a shaker.   This mixed solution is 2.5 × 20 cm of Spectra-Gel ACA202 (Spectrum) resin. Apply to the column, 50 mM sodium phosphate, pH 6.7, at 2.0 ml / min. Eluted. The absorbance at 280 nm was followed and 2.0 ml fractions were collected. Mosquito Fractions corresponding to the stomatal volume of rum (oxidized IgG) were collected.   0.66 ml of the above 15 mg / ml LALF solution was added to 1.3 ml of oxidized IgG (2. 1 mg / ml) while being added dropwise. This mixed solution is 1 using a rotary shaker. Incubated at 80 rpm for about 18 hours. Mix 39 μl of 1.0 N HCl While adding. Incubate this mixture at 180 rpm for 3 hours at ambient temperature. I got it.   Apply this mixture to a 1.5 x 20 cm column of Spectra-Gel ACA202 resin. Then, add PBS + 0.1% Tween 20 to 1.0 ml / Eluted in minutes. The eluate corresponding to the stomatal volume containing LALF: IgG conjugate was collected. The MIC of this LALF: IgG conjugate for E. coli HB101 is 3 It was determined to be 00 mg / ml.   b) LPS binding of LALF: IgG conjugate and E. coli   HB101 binding assay   E. coli 0111: B4 lipopolysaccharide (LPS) was obtained from Sigma and was added to PBS + 0.0. Dissolved at 0.02 mg / ml in 05% thimerosol. E. coli HB101 Diluted to 10,000,000 CFU / ml with PBS. LPS solution, E. coli HB Falcon Pro-Bind 96 with 100 μl aliquots of 101 suspension or PBS Wells were added to microtiter plate. Incubate the plate at 2-8 ° C for 18 hours. Incubated. Wells were washed 3 times with PBS. 100 μl PBS + 5 m Add g / ml BSA (Sigma Chemical Co.) to each well of the plate, The plates were incubated for 2 hours at room temperature. Decant those plates Add 100 μl of sample (eg, conjugate, antibody, etc.) per well and plate Were incubated for 2 hours at ambient temperature. Add the wells to BBS (0.125 sodium borate). Thorium, 1.0 M NaCl (pH 8.3)) + 0.5% Tween 20, 6 times, 5 It was washed 3 times with 0 mM sodium carbonate (pH 9.5). 3 Sigma 104 phos Phatase substrate tablets in 15 ml of 50 mM sodium carbonate buffer + 10 mM M gCl₂Dissolved in and added at 100 ml per well. About 2 at ambient temperature After 0 minutes, the absorbance of each well at 410 nm was measured. The result It shows in Table 40.   Separate LALF: IgG and PMB: IgG conjugates prepared as above did. PMB: IgG to LPS coated and uncoated (PBS) plates, Binding data for LALF: IgG and control IgG are shown in the table below. LPS coated play The binding of both conjugates to the cytoplasm was significant, and LP at 4 μg / ml and 0.8 μg / ml Higher binding of LALF: IgG to S. No control to LPS coated plate Poor binding of conjugated IgG and binding of both conjugates to PBS coated plates So, LALF: IgG conjugate and PMB: IgG conjugate specifically bind to LPS To do. Thus, binding is mediated by the LALF and PMB portions of the conjugate . Example 39 Neutralization of in vivo action of endotoxin by LALF-IgG   Confirm whether LALF-IgG conjugate can neutralize the lethal action of endotoxin In order to use the mouse model of endotoxin shock described in Example 16 (see above) I was there. Neutralization of endotoxin lethality was assessed by LALF-IgG. Minimum efficacy of endotoxin The lethal dose was 15 ng and the injection volume was 200 μl / mouse.   Diluent used was PBS and 0.1% Tween 20 (without BSA) As described in Example 23 (see above), E. coli 0111: B4 endotoxin (Sigm a) was prepared. Endotoxin and various amounts of LALF-IgG were used in Example 16 (b) (above). Incubated with varying amounts as in (cf. The results are shown in Table 42. . table 42, LALF-IgG conjugates showed 15 ng endotoxin per mouse. 5 μg per mouse was 100% effective in neutralizing the lethal dose of.

[Procedure Amendment] Patent Law Article 184-7, Paragraph 1 [Submission date] June 8, 1994 [Correction content]                                The scope of the claims 1. Antibiotics covalently bound to a non-specific immunoglobulin having an Fc region An antibiotic-antibody bond, which comprises an antibiotic-antibody bond capable of binding to bacteria through the antibiotic body. 2. The conjugate according to claim 1, wherein the immunoglobulin is IgG. 3. The IgG is capable of binding to phagocytes via the Fc region. Combination of. 4. The conjugate according to claim 1, wherein the conjugate is bacteriostatic. 5. The conjugate of claim 1, wherein the conjugate is bactericidal. 6. The conjugate according to claim 1, wherein the bacterium is a Gram-positive bacterium. 7. The conjugate according to claim 1, wherein the bacterium is a Gram-negative bacterium. 8. Wherein the conjugate is capable of binding lipopolysaccharide on Gram-negative bacteria Item 7. A conjugate according to item 7. 9. 8. The conjugate of claim 7, wherein the conjugate is also capable of binding free endotoxin. Combination of. Ten. 10. The combination according to claim 9, wherein the conjugate is capable of neutralizing free endotoxin. Coalescing. 11. 11. The conjugate according to claim 10, wherein the antibiotic is polymyxin. 12. The conjugate according to claim 11, wherein the polymyxin is polymyxin B. 13. Endotoxin binding covalently bound to a non-specific immunoglobulin having an Fc region An antibiotic-antibody conjugate consisting of a compound. 14. 14. The conjugate according to claim 13, wherein the immunoglobulin is IgG. 15． IgG can bind to phagocytes via the Fc region, The conjugate according to claim 14. 16. The method of claim 13, wherein the conjugate is capable of binding Gram-negative bacteria. Union. 17． 17. The conjugate according to claim 16, wherein the conjugate is bacteriostatic. 18. 17. The conjugate of claim 16, wherein the conjugate is bactericidal. 19. 14. The conjugate of claim 13, wherein the endotoxin binding compound is polymyxin. 20. 20. The polymyxin conjugate is also capable of binding free endotoxin. The conjugate according to. twenty one. Treatment with surface-active antibiotics covalently linked to non-specific immunoglobulins A medicinal preparation which is bactericidal against both Gram-positive and Gram-negative bacteria, A therapeutic preparation having a circulating serum half-life of 48 hours or more after administration. twenty two. Antibiotics selected from the group consisting of cephalosporins and penicillins The therapeutic preparation according to claim 21. twenty three. i) consist of a first antibiotic covalently linked to a non-specific immunoglobulin A first conjugate, and     ii) consists of a second antibiotic covalently linked to a non-specific immunoglobulin A second conjugate,   22. The therapeutic formulation of claim 21, further comprising: twenty four. The first antibiotic is polymyxin and the second antibiotic is bacitracin, The therapeutic preparation according to claim 23. 25. Two different antibiotics are covalently attached to the same immunoglobulin molecule The therapeutic formulation according to claim 21, wherein the therapeutic formulation is 26. One antibiotic can bind to Gram-positive bacteria and the other The therapeutic formulation according to claim 25, wherein the antibiotic is capable of binding to Gram-negative bacteria. . 27. a) prepare a mammal for treatment,     b) Surface-active antibiotic covalently bound to non-specific Ig via a cross-linking agent A therapeutic formulation containing     c) administering the formulation to the mammal prior to the appearance of signs of bacterial infection,   A method of treatment, including: 28. 28. The method of claim 27, wherein the endotoxin binding compound is polymyxin. 29. 28. The method of claim 27, wherein the protein is a non-specific immunoglobulin. . 30. 28. The method of claim 27, wherein the immunoglobulin is IgG. 31. 28. The method of claim 27, which is administered intravenously. 32. a) prepare a mammal at risk of developing sepsis,     b) an anti-antibody capable of binding to a microorganism covalently bound to a non-specific immunoglobulin Prepare a therapeutic formulation containing a biological substance, and     c) administering the formulation to the mammal before the onset of signs of sepsis,   A method of treatment, including: 33. Newborn calves and newborn horses are at risk of developing sepsis 33. The method of claim 32, selected from the group consisting of: 34. Mammals at risk of developing sepsis undergo surgery and trauma 33. The method of claim 32, selected from the group consisting of: and a victim of burns. 35. A mammal at risk of developing sepsis is an immunocompromised patient 33. The method of claim 32. 36. 33. The method of claim 32, which is administered intravenously. 37. The mammals described above may develop sepsis because they may be exposed to Gram-negative bacteria. 33. The method of claim 32, wherein the method is at risk of birth. 38. 33. The method of claim 32, wherein the antibiotic comprises a D-amino acid. 39. 39. The method of claim 38, wherein the antibiotic is polymyxin. 40. 40. The method of claim 39, wherein the polymyxin is polymyxin B. 41. 39. The method of claim 38, wherein the non-specific immunoglobulin is immunoglobulin G. . 42. The above mammals may develop sepsis because they may be exposed to Gram-positive bacteria. 33. The method of claim 32, wherein the method is at risk of birth. 43. 43. The method of claim 42, wherein the therapeutic formulation is capable of binding Gram positive bacteria. Law. 44. Bacitracin covalently bound therapeutic formulation to non-specific immunoglobulin 44. The method of claim 43, including. 45. The mammal may be exposed to Gram-negative and Gram-positive bacteria. 33. The method of claim 32, wherein the method is at risk of developing sepsis. 46. Therapeutic formulation is a bactericidal antibiotic against both Gram-negative and Gram-positive bacteria 46. The method of claim 45, comprising a substance. 47. Antibiotics selected from the group consisting of cephalosporins and penicillins The method of claim 46. 48. Therapeutic formulation covalently bound to non-specific immunoglobulin Consists of an antibiotic capable of reacting with surface components present on Gram-positive bacteria The first conjugate and a Gram-negative cell covalently linked to a non-specific immunoglobulin. Second conjugate consisting of an antibiotic capable of reacting with surface components present on the fungus 47. The method of claim 46, comprising a mixture with. 49. a) i) an antigen on the surface of a pathogen immobilized on a solid support,         ii) Surface-active antibiotics covalently bound to non-specific immunoglobulins A conjugate consisting of, and         iii) preparing a competitor consisting of the surface antigen present in a dissolved state,     b) incubating the immobilized antigen and the conjugate in the presence of the competitor,     c) washing the immobilized surface antigen to remove unbound binder and competitor, and     d) detecting the presence of the conjugate bound to the immobilized surface antigen,   A diagnostic method comprising: 50. Immobilization of said surface antigens onto said wells of a microtiter plate 50. The method of claim 49, which is performed by the attachment of an antigen. 51. 51. The method of claim 50, wherein the surface antigen is isolated from bacteria. 52. 52. The method of claim 51, wherein the bacterium is a Gram negative bacterium. 53. 53. The method of claim 52, wherein the Gram-negative bacterium is E. coli. 54. The method of claim 49, wherein the surface antigen comprises lipopolysaccharide isolated from E. coli. Method. 55. 54. The competitor is a lipopolysaccharide isolated from Gram-negative bacteria. The method described in. 56. Gram-negative bacteria are Escherichia coli and Salmonel la typhimurium), Pseudomonas aeruginosa, Vibrio cho lerae), Shigella flexneri, Klebsiella p. neumoniae), Salmonella enteritiditis, Serratia marcesce ns) and Rhodobacter sphaeroides 56. The method of claim 55, selected from the group consisting of: 57. a) reacting the antibiotic with a crosslinker to form a derivatized antibiotic, do it     b) reacting the derivatized antibiotic with a non-specific immunoglobulin to bind Form the body,   A method for synthesizing a conjugate comprising each step. 58. 58. The method of claim 57, wherein the antibiotic binds to the surface of the microorganism. 59. 59. The method of claim 58, wherein the antibiotic is a peptide. 60. 60. The method of claim 59, wherein the peptide is a Limulus anti-lipopolysaccharide factor. 61. 60. The method of claim 59, wherein the peptide is a D-amino acid containing peptide. 62. 62. The method of claim 61, wherein the peptide binds endotoxin. 63. 63. The method of claim 62, wherein the antibiotic is polymyxin. 64. 64. The method of claim 63, wherein the polymyxin is polymyxin B. 65. 65. The method of claim 64, wherein the antibiotic is bactericidal against Gram-negative bacteria. 66. The method of claim 57, wherein the antibiotic is bactericidal against Gram-positive bacteria. The method described. 67. 67. The method of claim 66, wherein the antibiotic is vancomycin. 68. 68. The method of claim 67, wherein the non-specific immunoglobulin consists of the Fc region. 69. a) reacting the non-specific immunoglobulin with the first modifying reagent to oxidize Forming a globulin preparation, and     b) reacting the oxidized immunoglobulin preparation with an antibiotic and a second modifying reagent. To form an antibiotic-immunoglobulin conjugate.   A method for synthesizing a conjugate comprising each step. 70. 70. The method of claim 69, wherein the immunoglobulin consists of the Fc region. 71. 70. The method of claim 69, wherein the first modifying reagent is an oxidizing agent. 72. 72. The method of claim 71, wherein the oxidant is periodate. 73. 70. The method of claim 69, wherein the second modifying reagent is a reducing agent. 74. 74. The method of claim 73, wherein the reducing agent is sodium borohydride. 75. a) reacting an antibiotic precursor with limited bactericidal activity with a first cross-linking agent Form a derivatized antibiotic precursor with increased bactericidal activity,     b) derivatized by reacting non-specific immunoglobulin with a second cross-linking agent Form immunoglobulins, and     c) A covalent bond between the derivatized antibiotic precursor and the derivatized immunoglobulin. Reacting a derivatized antibiotic precursor with a derivatized immunoglobulin to form , Form a conjugate,   A method for synthesizing a conjugate comprising each step. 76. The antibiotic precursors are 7-aminocephalosporanic acid and 6-amino acid. 76. The method of claim 75, selected from the group consisting of minopenicillanic acid. 77. 77. The method of claim 76, wherein the first crosslinker is difunctional. 78. The first bifunctional crosslinker is m-maleimidobenzoyl-N-hydroxysulfos 78. The method of claim 77, which is a succinimide ester. 79. 76. The method of claim 75, wherein the second crosslinker is difunctional. 80. 80. The method of claim 79, wherein the second bifunctional crosslinker is iminothiolane.

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 08 / 169,701 (32) Priority date December 17, 1993 (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), AU, CA, JP, US (72) Inventor Filka, Joseph Earl.             United States 60061 Ve, Illinois             Vernon Hills, Deer Pass Drive               Address 123 (72) Inventor Pew, Charles S. Gee.             United States 53711 Wisconsin             Madison, Hammer's Ray Road             No. 5817 (72) Inventor Pad High, Nisha Vicas             United States 53719 Wisconsin             21 Laramie Court, Madison, PA             Ground

Claims (1)

[Claims] 1. Consisting of an antibiotic covalently linked to a non-specific immunoglobulin, said antibiotic Antibiotic-antibody conjugate capable of binding bacteria through substances. 2. The conjugate according to claim 1, wherein the immunoglobulin is IgG having an Fc region. . 3. The IgG is capable of binding to phagocytes via the Fc region. Combination of. 4. The conjugate according to claim 1, wherein the conjugate is bacteriostatic. 5. The conjugate of claim 1, wherein the conjugate is bactericidal. 6. The conjugate according to claim 1, wherein the bacterium is a Gram-positive bacterium. 7. The conjugate according to claim 1, wherein the bacterium is a Gram-negative bacterium. 8. Wherein the conjugate is capable of binding lipopolysaccharide on Gram-negative bacteria Item 7. A conjugate according to item 7. 9. 8. The conjugate of claim 7, wherein the conjugate is also capable of binding free endotoxin. Combination of. Ten. 10. The combination according to claim 9, wherein the conjugate is capable of neutralizing free endotoxin. Coalescing. 11. 11. The conjugate according to claim 10, wherein the antibiotic is polymyxin. 12. The conjugate according to claim 11, wherein the polymyxin is polymyxin B. 13. Consisting of an endotoxin-binding compound covalently linked to a non-specific immunoglobulin , Antibiotic-antibody conjugates. 14. 14. The immunoglobulin according to claim 13, wherein the immunoglobulin is an IgG having an Fc region. The conjugate as described. 15． 15. The IgG of claim 14 capable of binding to phagocytes via the Fc region. Combination of. 16. The method of claim 13, wherein the conjugate is capable of binding Gram-negative bacteria. Union. 17． 17. The conjugate according to claim 16, wherein the conjugate is bacteriostatic. 18. 17. The conjugate of claim 16, wherein the conjugate is bactericidal. 19. 14. The conjugate of claim 13, wherein the endotoxin binding compound is polymyxin. 20. 20. The polymyxin conjugate is also capable of binding free endotoxin. The conjugate according to. twenty one. Comprising a surface-active antibiotic covalently bound to a non-specific immunoglobulin, A therapeutic formulation that is bactericidal against both Gram-positive and Gram-negative bacteria. twenty two. Antibiotics selected from the group consisting of cephalosporins and penicillins The therapeutic preparation according to claim 21. twenty three. i) consist of a first antibiotic covalently linked to a non-specific immunoglobulin A first conjugate, and     ii) consists of a second antibiotic covalently linked to a non-specific immunoglobulin A second conjugate, 22. The therapeutic formulation of claim 21, further comprising: twenty four. The first antibiotic is polymyxin and the second antibiotic is bacitracin, The therapeutic preparation according to claim 23. 25. Two different antibiotics are covalently attached to the same immunoglobulin molecule The therapeutic formulation according to claim 21, wherein the therapeutic formulation is 26. One antibiotic can bind to Gram-positive bacteria and the other antibiotic 26. The therapeutic formulation of claim 25, which is capable of binding Lam-negative bacteria. 27. a) prepare a mammal for treatment,     b) A therapeutic product containing a surface-active antibiotic covalently bound to a non-specific 1 g. Prepare the agent, and     c) administering the formulation to the mammal prior to the appearance of signs of bacterial infection, A method of treatment, including: 28. 28. The method of claim 27, wherein the endotoxin binding compound is polymyxin. 29. 28. The method of claim 27, wherein the protein is a non-specific immunoglobulin. . 30. 28. The method of claim 27, wherein the immunoglobulin is IgG. 31. 28. The method of claim 27, which is administered intravenously. 32. a) prepare a mammal at risk of developing sepsis,     b) an anti-antibody capable of binding to a microorganism covalently bound to a non-specific immunoglobulin Prepare a therapeutic formulation containing a biological substance, and     c) administering the formulation to the mammal before the onset of signs of sepsis, A method of treatment, including: 33. Newborn calves and newborn horses are at risk of developing sepsis 33. The method of claim 32, selected from the group consisting of: 34. Mammals at risk of developing sepsis undergo surgery and trauma And a claim selected from the group consisting of burn victims The method according to Item 32. 35. A mammal at risk of developing sepsis is an immunocompromised patient 33. The method of claim 32. 36. 33. The method of claim 32, which is administered intravenously. 37. The mammals described above may develop sepsis because they may be exposed to Gram-negative bacteria. 33. The method of claim 32, wherein the method is at risk of birth. 38. 33. The method of claim 32, wherein the antibiotic comprises a D-amino acid. 39. 39. The method of claim 38, wherein the antibiotic is polymyxin. 40. 40. The method of claim 39, wherein the polymyxin is polymyxin B. 41. 39. The method of claim 38, wherein the non-specific immunoglobulin is immunoglobulin G. . 42. The above mammals may develop sepsis because they may be exposed to Gram-positive bacteria. 33. The method of claim 32, wherein the method is at risk of birth. 43. 43. The method of claim 42, wherein the therapeutic formulation is capable of binding Gram positive bacteria. Law. 44. Bacitracin covalently bound therapeutic formulation to non-specific immunoglobulin 44. The method of claim 43, including. 45. The mammal may be exposed to Gram-negative and Gram-positive bacteria. 33. The method of claim 32, wherein the method is at risk of developing sepsis. 46. Therapeutic formulation is a bactericidal antibiotic against both Gram-negative and Gram-positive bacteria 46. The method of claim 45, comprising a substance. 47. Antibiotics selected from the group consisting of cephalosporins and penicillins The method of claim 46. 48. The therapeutic preparation is covalently linked to a non-specific immunoglobulin First conjugate comprising an antibiotic capable of reacting with surface components present on fungi And on gram-negative bacteria covalently bound to non-specific immunoglobulins A mixture with a second conjugate consisting of an antibiotic capable of reacting with a surface component 47. The method of claim 46, including. 49. a) i) an antigen on the surface of a pathogen immobilized on a solid support,         ii) a surface covalently bound to a non-specific immunoglobulin             A conjugate consisting of an active antibiotic, and        iii) a competitor consisting of the surface antigen present in a dissolved state, Prepare     b) incubating the immobilized antigen and the conjugate in the presence of the competitor,     c) washing the immobilized surface antigen to remove unbound binder and competitor, and     d) detecting the presence of the conjugate bound to the immobilized surface antigen, A diagnostic method comprising: 50. Immobilization of said surface antigens onto said wells of a microtiter plate 50. The method of claim 49, which is performed by the attachment of an antigen. 51. 51. The method of claim 50, wherein the surface antigen is isolated from bacteria. 52. 52. The method of claim 51, wherein the bacterium is a Gram negative bacterium. 53. 53. The method of claim 52, wherein the Gram-negative bacterium is E. coli. 54. The method of claim 49, wherein the surface antigen comprises lipopolysaccharide isolated from E. coli. Method. 55. The competitor is lipopolysaccharide isolated from Gram-negative bacteria. 55. The method of claim 54, wherein 56. Gram-negative bacteria are Escherichia coli and Salmonel la typhimurium), Pseudomonas aeruginosa, Vibrio cho lerae), Shigella flexneri, Klebsiella p. neumoniae), Salmonella enteritiditis, Serratia marcescen s) and Rhodobacter sphaeroides 56. The method of claim 55 selected from the group. 57. a) reacting the antibiotic with a cross-linking agent to form a derivatized antibiotic, and     b) reacting a derivatized antibiotic with a non-specific immunoglobulin to form a conjugate Let A method for synthesizing a conjugate comprising each step. 58. 58. The method of claim 57, wherein the antibiotic binds to the surface of the microorganism. 59. 59. The method of claim 58, wherein the antibiotic is a peptide. 60. 60. The method of claim 59, wherein the peptide is a Limulus anti-lipopolysaccharide factor. 61. 60. The method of claim 59, wherein the peptide is a D-amino acid containing peptide. 62. 62. The method of claim 61, wherein the peptide binds endotoxin. 63. 63. The method of claim 62, wherein the antibiotic is polymyxin. 64. 64. The method of claim 63, wherein the polymyxin is polymyxin B. 65. 65. The method of claim 64, wherein the antibiotic is bactericidal against Gram-negative bacteria. 66. 58. The method of claim 57, wherein the antibiotic is bactericidal against Gram positive bacteria. 67. 67. The method of claim 66, wherein the antibiotic is vancomycin. 68. 68. The method of claim 67, wherein the non-specific immunoglobulin consists of the Fc region. 69. a) Immunity that is oxidized by reacting a non-specific immunoglobulin with a first modifying reagent Forming a globulin preparation, and     b) reacting the oxidized immunoglobulin preparation with an antibiotic and a second modifying reagent. To form an antibiotic-immunoglobulin conjugate, A method for synthesizing a conjugate comprising each step. 70. 70. The method of claim 69, wherein the immunoglobulin consists of the Fc region. 71. 70. The method of claim 69, wherein the first modifying reagent is an oxidizing agent. 72. 72. The method of claim 71, wherein the oxidant is periodate. 73. 70. The method of claim 69, wherein the second modifying reagent is a reducing agent. 74. 74. The method of claim 73, wherein the reducing agent is sodium borohydride. 75. a) reacting an antibiotic precursor with limited bactericidal activity with a first cross-linking agent Form a derivatized antibiotic precursor with increased bactericidal activity,     b) reacting the non-specific immunoglobulin with a second cross-linking agent to derivatize the immunoglobin. Form a roblin, and     c) A covalent bond between the derivatized antibiotic precursor and the derivatized immunoglobulin. Reacting a derivatized antibiotic precursor with a derivatized immunoglobulin to form Form a conjugate, A method for synthesizing a conjugate comprising each step. 76. The antibiotic precursors are 7-aminocephalosporanic acid and 6-aminopenicillanes 76. The method of claim 75, selected from the group consisting of acids. 77. 77. The method of claim 76, wherein the first crosslinker is difunctional. 78. The first bifunctional crosslinker is m-maleimidobenzoyl-N-hydroxysulfos 78. The method of claim 77, which is a succinimide ester. 79. 76. The method of claim 75, wherein the second crosslinker is difunctional. 80. 80. The method of claim 79, wherein the second bifunctional crosslinker is iminothiolane.