JP2001517921A - Early diagnostic tests - Google Patents

Abstract

  (57) [Summary] Disclosed is a method of discriminative testing in the initial stage of infection of lime borreliosis and other infectious diseases. Between the heparinized whole blood of infected and uninfected animals, prior to a measurable antibody response in surface binding of cultured pathogens to naturally occurring polymorphonuclear leukocytes (PMNs) (eg, neutrophils) It has been found that there are dramatic qualitative and quantitative differences that exist early in the disease. This coupling reaction is quickly and accurately discriminated and quantized using various imaging techniques. The method involves visualizing the surface binding of cultured pathogens to naturally occurring PMNs to detect infection.

Description

DETAILED DESCRIPTION OF THE INVENTION CROSS-REFERENCE This application of early diagnostic tests related application is a continuation-in-part application of US application Ser. No. 08 / 487,188, filed on July 7, 1995. In particular, references relating to the amino acid and nucleic acid sequences of Borrelia burgdorferi, such as OspA, can be found in 08 / 087,601, filed June 23, 1993, No. 08 / 320,416 filed in No. 08 / 375,993 filed in No. 08 / 137,175 filed in No. 08 / 262,220 filed on Oct. 18, 1989, and WO 90/04411 (Symbicom AB). TECHNICAL FIELD The present invention relates to diagnostic tests, and in particular to a method for quickly and reliably detecting the presence of pathogens or other foreign substances in humans or animals. A list of references that more fully describes the prior art, to which this invention pertains, and which is one aspect of the invention, is at the end of the specification and immediately preceding the claims. The contents of these references and the references cited herein are hereby incorporated by reference in their entirety. BACKGROUND ART Lyme borreliosis is the most prevalent tick-borne disease in the United States and one of the most important tick-borne communicable diseases in the world. Lyme borreliosis is an endemic disease in the northwestern United States, Minnesota and Wisconsin. Bacterial spirocheta Borrelia burgdorferi is the causative agent of Lyme disease. Infection with Borrelia burgdorferi causes local or systemic symptoms. Local signs that appear early in the infection include illnesses such as skin damage, fever and flu, called chronic erythema migrans (ECM) in places where mites have been killed. Within weeks to months after infection, they develop systemic symptoms, including rheumatic, cardiac and neurological symptoms. In the early localized stages of Borrelia burgdorferi infection, it is easily treatable with antibiotics. However, at a later systemic stage, it is known to be more refractory to antibiotics. SUMMARY OF THE INVENTION Measurable between heparinized whole blood of infected and uninfected animals on surface binding and phagocytosis of cultured Borrelia burgdorferi to naturally occurring PMNs (eg, neutrophils) The dramatic qualitative and quantitative differences that exist in the early stages of the disease, prior to a severe antibody response, have become remarkable. This coupling reaction is quickly and accurately discriminated and quantized using various imaging techniques. This finding has been used to develop a rapid and inexpensive method of discriminating tests for early Lyme borreliosis. In addition, based on this difference from this finding, it has been discovered that a distinct and unique outbreak occurs with respect to another foreign body from Borrelia burgdorferi, thereby allowing virtually any pathogen (eg, bacteria, virus, It provides a quick and inexpensive method for the discrimination test of yeast (or some drugs). BRIEF DESCRIPTION OF THE DRAWINGS In the following detailed description, reference is made to the following figures. FIG. 1 shows the total blood in Balb / C mouse vaccinated with Borrelia burgdorferi, Balb / C mouse vaccinated with Freund's adjuvant, and control group blood of Balb / C mouse vaccinated only with BSK media. FIG. 2 is a graph showing the percentage of neutrophils in white blood cells. FIG. 4 is a graph showing the percentage of white blood cells bound to Borrelia burgdorferispirochetes in the total white blood cells in the blood of a control group of Balb / C mice. DETAILED DESCRIPTION OF THE INVENTION As described above, the present invention relates to a method for quickly and reliably detecting the presence of a pathogen or other foreign substance in a human or animal. Neutrophils are a type of polymorphonuclear leukocyte (PMN) whose known function is the release of drugs for phagocytosis (uptake and destruction of particulate matter) and inflammation. They make up almost 50-70% of the total white blood cells (white blood cells) in normal human blood. It is known that neutrophils solidify large numbers of spirochetes when whole blood of seropositive animals crosses cultured Borrelia burgdorferi. This reaction was observed in dog, horse, cow, goat and rat blood. Blood smears are stained with chromosomes that visualize binding such as DAPI, Hoechst (eg Hoechst 33258), acridine (eg acridine orange) and other fluorescent DNA chromosomes. Antibodies, such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, combined with fluorescent dyes or colorigenic agents are also suitable. Since the binding involves white blood cells, especially neutrophils, the method of the invention is not limited to the use of whole blood, for example urine, respiratory secretions, CSF, exudates from skin trauma or abscesses, tears Includes examination of smears of any bodily fluids or tissues where white blood cells, such as exudate from the sac, synovial fluid, are found. Examples Case 1 Determination of the specificity of binding of spirochetes of three strains of Borrelia burgdorferi to white blood cells of the mouse. This experiment was performed to determine the specificity of the binding of spirochetes of three Borrelia burgdorferi sp. To white blood cells of a mouse, inoculated with one of the three species. Seven-week old female Balb / C mice were used (Jackson Laboratories, Bar Harbor, ME). There were four inoculation groups: strain 2684, strain N-40, strain 25550, and BSK media control. The cultivation of the three strains started from freeze culture. Cultures were performed in the morning of the inoculation in the Petrov-Hauser room. Bacterial strain N-40 had a passage 3, a 90% motile, and a concentration of 7.1 × 10 ⁷ spirochetes / ml. Bacterial strain 2684 was passage 3 inoculated, 90% motile, at a concentration of 6.0 × 10 ⁷ spirochetes / ml. Bacterial species 25550 had a concentration of 3.0 × 10 ⁷ spirochetes / ml at passage 4, 70% motile. Cultures of Borrelia burgdorferi were grown in BSK media at 33-34 ° C.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] April 7, 1998 (1998.4.7) [Correction contents]                                  Specification Early diagnostic tests Cross-reference of related applications   This application is a continuation-in-part of US patent application Ser. No. 08 / 487,188 filed Jul. 7, 1995. Application.   In particular, Borrelia burgdorferi such as OspA Regarding the amino acid sequence and the nucleic acid sequence, see 08 / 079,6, filed June 23, 1993. No. 01, No. 08 / 320,416 filed in No. 08 / 375,993 filed in , No. 08 / 137,175, filed in 08 / 262,220, filed 19 No. 07 / 422,881, filed Oct. 18, 1989, and WO 90/04411 (Symbicom (S ymbicom) AB).   In particular, recombinant Borrelia proteins, their expression by vectors , And their nucleic acid sequences, see 07 / 973,3, filed October 29, 1992. No. 38, No. 08 / 373,455, filed Jan. 17, 1995 (No. 07 / 973,3 No. 38), No. 08 / 211,891, filed October 16, 1992 (PCT / US92 / 086 97 national processing stages), 07 / 888,765, filed May 27, 1992, and 1991. No. 07 / 779,048 filed on Oct. 18, 1998.   Each of the above applications is incorporated by reference in this disclosure.                                Field of the invention   The present invention relates to diagnostic tests, particularly to pathogens or other foreign substances (exogenous Substance) is detected quickly and reliably. The present invention relates to and For a listing of references that more fully describe certain aspects of the invention, see At the end, immediately before the claims. The contents of these references and the present The references cited in the specification are incorporated herein by reference.                                Background of the Invention   Lyme borreliosis is the most prevalent tick-borne disease in the United States. And is one of the most important tick-borne communicable diseases in the world. Lime Bo Leliosis is found in the northeastern United States, namely, Minnesota, Wisconsin, and Taihei Endemic in some parts of the northeastern coast. Bole, a bacterial spirochete Borrelia burgdorferi is the causative agent of Lyme disease. Bole Local or complete infection with Borrelia burgdorferi Develop physical symptoms. The local signs that appear early in the infection are the places where the mites bite Skin lesions called chronic erythema migrans (ECM) in children and fever and flow Including illness like feeling. Weeks to months after infection, rheumatic, cardiac, nervous It manifests systemic symptoms, including sexual symptoms. Borrelia b urgdorferi) In the early localized stages of infection, it is easily treatable with antibiotics. I However, at a later systemic stage, it becomes more refractory to antibiotics. It is known that there is.   Borrelia burderi in animal and human model systems gdorferi) Immune response to infection and early phagocyte response and subsequently How the resulting humoral and cellular responses affect disease development Much effort has been put into research that does. Against the invading spirochetes First, the successful action of phagocytic cells influences the progress of subsequent diseases It is certain that further research is needed.   There are many reports suggesting a cellular response to infection. Some research smells Neutrophils and monocytes may have opsonized and non-opsonized Binds to all passage strains of Borrelia burgdorferi and feeds It has been shown to germ. See, for example, Peterson et al., Infect. Im mun. (1984) 46: 608-611. Spirochetes using specific immune serum Psoninization significantly amplifies phagocytosis. This effect depends on the ability of the polymorphonuclear Fc receptor. It depends on force and appears to be independent of heat-dependent serum factors. Spirochetes treatment In the case of using normal serum (Benach et al., J. Infect. Dis. 984) 150: 497-507) or when using normal whole blood (non-specific opsonization) (ba J. Banfi et al. Applired Bact. (1989) 67: 37-45). In the case of no sonin treatment and the case of opsonin treatment with immune serum Between. Benach et al. (Yale J. Biol. Med. (1984) 57: 599-605). As reported more, experimentally derived skin in animal models and humans Phagocytes are observed in the infiltrate of the skin lesion. Cells involved in the immediate response are It is speculated that this may be different from cells circulating late in the cycle. Further Immediately after infection, natural killer (NK) cells increase in number, but It has been shown that the activity of NK cells in reduced individuals is reduced. this thing Appears to be associated with early expression of suppressor cell activity. The cellular response is Begin early, prior to the humoral response.   For best results in the treatment of Lyme borreliosis, Early in the disease if the phagocytic response of thalassemia and polymorphonuclear leukocytes (PMN) is dominant Administer antibiotics at the stage. Often seen with early, appropriate treatment Can prevent progression to more severe cardiovascular disease and arthritis. healing In addition to maximizing rates and minimizing patient distress, long-term intravenous Treatment of Late-Onset Arthritis and Neurosis in Lyme Disease Including Antibiotic Therapy Early treatment is less costly than treatment cannot be expected. Therefore, Early diagnosis of Lyme disease is essential.   At present, definitive diagnosis of Lyme disease is difficult. Early symptoms Often non-specific. Furthermore, Borrelia burdoferi (Borrelia bur) gdorferi) is a major source of ticks, Some patients do not, and 25-30% do not express ECM. Early stages of the disease Detection of Borrelia burgdorferi in Japan Screening tests allow for early diagnosis and treatment, allowing physicians to Eliminates the need for treatment based on floor findings only.   However, currently available diagnostic tests are inadequate. Infected animals And Borrelia Burgdorf in human blood, urine, cerebrospinal fluid (CFS) and synovial fluid Even though there are Berry (Borrelia burgdorferi), their numbers are very small, In addition, because the morphology is varied, a reliable diagnosis can be obtained by the conventional dark field test. I can't. Culture of these spirochetes from body fluids is Low numbers, and of Borrelia burgdorferi The very low growth rate of wild strains during culture (cultivation takes 5 weeks) Difficulty It is. Bacterial contamination of the sample is also a common problem. Borrelia Barber rich in nutrients necessary for growing Borrelia burgdorferi -Stainer-Kelly (BSK) medium also promotes the growth of contaminated cells and suppresses contaminated cells Only a few antibiotics are used to produce Borrelia burgdorferi (Borr elia burgdorferi) also slows down the growth.   The most commonly used diagnostic test for Lyme disease is enzyme-linked immunosorbent assay. The standard method (ELISA) and the immunofluorescent antibody (IFA) method are used for Borrelia burgdorferi. (Borrelia burgdorferi). Some reasons Therefore, these tests are often inaccurate. Clinical symptoms of Lyme disease can be measured Often appear prior to the development of a unique antibody response. Produces measurable amounts of IgM Requires 3-6 weeks post-infection and several months to reach detectable levels of IgG. In short, however, clinical signs develop within 2 to 20 days after infection. In addition, detection Some patients do not show a definitive antibody response, and antibody titers increase early in the antibiotic response. Attenuated by use and the sensitivity of antibody testing, especially for IFA There is a large variation due to Borrel as a antigen ELISA test results vary due to the use of different strains of ia burgdorferi) There is Finally, other spirochetotic diseases (eg, Leptospira or Treponema etc.) or other borreliosis (eg Borrelia helm) Specificity in patients with serologic tests (eg, Borrelia hermseii) Is missing. As a result, using current diagnostic tests, patients with syphilis May be false positive for syphilis in patients with Lyme disease . Different treatments for each of these diseases, as well as sexually transmitted Social stigmatization of syphilis as a disease is also problematic.   Therefore, in all patients in the early stages of the disease, Borrelia burgdorferi (Borr elia burgdorferi) The need for diagnostic tests to reliably detect infections still exists You.   For many other diseases, diagnostic tests for use in the early stages of the disease are desirable. Or required, but this provides direct evidence of the determination of the pathogen detected. Treatment is difficult or fatal before This is because   For example, acute bacterial meningitis, especially meningococcal meningitis (causative bacteria A meningitidis (Neisseria meningitidis) Is fatal within 1 hour, sore throat, fever, headache, torticollis and nausea Symptoms progress rapidly from symptoms such as, leading to drowsiness, loss of sensation, coma, and death. Early If the disease is confirmed in the early stage, antibiotics can be used to Although the mortality rate of meningitis can be reduced to less than 10%, it can be fatal if the diagnosis is delayed. And many. Current diagnostic methods include lumbar puncture followed by cerebrospinal fluid (CFS) culture and Includes checking for the presence of Cteria. However, such tests take time Lumbar puncture damages nerves if cerebral abscess or lesion is present There are cases. In addition, if no bacteria are found in the culture, the virus It is difficult to determine whether a patient has meningitis or bacterial meningitis. Thus, it is also difficult to determine how to treat a patient. Disease progression Early, if there is a strong suspicion of bacterial meningitis, the conclusion of such a diagnostic test Antibiotics should be started before the fruit is produced.   Similarly, sepsis (invasion of pathogenic bacteria and other toxins into the circulation) Characterized by acute circulatory failure and multiple organ failure including kidney, lung and heart Cause septic shock. Mortality is 25-90% and treatment is started early If not, higher. Therefore, antibiotics may be used before blood culture results are known. Quality administration must begin. This is a guess based on the doctor's experience. Means that treatment may be selected based on the I taste. In addition, especially in patients who have previously received antibiotic treatment, The result may be negative, but a negative culture is not a sepsis. I do not show it.   Sexual contact infection caused by the bacterium Neisseria gonorrhoerae Gonorrhea, if left untreated, can cause bacteremia and gonorrhea Presents complex symptoms such as flame. However, this disease does not involve culture. There is no quick diagnostic test. Gram stain of the urethra indicates that causative bacteria are present in about 90% of men. Can be identified, but only about 60% of the cervical Gram stain I do not respond. There is currently no definitive serological diagnostic test for gonorrhea.   Syphilis is caused by the spirochete Treponema pallidum. Happen. Serologic tests used to diagnose this sexually transmitted disease include, There are two types: non-specific screening to detect substances called syphilis reactants, And a specific test to detect anti-treponema antibodies. Screening test Easy and inexpensive to perform, but with a high rate of false positives, 3-6 weeks after initial infection If not, it will not be positive. Treponemal test is more accurate, but 3-4 post-infection It does not become positive until weeks have passed. For a quick diagnosis of syphilis, To check for Treponema pallidum in the fluid of the lesion Therefore, a technique for collecting and accurately identifying the pathogenic bacteria is required.                                Summary of the Invention   This time, surprisingly, Borrelia burgd orferi) surface-associated and phagocytosed by naturally occurring PMNs (eg, neutrophils) Dramatic qualitative changes between heparinized whole blood of infected animals and that of uninfected animals This phenomenon can be measured at an early stage of infection by measuring the antibody response. I found the fact that it existed before. This binding reaction can be used for various images (image formation). Using the technique, it is possible to quickly and accurately determine and quantify. This discovery was linked to early lime It was used to develop a rapid and inexpensive method for screening for borreliosis. Sa Furthermore, this finding shows that this difference is due to the Borrelia burd gdorferi) is clearly and specifically caused by foreign substances other than And thereby substantially any pathogen (eg, bacteria, virus) , Yeast, or certain drugs) Also, a method for performing the operation at low cost is provided.   Accordingly, the present invention relates to animal or human tissues or fluids containing polymorphonuclear leukocytes. Including exposing a foreign substance to a human or animal, including the step of taking a sample. Provide a way to detect. The sample is contacted with a foreign substance of interest. Sun Before, during or after contacting the pull with foreign material, leukemia So that spheres and foreign substances (such as DNA fluorescent dyes) can be detected Label the bodily fluids or tissues using a suitable label or higher. Use the right method (example e (Eg, fluorescence microscopy) to observe the degree of foreign substance binding to leukocytes . Due to the degree of this binding, exposure to foreign substances, especially when compared to controls Or the presence of foreign substances.   In a preferred embodiment, the leukocytes are neutrophils. Neutrophils can be animal or It can be obtained from any human body fluid or tissue. As a foreign substance, Such pathogens (eg, bacteria, viruses, yeast, fungi, protozoa, parasites) Lipoprotein (eg, in a preferred embodiment) OspA or OspC of Borrelia burgdorferi ), Biological molecules including lipopolysaccharides or glycoproteins.   In another preferred embodiment, the foreign substance is Borrelia sp. Enterobacteriaceae (E. coli), Bordetella pertussis (B. pertussis) ussis), Neisseria, Staphylococcus Genus, Leptospira, Treponema, Listeri Select from the group consisting of the genus Listeria and the genus Mycoplasma Bacteria.   In a further preferred embodiment, the foreign substance is parvovirus, herpes Virus, canine distemper virus, papovavirus, and parainflue A virus selected from the group consisting of anzaviruses.   In yet another preferred embodiment, a body fluid or set from an animal or human Textiles are blood, airway secretions, urine, cerebrospinal fluid, exudates from skin lesions or abscesses , Exudate from the lacrimal sac and synovial fluid.   Thus, the method can be used to determine whether an animal or human has been exposed to a foreign substance, for example, Whether the animal or human is infected or inoculated with the antigen, immunogen or pathogen, Provide an accurate way to determine otherwise exposed.   These and other advantages and embodiments are disclosed in the following description. Or will be obvious from them.                             BRIEF DESCRIPTION OF THE FIGURES   In the following detailed description, reference is made to the following figures.   Figure 1 shows Balb inoculated with Borrelia burgdorferi Balb / C mice inoculated with the blood of Freund's adjuvant White blood in the blood of Balb / C mice inoculated with BSK medium alone as control It is a graph which shows the ratio (%) of a neutrophil in a blood cell.   Figure 2 shows Balb inoculated with Borrelia burgdorferi. Balb / C mice inoculated with the blood of Freund's adjuvant White blood in the blood of Balb / C mice inoculated with BSK medium alone as control Among blood cells, Borrelia burgdorferi spiroche 4 is a graph showing the percentage (%) of leukocyte cells to which the erythrocyte is bound.   Figure 3 shows Borrelia burgdorferi N-40 strain, 2684. Out of the leukocytes of mice inoculated with the strain or one of the 25550 strains. Indicates the ratio (%) of binding of N-40 strain of Gudoferi (Borrelia burgdorferi) It is a graph.   Figure 4 shows the Borrelia burgdorferi N-40 strain, 2684. Out of the leukocytes of mice inoculated with the strain or one of the 25550 strains. Indicates the ratio (%) of binding of 2684 strains of Gudoferi (Borrelia burgdorferi) It is a graph.   Figure 5 shows the inoculation of N-40 strain of Borrelia burgdorferi Borrelia burgdorferi ) Of 25550 strains bound (%); Borrelia burgundifer burgdorferi) was inoculated with leukocytes from mice inoculated with Borrelia burgdorferi. Rate of binding to 25550 strains of Borrelia burgdorferi (%); Leukocytes from mice inoculated with 25550 strains of Borrelia burgdorferi Of which 25550 strains of Borrelia burgdorferi bind It is a graph which shows the rate (%) which performs.   FIG. 6 shows a mouse inoculated with Borrelia burgdorferi. Of neutrophils in total leukocyte cells in the blood of human vs. mice inoculated with BSK medium It is a graph which shows the ratio (%) of the neutrophil in the total leukocyte cell in blood.   Figure 7 shows Borrelia burgdorferi among leukocytes from mice inoculated with BSK medium. Percentage (%) of N-40 strain of li (Borrelia burgdorferi) spirochetes bound; S Among leukocytes from mice inoculated with the genus Staphylococcus, The N-40 strain of Borrelia burgdorferi spirochetes binds % Of the white blood cells of mice vaccinated with OspA vaccine B-40 spirochete strain of Borgelia ferri (Borrelia burgdorferi) is bound It is a graph which shows a ratio (%).   Figures 8a and 8b show distemper, herpes, parvo and influenza. Of the leukocytes of mice inoculated with the pathogen, Borrelia burgdorferi  burgdorferi) A graph showing the percentage (%) of spirochetes to which the N-40 strain is bound. is there.   FIG. 9 shows binding to leukocytes of mice inoculated with the genus Leptospira. In relation to Leptospira and Borrelia b. urgdorferi).   Figure 10 shows that Borrelia burgdorferi in male mice inoculated with strain N-40. Percentage of cells to which the N-40 strain of Borrelia burgdorferi spirochetes is bound ( %).   Fig. 11a shows inoculation of Borrelia burgdorferi intradermally Borrelia burgdorferi sp. In isolated mice It is a graph which shows the ratio (%) of the neutrophil with which the N-40 strain of pyrochete is bound.   Fig. 11b shows inoculation of Borrelia burgdorferi intradermally 2 shows an immunoblot analysis of the isolated mice.   FIGS. 12a and 12b show Borrelia burgdorferis From the neutrophils of mice inoculated with N-40 strain of pyrochetes, blood was collected sequentially by fundus bleeding. In blooded mice (FIG. 12a) or mice killed sequentially by decapitation (FIG. 12b). 7) is a graph showing the ratio (%) of binding in ()).   Figure 13 shows natural exposure to Borrelia burgdorferi Binding in exposed or unexposed (control) human volunteer subjects It is a graph which shows the ratio (%) of the cell which has.                             Detailed description of the invention   As mentioned above, the present invention provides for the identification of pathogens or other foreign substances in humans or animals. It relates to a method for quickly and reliably detecting presence.   Neutrophils are a type of polymorphonuclear leukocyte (PMN) whose main known function is phagocytosis Use (ingestion and destruction of particulate matter) and release of chemicals related to inflammation. Through It accounts for almost 50-70% of all white blood cells (white blood cells) in normal human blood. Seropositive (Seropositive) Animal whole blood cultured in Borrelia burgdorferi (Borrelia bur) gdorferi) when mixed with neutrophils are found to solidify large numbers of spirochetes. Was done. This response has also been observed in dog, horse, cow, goat and mouse blood. I was guessed. Blood smears are available from DAPI, Hoechst (eg Hoechst 33258), acridines (eg, acridin orange (Acridi) ne Orange)) and other fluorescent DNA stains Can be dyed. Fluorescent dyes or colorigenic agents For example, fluorescein isothiocyanate (FITC), rhodamines, texa Antibodies conjugated to threads (Texas Red) are also suitable. Leukocytes for binding Because they contain cells, especially neutrophils, the method of the invention is not limited to the use of whole blood, Urine, airway secretions, CSF, exudate from skin trauma or abscess, oozing from lacrimal sac Smear of any body fluid or tissue in which leukocyte cells, such as exudates, synovial fluid, etc. are found Including book inspection.   The origin of the specificity of the binding response and the origin of the binding cells are not yet known. S The increase in the number of neutrophils binding to pyrochetes is only a fraction of the total neutrophil count or fraction. It only responds to the increase. In other words, the host's response to early Lyme disease In related syndromes, neutrophils are not an essential component. Therefore, cells It seems that from some source it can recruit recognition and / or binding factors is there. Also, an increase in the number of reactive cells is produced by the response to infection. It doesn't seem to be due to the new cells that were born.   Apply the material on a glass slide, air dry, fix, stain, and prepare using a microscope. Bell. Next, the presence and degree of bound cells are observed by an indirect method. preferable In one embodiment, the protocol for the simple hematocrit test is Change to mix reaction reagents prior to centrifugation. Cell binding exists In some cases, enzyme labels or fluorescent labels appear and quantitative evaluation is possible. Another favored In a preferred embodiment, the colorimetric reaction, which can be read in serum, or Enzymes are combined to produce a precipitate that appears in the layer of vesicles. High performance reading of this method The picking device is commercially available from Becton Dickinson. There are QBC devices that are being used. Flow cytometry, for example, fluorescence activated cell Fluorescent cell sorting using FACS (FACS) is useful for the method of the present invention. fill Readers and other schemes can also be used in the method of the present invention. For the exam The use of fluorescent or enzyme-linked labels as qualitative and quantitative markers The advantage is that the internal controls and standards can be incorporated. That is, other By adding an antibody that binds to the cellular components of the Make sure blood is stored correctly, mixing is complete, etc. .   In a preliminary experiment, Balb / C mice were given Borrelia b. urgdorferi), Freund's adjuvant or BSK culture as control The land was inoculated. Mice were bled three times a week for two weeks after inoculation. Whole blood Mix with cultured Borrelia burgdorferi spirochetes Was. Prepare a thin blood smear, stain it with DNA fluorescent dye, and And Borrelia burgdorferi spirochetes visualized did.   From the results shown in Figure 2, Borrelia burgdorferi Borrelia burgundifer (Borrelia bur) on neutrophils in the blood of inoculated mice gdorferi) is bound to mice inoculated with Freund's adjuvant. And increased compared to control mice. The binding response was 2 ~ 4 days early (measurable antibody response in mice taking about 1 week) Answer). These results indicate that the binding response is specific and very It suggests that it happens early.   Characterize the background binding observed in controls in these experiments There are both quantitative and qualitative differences. Most backgrounds Binding is less than 5%, and spirochetes bind to platelets, followed by macrophages. This is the result of the image taking the rest. By using a phase contrast microscope, The plate is clearly visible and the buffer is added by adding a small amount of spirochet to the test medium. It has been shown that background can be reduced. Background tie If any, such as physically separating platelets and associated residues from whole cells. Can be significantly reduced by the method (eg, using a QBC device).   Sensitivity using different Borrelia burgdorferi strains Whether staining results in an equivalent binding response and whether the infected host white blood cells Does it bind to different Borrelia burgdorferi strains? To check if it ’s Borrelia burgdorferi Mice were infected with one of three isolates (N-40, 2684 or 25550). I let you. The control group was inoculated with BSK medium. Mice draw blood twice a week for one month did. Aliquots of each whole blood sample were taken from Borrelia burdoferi gdorferi) was incubated with one of the three strains. A smear of blood Prepare and culture Borrelia burgdorferi spirochetes The binding characteristics of white blood cells to liposome were quantified. Using Western blot analysis The comparison of the response was also performed. Neutrophils in white blood cells of infected and control mice. The ratio (%) is shown in FIG. Borrelia burgdorferi Culture of Borrelia burgundum on white blood cells of mice inoculated with each of the three strains The percentage (%) of each strain of ferri (Borrelia burgdorferi) bound is shown in FIGS. And 5. As shown in these figures, using any of the three strains Infected mice also responded. In addition, the different Borrelia burgdorferi strains differ in binding Is shown. Binding occurs from day 3 post-infection, peaking at days 7-10, and at day 14 Decreased to preinfection levels.   A double-blind study (to rule out observers' biases) was carried out, Does not cross react with Borrelia burgdorferi It is for showing that. All administered substances should be above background. Did not cause binding to spirochetes. Therefore, secondary infection by other pathogens Or conclude that specificity is not compromised by concomitant infection You.   The method of the present invention is not limited to the detection of Borrelia infection. No. Responder cells have been shown to recognize fungi other than Borrelia You. For example, Escherichia coli (E. coli) . active binding to E. coli) was observed.   Furthermore, the cell recognition reaction was also induced by the isolated protein. In an experiment Lipoproteins cloned and expressed by E. coli The mouse OspA was administered to the mice. OspA is Borrelia Bourg-de-Ferry (Borr elia burgdorferi), the major surface protein used in Lyme disease vaccines. It has been the control of many studies as a protective antigen used, for example, International Patent Application No. WO 92/14488. Blood collected from mice Mixed with live Borrelia burgdorferi and tested The percentage (%) of bound leukocytes was calculated. The results are shown in FIG. Spirochetes Neutrophils that are able to bind to Was shown to be similar to This means that the whole bacterium is needed to elicit a response. Does not exist. Therefore, live bacteria are killed, for example, by ethanol. Culture material or glycoprotein, lipopolysaccharide or lipoprotein (Borelli A burgdorferi (such as OspA or OspC from Borrelia burgdorferi), or Is a mixture of them, isolated from bacteria of interest for in vitro analysis It can be replaced by something like that. More importantly, this response It happens to almost any form of substance. Therefore, this test Molecular properties such as ceria, viral, fungal, protozoan and certain drugs Useful for detecting the presence of a substance.   For example, the method of the present invention provides a rapid and accurate diagnosis of early stage human and animal diseases. It can be used for cutting. Such diseases include bacterial infections (eg, , Sepsis), bacterial meningitis, gonorrhea, syphilis and other sexually transmitted diseases Disease, Streptococcal infection (S. pneumoniae, S. pyogenes) Fungi (including those caused by S. pyogenes and S. mutans), Taphylococcus infections (S. aureus and S. epidermidis) (Including those caused by S. epidermidis), listeriosis and livestock in humans Rotation disease in both (both are caused by the genus Listeria and are the main pathogens) Is in the genus Listeria monocytogenes (L. monocytogenes) and Hemophilus Infection Disease (H. influenzae), H. parainfl uenzae), H. aphrophilis, H. aphrophilis H. a, egyptius, H. ducreyi and hemophil S. somnus (including those caused by H. somnus), relapsing fever, Gel, strawberry tumor and tropical vitiligo skin disease), leptospirosis, mycopla Zuma infections, including those caused by Mycoplasma pneumoniae ), Chlamydia infections (Chlamydia psittaci) and Trachoc Pathogens (including those caused by Chlamydia trachomatis).   Such diseases include yeast or fungal infections such as candidiasis, Also viral infections such as blastosis and histoplasmosis, for example papilloma Includes things such as those caused by herpes virus or papova virus You.   The methods of the present invention also include protozoan infections (eg, tricomonads and opari Infections (roundworm, tapeworm, filamentous worm, Detection of the presence and exposure of amblystomes and nematodes Useful for   The method of the present invention may be used to monitor any label or neutrophil binding of a foreign substance. And using any suitable imaging method. Preferably, the cells are Hoechst ( Hoechst) stain using a fluorescent DNA stain such as 33258 or DAPI, Observe under a microscope using an external light source. However, other methods are available For example, phase contrast microscope, Nomarski phase contrast microscope, Hoff Hoffman phase contrast microscope, dark field microscope, micro interferometer, or low scan Alternatively, there is a transmission electron microscope.   It has been found that the method of the invention can be automated. For example, the method of the present invention And an automatic digital imaging device can be used. Sample is a slide glass Prepare above and place it on a microscope-operated objective. Focus automatically After that, the image in the field of view of the microscope is captured by a video camera. The scanning process is as follows Do: the camera image is digitized by an analog-to-digital converter , And save it to computer memory. By scanning characteristic image parts , A computer program analyzes the image. If any positive elements are found If so, record the position of the microscope objective and the focal length. The computer is next Adjust the position and / or focus of the objective. By repeating the above steps A complete scan of all mounted samples is performed.   The following examples illustrate the invention, which are limited to this description. Do not mean.                                  Example Example 1   Borrelia burgdorfe against white blood cells of mice ri) Determination of the binding specificity of the spirochetes of the three strains   This experiment was performed on white blood cells of mice inoculated with one of three strains. Spirogee of three strains of Borrelia burgdorferi Performed to determine the specificity of binding of the protein. 5-7 week old female Balb / C mice Used (Jackson Laboratories, Inc., Bas., Maine) -Harbor). Four inoculation groups were established, with 2684, N-40 and 2555 strains 0 and a control group using BSK medium. Cultivation of three strains starts from frozen culture Awake. In the morning of the inoculation day, the culture is transferred to a Petroff-Hauser chamber. user Chamber). N-40 strain is 3 passages, 90% motile, The concentration is 7.1 × 10⁷Spirochetes / ml. 2684 strains are 3 passages, 90% exercise type , Concentration is 6.0 × 10⁷Spirochetes / ml. 25550 strains, 4 generations, 70% luck Dynamic type, concentration is 3.0 × 10⁷Spirochetes / ml. Borrelia Burgdorferi (Borrelia burgdorferi) cultures were grown in BSK medium at 33-34 ° C.   Before inoculation, mice are methoxyflurane (Pitman-Moore) ), Shaving the left lower abdomen and sterilizing with Betadine, followed by Sterilized with 95% ethanol. Next, use a 27 gauge tuberculin syringe to Under bacterial conditions, 0.1-0.3 ml was inoculated intraperitoneally into mice. Several hours after inoculation, anesthesia and The mice were carefully observed to see the effects of injection and inoculation.   Each group consisted of 28 mice. 0, 3, 7, 10, 14, 21, 24 and 28 days Three mice were killed in each eye. The remaining mice were killed on day 36. of mouse Sampling was performed on the day of inoculation and every 3 to 4 days after inoculation. Respectively On the day of sampling, three mice from each inoculation group were killed. Two of the three Whole blood was used for the test, and the remaining one serum was kept for Western blotting (Example 2). Existed. Mice are deeply anesthetized with methoxyflurane and cut using surgical scissors. Using a funnel, add blood to a small beaker with a drop of heparinized saline. Collected. Take a part of the blood and add 1.0ml Nunk Cryotube yotubles) (USA Scientific, Oka, Florida) La).   Cultures were counted quickly before incubation. For each of the three strains Incubation. Spirochete concentration of strain N-40 is about 2-6 × 10⁷S Pyrochetes / ml were performed for 3 passages. Spirochete concentration of 2684 strains is about 2.5-4.5 × Ten⁷Spirochetes / ml. Four passages were performed. Spirochete concentration of 25550 strains Is about 1.0-6.1 × 10⁷Spirochetes / ml and were passed for 4-5 passages. Inn For the incubation, 100 μl of blood was mixed with 200 μl of culture. Test tube Add the culture of Borrelia burgdorferi to the pan , Placed in a Coulter blood mixer and left for 30 minutes with shaking. Suitable It was checked regularly that it was mixed well. After 30 minutes, remove the test tube, Corning Single-Frosted machines from each 4-6 blood on microslide (Corning, NY) Smears were prepared, but the microslides were soaked overnight in distilled water and Rinse for at least 1 hour with tanol and wipe several times with cheesecloth. Paint M specimens were fixed in 95% ethanol for 15 minutes within 3 days. 4-6 sheets in each group One of the rides was selected and stained.   Staining was performed immediately before reading. Slides in phosphate buffered saline (PBS) For 5 to 15 minutes. When the time is up, remove the PBS to a final concentration of 5 μg / ml Hoechst's stain was added as described above. Slide 30 in the dark Let stand for minutes. When the time is up, remove the slide from the staining solution and immediately cover the slide. With clear nail enamel, rubber cement or mounting agent. Sealed.   The slides were mounted on a Zeiss Axioplot epifluorescence microscope (Axiop lot epifluorescence microscope), AH-2 epifluorescence from Olympus Microscope (epifluorescence microscope) or Olympus BX-60 Use an epifluorescence microscope, 40x, 60x, or 10x Observed at 0x. The entire smear was observed. Total number of leukocytes and neutrophils And the number of spirochetes bound to each cell (1-3 or 3 Above) were counted.   Put the data into a spreadsheet program and, of all neutrophils and all cells, Percentage of neutrophils with 3 or more spirochetes bound, total neutrophils and total Percentage of neutrophils to which one or more spirochetes are bound in cells ( %) And the percentage of leukocytes to which one or more spirochetes are bound ( %) Was calculated. Obtained from two mice in each sampling day and each treatment group The values were averaged and the standard deviation was calculated. If the standard deviation is large, two mice Read the second slide in each, and from that group a total of 4 slides Was used. If the value of one slide is far apart from other slides In that case, discard the data for that slide and average the data for the remaining three slides. did.   Since mice inoculated with BSK show a baseline of non-specific binding, The values for these mice were averaged for each series of experiments and one standard deviation was calculated. Was. In the first experiment, the standard deviation was 3 treatment groups (ie 2684 strains, N-40 And mice that received 25550 strains) Indicates that they are similar enough to make a match, One standard deviation was calculated at the sampling date. This is the N-40 strain (Figure 3) And 2684 strains (Fig. 4) were incubated. ) Was not performed.   Example 2   Western blot analysis of antibody production   Using Western blot analysis, specific Borrelia burgundy in mice Production of antibodies against ferri (Borrelia burgdorferi) protein was confirmed.   The method described by Laemmli et al. (Laemmli SE 600 Vertical Slab gel electrophoresis unit, Hoefer Sci entific) Inc., San Francisco, California) ・ Dodecyl sulphate was isolated from an isolated mite of the burgdorferi mite (Borrelia burgdorferi). Sodium acid polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Borrelia 15 out of all cells in the logarithmic growth phase of Burgrell burgdorferi 0 ml of 10,000 × g, 15% in a 0.1% solution of methiolate and 1 × PBS. Spirochetes were prepared by washing three times by centrifugation for minutes. Re-cell Suspension and the method of Bradford et al. (Bradford ), 1976).   About 300 μg Borrelia burgdorferi, freshwater rep Tospira (L. biflexa), Treponema branti (T. bryantii) and E. coli protein was converted to SDS sample buffer (187 μl) (0.25 mol / L) Tris-HCl, 40% glycerol, 2% sodium dodecyl sulfate (SDS), 2% 0% 2-mercaptoethanol, 0.25% bromophenol blue) Denatured by boiling for 5 minutes. Protein and molecular weight standards (by manufacturer (Prepared according to Bio-Rad (Richmond, CA)) Was performed using an 11% degradation gel and a 4% stacking gel. Electrophoresis was performed at a constant current of 100 mA until the staining tip reached 1 cm from the bottom of the gel. I went in the flow. The protein was prepared using a transcription buffer (25 mM Tris base, 38 mM glycine, 20 mM % Methanol, pH 8.3) for 30 minutes prior to equilibration and then towbin (Towbi n) 0.45 as described by et al. (Towbin et al., 1979). Transferred onto a μm nitrocellulose membrane.   Following transfer, the nitrocellulose membrane was stained with Ponceau's stain. ) (Sigma, St. Louis, MO) for about 10 minutes. It was confirmed whether the protein was properly transcribed. Slice this membrane into small pieces Put in tray, 2% calf serum albumin fraction IV (BSA) and 1% horse serum Block for 1 hour at room temperature with shaking using Tris-buffered saline containing Was. Strips are buffered (150 mM sodium chloride, 10 mM Tris, 0.05% Tween) (Tween) 20) and washed in (10 minutes x 3 times), and experimental calf serum (1: 100 dilution) Before Incubation was performed for 2 hours in the same manner as in the above. Remove serum and wash strips as described above. Phosphatase-labeled conjugate of goat anti-mouse long and short chain IgG Gate (Kirkegaard & Perry), Gay, MD (Saasberg) (1: 5010 dilution) was added and incubated for 1 hour. Before the strip Wash as before until protein band appears most intense (1-10 minutes) BCIP / NBT (phosphatase substrate) (Kirkegaard & Per ry), Gaithersburg, MD). Removing substrate The reaction was stopped by washing with distilled water several times.   To determine the molecular weight of the protein subunit that reacted with calf serum, The calculated curve of the relative transfer (Rf) value of the quality standard was plotted against its molecular weight. Rf Values were determined by dividing the distance traveled by the protein by the distance traveled by the tracking dye. Once not Once the Rf value of a known protein has been determined, its molecular weight can be determined from a standard curve. Was completed.   Example 3   Borrelia burgdorferi on white blood cells of mice treated with other substances Determination of binding specificity of Borrelia burgdorferi)   This experiment was performed to confirm the specificity of the test. Because there are many treatment groups, The experiment was divided into several parts. Except where noted, all Balb / C mice ( Jackson Laboratories, Bar Harbor, Maine -) Was used.   Treatment and dosage are as follows:   1. Canine Parvovirus MLV (Solvay, Lot No. 83000, Dog 1/10 of the dose in 0.1 = 0.1 ml)   2. Equine herpes virus (Rhinopneumonitis-ML) V) (0.1 ml; 1/20 of the dose in horses = 0.05 ml)   3. Canine Distemper MLV (Solvay, Lot No. 81000A, 0.1m l, 1/10 of the dose in dogs)   4. Staphyrococcus genus (cow) without coagulation-promoting enzymes (1.5 x 10)⁸0.3 ml of CFU / ml)   5. C57BL / 6J mouse (Jackson Laboratories) Borrelia burgdor to Bar Harbor, Maine feri) N-40 strain inoculation (2.07 × 10⁶Spirochetes / mouse)   6. BSK control (0.3ml)   7. Borrelia burgdorferi N-40 strain control (1.6 × 10⁷Spirochetes / mouse)   8. E. coli (1.0 × 10^FourCFU / mouse)   9. Borrelia burgdorferi OspA vaccine Note (Connaught) (5 μg / mouse in PBS)   Ten. Freshwater Leptospira (L. biflexa) serotype PATOC (1.4 × 10⁶Spirochetes / Mouse; 90% cultured)   11． T. bryantii (8 × 10⁷Spirochetes / Ma Mouse; 95% of culture killed)   12． BSK control   13. Borrelia burgdorferi N-40 strain control   14. Mouse Adaptable Influenza A / Shanghai / 16/89 (Kono (Connaught)   15． Freshwater Leptospira (L. biflexa) serotype PATOC   16． T. bryantii   Cultures were counted on the morning of the inoculation day. Syringes from treatments 1-9 receive 30-12 inoculations. Filled 0 minutes before. The syringes of treatments 10 and 11 were filled just before inoculation. Mau Were anesthetized and inoculated as described in Example 1.   Each treatment group used 15 mice. As described in Example 1, 0, 3 On days 7, 7, 10, and 14, three mice were decapitated and killed, and blood was collected.   Three strains of Borrelia burgdorferi (N-40, 26 84 and 25550). Cultures are incubated in Counted immediately prior to ovulation. Spirochete concentration of strain N-40 is 1.5-3.5 × 10⁷S Pyrochetes / ml and passage number was 4-7. 2684 strains are 1-3 × 10⁷Spy Lochate / ml and passage number was 3-9. 25550 strains are 0.75-2.5 × 10⁷S Pillow Per ml and the number of passages was 4-6. For incubation, 150 μl of blood was mixed with 150 μl of culture. As described in Example 1, A sample was prepared and the slide was fixed.   Incubation with Borrelia burgdorferi In addition to the selected mice, E. coli and staphylococcal Incubation with Staphyrococcus was also performed. These incubes All experiments were performed on the mouse blood on day 10. E. coli K-1 Two strains were obtained from the blood and incubate of mice inoculated with BSK, N-40 and E. coli. I was vate. Staphyrococcus (Staphyrococcus) Staphyrococcus) and incubated with the blood of mice inoculated. 15μ of culture l (concentration is 1 × 10⁸CFU / ml) was incubated with 150 μl of blood.   Example 4   Borrelia burgdorferi on white blood cells of mice inoculated with OspA protein (Borrelia burgdorferi) Spirochetes binding   According to the method described in Example 1, three groups of Balb / C mice were inoculated. First group Were inoculated with OspA (Connaught), and the second group was Staphylococca. And a third group (control) was inoculated with a BSK medium.   Mice were killed on days 0, 3, 7, 10, and 14 as described in Example 1. And blood was collected.   Incubation, Borrelia burgdorferi Using the N-40 strain. Prepare a smear as described in Example 1, The slide was fixed. Observe the slides under an epifluorescence microscope and determine the binding within each inoculation group. The percentage of leukocytes was calculated. FIG. 7 shows the results. The dotted line in the OspA graph This indicates that there was variation between the pulls, and the values at these points are Not standing.   Example 5   Binding of OspA-L and OspA-NL modified Dynabeads to animal blood The specificity of the combination   250 μl aliquots from several washes with PBS in 1.5 ml tubes G (For example, 10⁸By washing three sets of beads obtained from -Dynal beads (Dynal Dynabeads) were prepared. Use a large magnet to remove fines The solution and beads were suspended in 750 μl of PBS. These beads are: Conditions were provided: (1) about 5 μg protein / 10⁷(2) dark Degree is about 10⁸Cells / ml; and (3) the approximate expected working concentration for the reaction is 4 cells / cell More than a vesicle. Thus, three groups of beads were prepared.   Borrelia burgdorferi (Kirkegaard & Perry) (Kirkegaard & Perry), Cessnacourt 2, Gaithersburg, MD No. 01-97-91) from goat serum immunized with whole cells A (Borrelia) antibody is prepared and adjusted to 1 mg / ml in a 50% glycerol solution. Suspended. Add 50 μl aliquots to Dynabeads and add 2 tubes Shake gently at room temperature for 0 minutes. Add BSA to a final concentration of 0.1%, The tubes were gently shaken at 37 ° C. overnight. BSA is an antibody complex Used to get "in place" on navy's. 0.1% BSA The beads were washed with PBS containing PBS, the antibody solution was removed using a magnet, and the beads were washed with Tris-salt. The cells were suspended in an acid buffer and incubated at 37 ° C. for 24 hours. The last step is on the beads Block remaining residues and wash away proteins that are not covalently attached to the beads. It is for dropping. If beads cannot be blocked efficiently, free Nonspecific binding to other residues on the cell that can form bonds, such as amines Coupling occurs and the specificity of the beads approaches zero.   The second and third groups of beads are either OspA-L (lipidated) or OspA-NL (non-lipidated). Denaturation). OspA-NL has an initial concentration of 700 μg / ml. A total of 70 μl was added to the beads, and OspA-L was used at an initial concentration of 1210 μg / ml. Used, a total volume of 50 μl was added to the beads. 37 ° C for both solutions, without the addition of BSA Shake gently for 24 hours.   In most cases, 70 μl or 140 μl of blood was collected and processed. If no live cells are added, BSK Was added instead. Beads were added either during or after mixing. A maximum volume of 25 μl of the bead suspension was added to the blood. Pipette into the blood sample. Before the addition, the beads were vortexed. All operations are performed sequentially at 4 ° C went. Wash the sample in PBS containing 0.1% BSA for several minutes with gentle shaking. Was cleaned. Place microfuge tube on carrier and bind to beads using bar magnet The cells were separated and placed in one side of the test tube. Liquid removed with Pasteur pipette . Ensure that the sample does not contain unbound cellular material (or Washes several times.   The sample was placed in the cell bucket. It has a structure with three wells Centrifuge all contents in the microfuge tube to 22 mm^TwoOn the surface of Can be done. Experimental and control samples were prepared on the same slide. As a result, the variation in staining between slides can be controlled. Bake The rotator was rotated at a very low speed and the resulting surface was uniformly spread with cells. Here, the connected cells are visualized and the nuclei can be seen in phase contrast. it can. Slides were air dried and fixed with methanol for storage. This method It is equivalent to aqueous light stain (Wright's stain), inside the cell bucket device. Excludes petroleum sealants used in   Blood was obtained 10 days after infection (see Example 3; Borrelia burgdorferi).  burgdorferi) N-40 strain, 10⁶) Of two animals (mice as in the above example) ) From the eye by blood sampling, prepared as described above, Photographs were taken using. Cellular material detected in control (control using medium) However, beads coated with OspA-L and OspA-NL showed strong binding And platelets were very actively cross-linked to the bead surface. Bacteria and anti-bore For samples containing both Borrelia-bound beads, spirochetes and beads And were added at the same time. Therefore, the binding sequence first attaches to the beads and then to the cells. And vice versa. Beads to which antibodies are bound are infected animals other than neutrophils The cells in the cells can be detected and used to monitor the whole blood conversion cycle.   In this experiment, animals killed by decapitation 7 days after inoculation (the same as the previous examples) Was repeated using blood collected from mice. Fine while shaking gently Spirochetes were added to the cells, followed by anti-borrelia-coated beads. Neutrophils, a cell type substantially free of any control, were isolated . Other cell types were found in the preparation when the slides were evaluated using fluorescence. Was. When beads and bacteria are added to the blood at the same time, the background is better It has grown. In these experiments and in all the accompanying reactions, BSA-containing BSKs were It appears to be a complicating factor when used. Obviously, the immune response is raised Then, the cells recognizing BSA used in the blocking reaction are activated.   Example 6   Determining the binding properties of various pathogens to animal neutrophils   Ten animals (mice as in previous examples) were divided into five groups of two animals each. For each group, any of the following was administered intraperitoneally at 5 μl / mouse: Modified pneumococcal surface protein A (PspA), non-lipidated PspA, influenza B / P Nama (Panama) hemagglutinin (detergent extracted HA), tetanus toxin (respectively, Note Laboratories (provided by Connaught Laboratories) and control As PBS. Animals were killed 7 days after inoculation. White by the method described in Example 1 Blood cells were analyzed. Coating Dynabeads with each of the above antigens, It was mixed with the test blood. There were many large cells in the blood of the experimental animals, There was no or substantially little presence in the control sample. Ie Antigen-coated beads bind to white blood cells from animals that have been inoculated with the same antigen. Was. Little binding was seen in blood from control animals. Lyme disease test The presence of the same type of neutrophils was positive in control experiments (lime Blood from stained mice is incubated with Borrelia and a similar procedure is performed. Determine cells using anti-Borrelia coated beads according to protocol ). Both whole blood and whole blood with platelets removed were used. Go In some cases, specificity between these materials has been found to be comparable. Was. OspA-L coated beads (platelet-depleted blood samples from challenged mice) (See Example 5) cross-reacted with any experimental or control material. Did not respond. There was no response to the positive control. When not removing platelets Showed extensive platelet binding to blood from lime, HA and PspA material. Breaking Although tetanus toxin showed a positive response, it was not easily distinguishable from control samples. This That means proteins lack efficiency in eliciting cell binding responses , Tongue The protein is not well bound to the beads or the protein is almost degraded It is considered to indicate that Unlabeled PspA is a control animal But bound to blood from both animals but with little specificity. Was.   Example 7   Infectious agents and Borrelia burgdorferi in the lime test (ELT) determination of potential cross-reactivity between lia burgdorferi) (Bb)   Each group of mice (as in the previous example) was intraperitoneally inoculated with one of the following: Ta: Staphylococcus aureus (0.45 × 10⁸pfu / mouse), large Enterobacteriaceae (E. coli) (1 × 10^Fourpfu / mouse), Treponema (Treponema) (9.0 × 1 0⁶pfu / mouse), Leptospira (5 × 10⁶pfu / mouse), in Fluenza virus (10⁶Individual / mouse), parvovirus (10⁶Pcs / mouse), f Lupes virus (10⁶Cells / mouse), paramyxovirus (canine distemper; 1 0⁶Cells / mouse), and BSK (control). Mice (two per group) Blood was collected by decapitation on days 7, 10 and 14, and the blood was lime as described in Example 1. The test (ELT) was used. The results are shown in FIGS. 8a and 8b. Inoculate these pathogens If ELT was performed on blood from the isolated mouse, Borrelia burgdorferi No remarkable binding of li (Borrelia burgdorferi) was observed.   Example 8   Determining the applicability of ELT to other infectious agents   10 days after inoculation of Leptospira inoculated mice (inoculated in Example 7) Blood was collected by decapitation on the day and exogenous Leptospira and Borrelia Monitor blood for reactivity with Borrelia burgdorferi Was. The results are shown in FIG. 9 where the data is the combined leukocyte count / total leukocyte count (% Cb) and three or more Borrelia burgdorferi ) Is a bar graph showing the ratio of neutrophils / total leukocyte count (% Nb> 3) You. Binding occurs between leukocytes and exogenous Leptospira Between white blood cells and exogenous Borrelia burgdorferi Did not occur. These results indicate that ELT has Even It is effective.   Example 9   Evaluation of alternative forms of test reagents for cell binding   Kill by fixation in formalin or methiolate, saline Run all Borrelia burgdorferi washed in buffer Used for ELT as described in Example 1. In the ELT, living Borrelia Borrelia burgdorferi in this state when compared to Lugudoferri (Borrelia burgdorferi) Borrelia burgdorferi is found to respond equally well Was. However, Borrelia burgdorferi killed by repeated freezing and thawing (Borrelia burgdorferi) caused separation and did not react well with ELT. Therefore In the ELT binding response, live exogenous Borreli burgdorferi a burgdorferi).   Example 10   Determining the presence of dimorphism in the development of cell binding   Borrelia burgdorferi or BSK (control) Administered intracavitally (6 each), on days 0, 3, 7, 14, 21, and 28 post inoculation Two mice (one inoculated and one control) were decapitated and bled for a total of 12 mice. ELT was performed on 7 month old male mice. The results are shown in FIG. Borrelia ELT in male mice inoculated with burgdorferi (Borrelia burgdorferi) Was found to be similar to the response seen in   Example 11   Determining the effect of intradermal versus intraperitoneal inoculation on ELT response   Mice (as in the previous example) were given Borrelia burgd orferi) (10^Three), And 2 animals each on days 0, 3, 7, 14, and 21 after inoculation Blood samples (inoculated) are collected and examined for response in ELT and immunoblot Was. The results are shown in FIGS. 11a and 11b. The ELT binding response is strong and the serological response (Measured by stun blot) occurred late and was very weak. These results This shows that the ELT binding response is distinct from the measurable antibody response.   Example 12   Borrelia burgdorferi blood in ELT results Evaluation of the effect on platelet bonding   Mice inoculated with Borrelia burgdorferi can be Increased blood platelet aggregation and background Borrelia burgdor (Borrelia burgdor) feri) -inoculated mice (after inoculation, blood is continuously collected from the fundus or decapitated on the same day Experiments to compare ELT directly between Counting was also performed. The results are shown in FIGS. 12a and 12b. In decapitated mice Shows a typical ELT binding response and was suppressed in mice with fundus bleeds. The answer was seen. From rigorous experiments on ELT smears, Borrelia In the group inoculated with Borrelia burgdorferi and collecting blood from the fundus, Borrelia burgdorferi A large number of platelet clumps bound by Borrelia burgdorferi, and Slight neutrophils were observed. Borrelia burgdorfe ri) Inoculated and decapitated mice have low levels of platelet aggregation I understood. In control mice (medium inoculated), no fundus or decapitation Even in the deviation, platelet aggregation was basically not observed. Mutated leukocyte count and The reticulocyte count was similar in the test and control groups. Borrelia Bull In mice inoculated with Gudoferi (Borrelia burgdorferi) and blood collected from the fundus, A slight increase in platelet aggregation was observed. Borrel ia burgdorferi) and platelets are activated by repeated vascular damage Which were exogenously added to exogenous Borrelia burgdorferi (B orrelia burgdorferi). Borrelia burgudofe bound to platelets Li (Borrelia burgdorferi) interferes with neutrophil binding and binds to white blood cells. The combined response will be inhibited.   Example 13   Evaluation of ELT in human subjects   Various contacts against Borrelia burgdorferi ELT and Western blot were performed on human volunteers (According to Example 1). FIG. 13 shows the results. Patient history and results are as follows Is:   LH: 4 years of chronic Lyme disease, Bell's palsy, arthritis, Westa Strong positive in blots, antibiotic treatment at the time of sampling, 3 types of ELT Strong binding response in all of   PM: Previously treated Lyme disease, Western blot not clear, ELT is moderately bound in% Cb and Nb <3 during relapse of symptoms at sampling response;   DH: Lyme disease symptom lasts for about 1 month, treated with amoxicillin for 10 days, antibiotics Symptom relapse when quality was discontinued, Western blot response only 41 kDa, ELT Nb <3 Only has a moderate binding response;   TM: No history of Lyme disease, Borrelia burgdorferi (Bo rrelia burgdorferi), ELT has only minimal binding response;   KC: No history of Lyme disease, Borrelia burgdor No contact with feri), ELT shows no binding:   AL: No history of Lyme disease, Borrelia burgdor No contact with feri), ELT shows no binding:   MB: No history of Lyme disease, Borrelia burgdor No contact with feri), ELT shows no binding:   Data are for the combined ratio of combined leukocytes / total leukocytes (% Cb), Neutrophils / whole leukocytes associated with Borrelia burgdorferi Percentage of spheres (% Nb> 3) and 1 to 3 Borrelia burgundiferi urgdorferi) and the percentage of neutrophils / total leukocytes (% Nb <3) bound in a bar graph Is shown.   Although only certain preferred embodiments of the invention have been described in detail, the invention Is not limited by the above specific description, but is defined by the appended claims. And these obvious variations are included in the scope of the present invention. The scope of the claims 1. A method for detecting a foreign substance in a human or animal,   Collecting a body fluid or tissue containing white blood cells from the human or animal,   Incubating the body fluid or tissue with the foreign substance,   The liquid or tissue is removed so that the white blood cells and the foreign substance can be detected.   Label with one or more labels,   Detect foreign substance binding to leukocyte cells using appropriate methods A method comprising the steps of: 2. The foreign substance is a bacterium, virus, yeast, fungus, protozoan, parasite   Group consisting of, proteins, lipoproteins, lipopolysaccharides and glycoproteins   2. The method according to claim 1, wherein said method is selected. 3. 3. The method according to claim 2, wherein the foreign substance is a bacterium.   Method. 4. The bacteria are of the genus Borrelia, E. coli, Bordete.   Bordetella pertussis, Neisseria   , Staphylococcus genus, Leptospira genus   , Treponema, Listeria and Mycop   Claims selected from the group consisting of the genus Razuma (Mycoplasma)   4. The method according to paragraph 3. 5. The bacterium is Borrelia burgdorferi.   5. The method according to claim 4, wherein the method comprises: 6. 3. The method according to claim 2, wherein said foreign substance is a virus.   . 7. The virus is parvovirus, herpes virus, canine distemperui   Selected from the group consisting of Rus, Papova virus, and Parainfluenza virus   7. The method according to claim 6, wherein the method is selected. 8. 3. The method according to claim 2, wherein the foreign substance is a lipoprotein.   the method of. 9. The lipoprotein is of Borrelia burgdorferi   9. The method according to claim 8, wherein the method is OspA or OspC. Ten. The fluid or tissue is blood, airway secretions, urine, cerebrospinal fluid, skin lesions or   Selected from the group consisting of exudate from abscess, exudate from lacrimal sac, and synovial fluid   The method of claim 1 wherein: 11． 11. The method according to claim 10, wherein the body fluid or tissue is blood.   Law. 12． The label is a fluorescent dye, and the imaging method is fluorescence microscopy.   The method of claim 1 wherein 13. A method for diagnosing a human or animal disease,     Collecting a body fluid or tissue containing leukocytes from the human or animal,     Incubating the body fluid or tissue with the pathogen;     The body fluid or tissue is collected so that the white blood cells and the foreign substance can be detected.   Label with one or more labels,     Detect pathogen binding to leukocyte cells using appropriate methods   A method comprising the steps of: 14. The pathogen is a bacterium, virus, yeast, fungus, protozoan, parasite,   From the group consisting of proteins, lipoproteins, lipopolysaccharides and glycoproteins   14. The method according to claim 13, wherein the method is selected. 15． The method according to claim 14, wherein the pathogen is a bacterium.   . 16． The bacterium is of the genus Borrelia, Escherichia coli,   La Pertusis (Bordetella pertussis), Neisseria (Neisseria) genus,   Staphylococcus genus, Leptospira genus,   Treponema, Listeria and Mycopra   Claims selected from the group consisting of the genus Mycoplasma   16. The method according to claim 15. 17． The bacterium is Borrelia burgdorferi.   17. The method according to claim 16, wherein: 18． 15. The method according to claim 14, wherein said pathogen is a virus. 19. The virus is parvovirus, herpes virus, canine distemperui   Selected from the group consisting of Rus, Papova virus, and Parainfluenza virus   19. The method of claim 18, wherein said method is selected. 20. The method according to claim 14, wherein the pathogen is a lipoprotein.   Method. twenty one. The lipoprotein is of Borrelia burgdorferi   21. The method according to claim 20, wherein the method is OspA or OspC. twenty two. The fluid or tissue is blood, airway secretions, urine, cerebrospinal fluid, skin lesions or   It may be selected from the group consisting of exudate from abscess, exudate from lacrimal sac and synovial fluid.   14. The method according to claim 13, wherein: twenty three. 23. The method according to claim 22, wherein the body fluid or tissue is blood.   Law. twenty four. The label is a fluorescent dye, and the imaging method is fluorescence microscopy.   14. The method according to claim 13, wherein twenty five. Borrelia burgdorferi in humans or animals   ) To detect infections,     Collecting a body fluid or tissue containing leukocytes from the human or animal,     The bodily fluid or tissue is cultured in Borrelia burgdorferi   ) And incubate     The white blood cells and Borrelia burgdorferi   Labeling the body fluid or tissue with a label for detection,     Borrelia burgdorferi on white blood cells using appropriate imaging methods   ia burgdorferi) binding   A method comprising the steps of: FIG.FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 8FIG. 9FIG. 10FIG. 11FIG. 11FIG.FIG.FIG. 13

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), AU, CA, FI, J P, NO (72) Inventor Bush Mitch, Sandra Lee             United States Connecticut             06248 Hebron Slowcam Road             96

Claims (1)

[Claims] 1. In a method for detecting a foreign substance in a human or an animal,     Obtaining a liquid or tissue containing white blood cells from the human or animal   A method comprising the steps of: