Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the application is related to United States
• the present invention is generally related to Streptolysin 0 and particularly to Streptolysin 0 antibodies of the monoclonal, polyclonal and recombinant- DNA derived types.
• the present invention relates to antibodies to Streptolysin 0 derivatives and variants produced by recombinant DNA technology.
• Streptolysin 0 has an approximate molecular weight of between about 65,000 and about 70,000 daltons. SLO belongs to a class of oxygen sensitive (“thiol-activated”) , cell destroying (“cytoly ic") toxin (“cy o oxin”) which are produced by gram- osi ive bacterial species belonging to four different genera ,(streptococcus, bacillus, clostridium and listeria) .
• SLO interacts with membrane cholesterol and exerts cytolytic-cytotoxic effects on a broad range of mammalian cells. Additionally, SLO has very potent cardiotoxic properties. One d ' f the toxic and pathogenic properties associated with SLO is its hemolytic activity, i.e. SLO will lyse red blood cells, resulting in the release of hemoglobin. SLO can be lethal to laboratory animals in relatively small doses. Injection of SLO into an animal typically results in its immediate death. Most typically, SLO is associated with rheumatic fever in humans, an acute infectious disease characterized by fever, profuse perspiration, painful inflammation and swelling of the joints, and often inflammation of the lining membrane of the heart (“endocarditis”) .
• SLO is produced by specified bacterial species, when these species '-invade" a mammalian host, the SLO released by the bacteria is treated by the host as a foreign protein.
• SLO is an antigen.
• Antigens are high molecular weight compounds which upon entry into the blood stream of a vertebrate stimulate the transformation of the small lymphocytes of the B-type into lymphoblasts. The lymphoblasts secrete antibodies specific to the antigen stimulator. The antibodies are proteins possessing reactive sites specifically complementary to a reactive feature or site on the stimulating antigen.
• Antibodies generally:__have:- he property of rendering the antigen harmless to the host organism by occupying the immunologically active sites, or "epitopes", on the antigen particles or molecules.
• Anti-SLO antibodies are therefore produced by the MISSING UPON FILING
• SLO Streptococcus pyogenes
• Monoclonal antibodies have a single antibody specificity and affinity, as well as a single immunoglobulin isotype (a protein fraction containing antibody activity is referred to as an "immunoglobulin") .
• Polyclonal antibodies contain a variety of antibody molecules directed against an antigen of interest, as well as antibodies which do not react with the antigen of interest. Because of the specificity and affinity associated with monoclonal antibodies, these are generally preferred over polyclonal antibodies when such specificity is needed, e.g., in purification protocols.
• the specificity of monoclonal antibodies allows one to identify and screen for monoclonal antibodies which comprise the specific binding characteristics of interest. For example, two monoclonal antibodies can be selected to bind to the same antigen, but at different locations.' Such antibodies-can be of significant value in, e.g., sandwic -based immunoassays. -5 -
• the production of monoclonal antibodies comprises the following steps: Immunization; Hybridization; Propagation; Screening; and Cloning.
• the immune response of an animal e.g. a mammal such as a mouse, injected with an antigen which it has never encountered before will elicit a "Primary Response", i.e., the animal's immune system will generate a small amount of antibody aga 5 inst the antigen. If after a time period the animal is reinjected with the antigen, the immune system will elicit a "Secondary Response", i.e., the response is faster, stronger (more antibody is made) , and qualitatively different from the Primary Response (antibody which binds with a higher affinity to the antigen will be generated) . Generally, it is preferred that a pure version of the antigen be used in order to reduce the number of irrelevant antibodies produced.
• soluble.antigens it is preferred to immunize the animal with a mixture of the antigen and an adjuvant.
• Adjuvants provide a pool of the emulsified antigen at the injection site, and this allows the antigen to be released slowly over an extended period of time. Immunization may be by the intraperitoneal, intravenous, subcutaneous or intramuscular routes.
• Hybridization consists of the preparation of a myeloma cell line, spleen cells and the process of fusing these cell lines.
• the myeloma cell line confers "immortality" to the antibody; this cell line has the ability to multiply indefinitely and secrete immunoglobulin.
• the spleen cells are derived from the spleen of the immunized animal. * The fusio — rocess, in essence, merges the immortal myeloma cells with the spleen cells containing antibody to the antigen. The result of the fusion process is typically referred to as a "hybrid” or, "hybridoma".
• the propagation phase encourages the growth of hybridoma colonies.
• hybridomas will grow: unfused myeloma cells and unfused spleen cells cannot propagate.
• Hybridoma colonies are placed into growth-stimulating medium and incubated until sufficient colonies have propagated.
• hybridoma colonies producing antibodyagainst are identified.
• any assay for antibody against the antigen can be used for screening.
• a labelled anti- immunoglobulin can be used to detect the presence of immunoglobulin.
• Cloning ensures that the cells producing antibody comprise a monoclonal population.
• the hybridomas selected in the screening phase may comprise antibodies which lack the requisite monoclonality.
• cloning involves the setting up of sample-cell cultures, each cell intended to grow into a colony of identical cells.
• Three different approaches may be used: limiting dilution; semisolid agar culturing; and selective isolation by flow sorting.
• Limiting dilution is typically preferred. This process comprises plating out in individual culture wells a suspension of hybridoma cells at a dilution such that, statistically, the number of cells in any particular well is from about 0 to about 10 cells.
• Re-cloning i.e. repeating the foregoing
• the antibody is analyzed by, e.g., electrophoresis or rate nephelome ry. - cooperate. ' .-
• the production of polyclonal antibodies comprises the following steps: Immunization and
• Purification involves the removal of a sample of the immunized animal's body fluid and purifying it by insolubilizing .the antigen on an appropriate affinity gel and pouring the body fluid over such gel; bound antibody can then be removed by, e.g., elution or displacement chromatography. Goats, rabbits or mice can b'e used as the animal, with rabbits being preferred.
• Antibodies can also be derived by recombinant
• the gene or relevant portion of the gene of a cell immunized against an antigen can be removed and inserted into a suitable cloning vector.
• the recombinant vector can then be transfected into an appropriate host under conditions • whereby recombinant antibodies are secreted.
• wild-type SLO is meant to indicate SLO that is naturally secreted by bacterial sources such as, for example, S. pyogenes.
• Monoclonal, polyclonal and recombinant-derived antibodies can be generated using the SLO derivatives and variants disclosed in the above-referenced and co-pending applications. Such antibodies are particularly applicable in at least the following areas: the purification of both wild-type SLO derived from, e.g., bacterial sources, as well as SLO derived by recombinant DNA technology whereby/ the antibodies are used to bind
• SLO from the solutions from which the antigenic material was generated; immunodiagnostic assays, including, for example, competitive assays, two-site or "sandwich” assays, and non-competitive assays, whereby the presence of SLO is examined; identification and localization of points of SLO infection whereby labelled antibodies are introduced to a host infected by SLO to determine the point(s) of SLO interaction; and therapeutic areas whereby the antibodies are introduced via a suitable pharmaceutical carrier.to a host infected by S. pyogenes in an effort to neutralize the resulting secretion of SLO.
• immunodiagnostic assays including, for example, competitive assays, two-site or "sandwich” assays, and non-competitive assays, whereby the presence of SLO is examined
• identification and localization of points of SLO infection whereby labelled antibodies are introduced to a host infected by SLO to determine the point(s) of SLO interaction
• therapeutic areas whereby the antibodies are
• an improvement in generating such monoclonal antibodies is provided whereby prior to the immunization step, the rSLO or mSLO is complexed with high density lipoprotein, or introduced to an agent capable or reducing a cysteine amino acid residue.
• the polyclonal antibodies should have substantially no cross-reactivity to antigenic substances secreted by S. pyogenes other than SLO, and most preferably, substantially no cross reactivity to antigenic substances other than rSLO and mSLO.
• the monoclonal antibodies have an affinity for wild- ype SLO, mSLO, and rSLO of greater than about 10 *6 , preferably greater than about 10" 7 , and more preferably greater than about 10" 8 .
• soluble SLO derivatives which are hemolytically active, recognized by ASO antibodies against wild-type SLO and which are derived via recombinant DNA techniques (hereinafter referred to as "rSLO")
• soluble SLO variants which are non-hemolytically active, recognized by ASO antibodies against wild-type SLO and which are derived via recombinant DNA techniques (hereinafter "mSLO”)
• the antibodies disclosed herein are based upon rSLO and mSLO.
• rSLO indicates an SLO derivative as defined above and having a percent wild-type SLO specific activity of less than about 75%, more preferably between about 5% and 50%, and most . preferably about 9%, based upon a wild- ype SLO specific activity of 4xl0 5 hemolytic units/mg wild-type SLO.
• mSLO indicates an SLO variant as defined above and having a percent wild-type SLO specific activity of less than about 1.5%, more preferably less than about 0.5%-, and mos ⁇ _.preferably less than about 0.11%, based upon a wild-type SLO specific activity of 4xl0 5 hemolytic units/mg wild-type SLO.
• the variant, mSLO.3/6, differs from the derivative, rSLO.3, by a single amino acid.
• the foregoing indicates that purified rSL0.3 is about 3.6% less hemolytically active than wild- ype SLO and that purified mSLO.3/6 is about 1.4xl0 "3 % less hemolytically active than wild-type SLO.
• mice were administered undiluted and diluted intravenous injections of rSL0.3 and mSL0.3/6. Undiluted and diluted control suspension buffer was administered to an equivalent number of mice. To improve the intravenous injections, the mice were warmed under a heat lamp for 20-30 minutes of pre- injection. Approximately 20 mice were used for each condition.
• each mouse received an approximate dosage of 17 mg/kg, while for the diluted rSL0.3 and mSL0.3/6, each mouse received an approximate dosage of 1000 ⁇ g/kg.
• Control solution buffer did not affect the control mice.
• mice receiving either diluted or undiluted rSLO.3 or mSLO.3/6 showed any ill effects from the intravenous administrations..
• Hybridomas capable of making monoclonal antibody with a specific affinity for rSLO and mSLO " were prepared.
• the materials utilized were as follows.-
• the myeloma cells used were derived from P3X63-AG8.653 myeloma cell line, a non-secreting mouse myeloma line described by Kearny et al. J ⁇ . Immunol, 123.:1548 (1970) .
• Myeloma cells can be derived from interspecies hybrids, such as, for example SMH-D33, a mouse-human "heteromyeloma" fusion partner.
• myeloma cell lines derived from humans offer the opportunity to develop human monoclonal antibodies; such would be preferred for use in, for example, therapeutic situations.
• Human myeloma cell lines are described in Nilsson et al J. Clin. EXP. Immunol. 2:477 (1970) ("U- 266") and Matsuoka et al Pr ⁇ c. Soc. Exp..Bl ⁇ l-Med. 125:1246 (1969) ("RPMI-8226”) .
• Spleen cells used were obtained from Balb/c mice immunized according to the procedure disclosed below. The growth media was DME low glucose .
• lOxlO ⁇ M hypoxanthine and 1.6xlO" 5 M thymidine both from GIBCO, Grand Island, N.Y.
• 10 units/ml insulin Eli Lilly, Indianapolis, Ind.
• the conditioned media was 50% growth media - 50% used media and 2.5xlO' 5 M b-mercaptoethanol (Sigma).
• Polyethylene glycol (“PEG”) with a molecular weight between about 1300 and 1600 (Sigma) was used.
• Injection media was DME low glucose with 100 units/ml penicillin-streptomycin solution (both from Irvine Scientific) .
• One-half milliliter of PristaneTM solution (2,6,10,14-tetramethylpentadecane; Aldrich) was injected intraperitoneally into each Balb/c mouse two weeks prior to hybridoma injection.
• Hybridomas were constructed in accordance with the method described by Kohler and Milstein, Nature 256:495 (1975) .
• the spleen from the immunized mouse was aseptically removed after cervical dislocation and was ground in a tissue sieve until a single-cell suspension was obtained. After washing, the cells were mixed with the washed 653.1 myeloma cells in a 2:1 ratio.of spleen to myeloma cells and then pelleted. The supernatant was removed and the PEG added slowly over one minute. PBS was added to bring the total volume to 22ml and the cells were then pelleted after 8 minutes from the initiation of - 14 -
• the pellet was resuspended in 200 ml of CHAT media and 0.2ml of the suspension was added to each well of ten 96-well (microtiter plates (960 wells total) .
• the wells were supplied with fresh CHAT on day 6 or 7 post-fusion.
• Alkaline phosphate substrate was prepared as follows. lOmg p-nitrophenyl phosphate (Sigma) was dissolved for each 1 ml of diethanolamine buffer.
• Diethanolamine buffer was prepared by dissolving .203 g MgClj (Mallinckrodt) in 100 ml double distilled water, followed by the addition of ' 95.87 ml of diethanolamine (Mallinckrodt) in 750 ml double distilled water; Ph was adjusted to 9.8 with HC1 and NaOH. Final volume of one liter was achieved by the addition of double distilled water.
• Working dilution is defined as Img p-nitrophenyl phosphate per 1ml of diethanolamine buffer and was achieved by diluting the p-nitrophenyl phosphate- diethanolamine buffer in diethanolamine buffer.
• Cloning was carried out by the limiting dilution method, whereby two 96-well plates were utilized, one with 5 cells per well in conditioned media and one with 1 cell per well in conditioned media.
• EIA enzyme immunoassay
• Immunization protocols were as follows. For the mice designated SLO ASH and SLO AS12, 20 ⁇ g of rSL0.3-RA (to be described infra) in Freund's Complete Adjuvant ("FCA") (1.5 ml Aracel A (Mannide Monooleate) ; 8.5 ml Bayol F (paraffin oil); 5 mg mycobacterium butyricum, killed and dried) was injected intraperitoneally followed at six weeks by intraperitoneal injection of 20 ⁇ g of rSL0.3-RA in Freund's Incomplete Adjuvant ("FICA"); FICA differs from FCA in that it does not include therein mycobacterium butyricum..
• FCA Freund's Complete Adjuvant
• FICA Freund's Incomplete Adjuvant
• lO ⁇ g of rSL0.3-RA in FICA was injected intraperitoneally.
• Three weeks later (three days prior to fusion) 5 ⁇ g of rSL0.3-RA in PBS was injected intraperitoneally.
• lO ⁇ g of rSL0.3-HDL (to be described infra) in FCA was injected intraperitoneally, followed at six weeks by intraperitoneal injection of 20 ⁇ g of rSL0.3-HDL in FICA.
• 5 ⁇ g of rSL0.3-HDL in PBS was injected intraperitoneally.
• Two of the monoclonal antibody candidates were identified as capable of blocking the hemolytic activity of rSL0.3. These two antibodies were derived, from SLO AS16 and SLO AS21, respectively. Identification protocol was as follows. To one-half milliliter of PBS (pH 7.0) was added .08 ⁇ g of rSLO.3 and .2 ⁇ l of delipidated ascites fluid. Delipidated ascites fluid was prepared by mixing in a 1:1 concentration ratio the ascites fluid from the above indicated hybridomas and Beckman Lipid Clearing Solution (Beckman Instruments, Inc.); this mixture was vortexed for at least 1 min. After 5 min.
• a positive result a result indicating blockage of hemolytic activity by binding of the monoclonal antibody to rSL0.3, was indicated by a clear supernatant and a pellet having a red color (i.e. the pellet comprised red blood cells) ; a negative result, a result not indicating blockage of hemolytic activity, was indicated by a reddish colored supernatant (i.e. lyses had occurred) .
• the microcapsules containing antibody secreted from the SLO AS16 and SLO AS21 evidenced a clear supernatant, i.e. a positive result.
• mice were immunized with either rSL0.3-RA or rSL0.3-HDL.
• Manipulation of rSL0.3 prior to immunization was conducted based on two properties of wild-type SLO. First, secreted wild-type SLO tends to initially bind to cholesterol within the host. Second, SLO has a single Cys amino acid. These manipulations involved the formation of rSLO-High Density Lipopr ⁇ tein fractions (“rSLO-HDL”) mixtures, and introduction of rSLO to a reducing agent (“rSLO-RA”) . These manipulations are also applicable to rSLO, mSLO, and mSLO.3/6.
• rSL0.3-HDL immunization with rSL0.3-HDL would lead the immune system of the host to recognize the HDL portion of the complex as a foreign substance, thus provoking an immune response.
• the immune system of the host would be "sensitized" to the presence of rSL0.3 in the complex and thus antibodies would also be generated by the host relative to the ,epitopic sites on the rSL0.3.
• rSLO-RA Applicants theorized that the reducing agent interacts with the Cys amino acid of rSLO.3, thus leading to the separation of two rSL0.3 protein structures joined via a single disulfide bond and the formation of -SH groups on each of the two rSL0.3 structures.
• These rSL0.3 structures would then be capable of forming mixed di ⁇ sulfide bonds between a single rSL0.3 protein structure and material in the adjuvant comprising an -SH group.
• Such an rSLO.3-adjuvant complex would be similar to the rSL0.3-HDL complex described. I.e., "similar" in the sense that the adjuvant portion of such a complex would be recognized by the host's immune system as a foreign substance, thus leading to sensitization of that system to the rSLO.3 portion of the complex.
• High density lipoprotein fractions can be from any mammalian source, and most preferably, the mammalian source is not the same as the mammalian host to be immunized. Accordingly, any HDL is applicable to the present invention, with Bovine HDL Cholesterol and Human HDL Cholesterol being particularly preferred. A most preferred HDL is Pentex ® Human Cholesterol Concentrate II (Miles Laboratories, ankakee, 111. Source: human plasma, about 24% HDL/cholesterol) . The weight ratio of rSLO to HDL (rSL0:HDL) is between about 10:1 to about 1:1, preferably between about 6:1 and about 1:1, and most preferably about 2:1.
• Reducing agents which can be utilized include mercaptoethanol, sodium borohydride, sodium cyanoborohydride, sodium bisulfite, sodium thiosulfate, ascorbic acid (vitamin C) , uric acid, dithioerythreitol, and dithiothretiol.
• dithiothretiol is a particularly preferred reducing agent.
• the concentration of the reducing agent is preferably between about 2.O M about 8.OmM, more preferably between about 3.OmM and about 7.OmM, and most preferably about 5.OmM.
• rSL0.3-RA 20 ⁇ g of rSL0.3 was dissolved in 5.0 mM DTT in PBS. This mixture was heated at 37°C for 30 minutes prior to immunization.
• rSLO.3 (1 mg/ml) was mixed with of Pentex ® Human Cholesterol Concentrate II in a 6:1 volume to volume ratio (rSLO.3:Pentex ® ) prior to immunization.
• the hybridomas prepared by this method were capable of producing monoclonal antibody with a specific affinity for rSLO.
• m ⁇ noclonal antibodies also had a specific affinity for wild-type SLO.
• DIFCO SLO was obtained from DIFCO Laboratories (Detroit, '.Michigan, Code 0482) .
• the DIFCO SLO is a desiccated, standardized filtrate of Streptolysin 0, in reduced form, prepared from group A streptococcus.
• the DIFCO SLO was rehydrated in water according to supplier directions just prior to use.
• a positive result that is, a result indicating blockage of hemolytic activity by binding of the designated monoclonal antibodies to the wild-type SLO, was indicated by a clear supernatant and a red-colored pellet.
• a negative result one not evidencing blockage of hemolytic activity, was indicated by a reddish-colored supernatant.
• Microfuge tubes containing the designated monoclonal antibodies evidenced a clear supernatant, i.e. a positive result.
• the hybridomas prepared in Example 2 were capable of producing monoclonal antibody with a specific affinity for wild-type SLO.
• SLO monoclona'l antibodies can be used in the purification of wild-type SLO and in assays to determine SLO. It is to be understood that the following are not limited solely to SLO monoclonal antibodies; SLO polyclonal antibodies and recombinant DNA derived SLO antibodies are equally applicable. Most preferably, monoclonal antibodies specific to SLO derivatives o SLO variants are utilized, more preferably monoclonal antibodies specific to rSLO.3, and most preferably, the monoclonal antibodies derived from the SLO AS16 and SLO AS21 hybridomas.
• Purification of wild-type SLO can be accomplished using monoclonal antibodies specific to, e.g. SLO derivatives, by coupling such monoclonal antibodies to a suitable solid support.
• SLO monoclonal IgG either freshly prepared or previously frozen and thawed, may be utilized.
• the monoclonal antibody may be bound to any material which itself does not have a high affinity ' -for protein, materials such as glass beads, agarose and derivatives thereof are preferred. Most preferred is Bio-Rad Affi- Gel 10.TM Methods for coupling monoclonal antibodies to such materials are known. For example, lOOmg of such monoclonal antibodies in 30ml of 0.1M MOPS buffer, pH. 7.5 is prepared and ten milliliters of unwashed Bio-Rad Affi-Gel 10 is then added thereto.
• the resulting slurry is allowed to react at room temperature for 2 hours, followed by centrifugation at 1500 rpm in, for example, a Beckman TJ-6 centrifuge. The supernatant is then removed and the remaining gel-pellet is washed exhaustively with L, e.g., distilled water, and then with PBS.
• L e.g., distilled water
• Removal of the bound wild-type SLO can be accomplished by methods well known to those in the art, although elution chromatography is preferred. For example, by flushing the column with deionized water, the ionic strength of the column is•lowered such that the wild-type SLO is eluted. Similarly, use of buffer having a pH of about 6.0 or less can be used for the elution of the wild-type SLO although a buffer having a pH of greater than about 6.0 may achieve similar results.
• Elution reagents are well known and can include, for example, high pH reagents (100mm triethyla ine, pH 11.5; 100mm phosphate acid, pH 12.5) ; low pH buffers (100mm glycine, pH 2.5; 100mm glycine, pH 1.8); high salt buffers (5m LiCl, 10mm phosphate, pH 7.2;.3__.
• the resulting purified wild-type SLO can then.be re-subjected to the same purification protocol.
• immunodiagnostic assays includes nephelometric or turbidimetric assays wherein at least two SLO monoclonal antibody specific for different epitopic regions of SLO are biotinylated. These biotinylated monoclonal antibodies (or fragments thereof) are admixed with a sample suspected of containing SLO, and avidin.
• Avidin is a relatively large macromolecular protein found in egg whites, and contains four subunits.
• Biotin is a relatively small, stable, water soluble vitamin.
• Each of the four avidin subunits of an avidin molecule is capable of specifically binding to a molecule of biotin.
• the biotinylated SLO monoclonal antibody conjugates can become bound to a molecule of avidin.
• nephelometer is the Beckman ICSTM nephelometer (Beckman Instruments, Inc.).
• the biotinylated SLO monoclonal antibodies will bind to avidin. If SLO is not present in the sample or is present in a limited titer, the formation of aggregates will be limited. However, if SLO is present in the sample, then the SLO monoclonal antibodies can bind thereto, thus increasing the formation of aggregates as follows:
• MAb ⁇ 1 and MAb 2 are SLO monoclonal antibodies specific for different SLO epitopic sites, and "n" indicates that the parenthetical is repetitive. It should be noted that the aforementioned technique may also be performed without the necessity of the use of avidin and biotin.
• SLO monoclonal antibodies can be coupled to, for example, latex particles for use in latex enhanced nephelometry.
• latex particles for use in latex enhanced nephelometry.
• Such a protocol is simil ' ar_..t ⁇ -the above described nephelometric assay whereby the SLO monoclonal antibody is coupled to latex particles (instead of biotin) . These can then bind to SLO in a sample, and thus increase the number of scatter centers. For - 25 -
• latex beads having carboxyl groups on the surface thereof (17-29nm, available from IDC) can be coupled to, e.g., SLO monoclonal antibody.
• the carboxyl groups can be activated with, e.g., a soluble carbodii ide, l-ethyl-3- (3-dime hylaminopropyl) carbodiimide (“EDC”) and stabilized with, e.g., n- hydroxysuccinamide (“NHS”); ' the concentrations thereof are lOmM and 70mM respectively.
• EDC soluble carbodii ide
• NHS n- hydroxysuccinamide
• Excess untreated EDC and NHS are removed by centrifugation and the "activated" beads are resuspended in an appropriate "attachment” mixture.
• An appropriate attachment mixture can comprise 5mg/ml of SLO monoclonal antibody in PBS and 1% (v/v) TWEEN-20. After an appropriate attachment period, the SLO monoclonal antibody:latex beads are washed twice with PBS by centrifugation, followed by resuspension in PBS.
• an inhibition assay to determine levels of anti-Streptolysin 0 can also be performed.
• a fixed nephelometric signal is determined by combining a fixed amount of SLO (preferably rSLO or mSLO) and a fixed amount of an SLO monoclonal antibody. Thereafter, a serum sample suspected of containing ASO would be combined with the fixed amount of SLO such that an immuno-reaction takes place. This is followed by the addition of a fixed amount of SLO monoclonal antibody would be added to the immuno-reactive mixture.
• SLO preferably rSLO or mSLO
• An inhibition signal is obtained different from the fixed signal to the degree that ASO is in the sample; i.e., as the amount of ASO in the sample increases, the signal for the immuno-reaction will be different than the fixed signal obtained from the SLO-SL0 monoclonal—antibody signal.
• SLO monoclonal antibodies can be incorporated, or coated onto a first region of a so-called "dip-stick"; these are capable of travelling along the regions of the dipstick.
• SLO derivatives such as, for example, rSL0.3 can be permanently affixed onto a second region above that of the first region, and labelled antibodies to the SLO monoclonal antibodies can be incorporated, coated, or permanently affixed, onto a ' third region above the second region.
• the dipstick which operate by capillary action, is inserted into a sample suspected of containing SLO such that the sample is able to travel from the first region, through the second region, and then the third region.
• SLO SLO monoclonal antibodies
• capillary action will carry both bound and unbound SLO monoclonal antibodies into the second region.
• Those that are unbound, however, will become bound to the immobilized SLO in the second region; thus, only SLO-SLO monoclonal antibody complexes will be capable of travelling to the third region.
• the labelled anti-SLO monoclonal antibodies will bind to those complexes such that, depending upon the label (e.g., radioactive, chemiluminescent, bioluminescent, enzymatic,etc.) , the labelled complexes can be either read directly or subjected to further chemical analysis prior to reading the label.
• Suitable dipsticks which can be utilized in conjunction with such monoclonal antibodies are well-known and will not be discussed herein in detail. See, for example, U.S. Patent No. 5,013,669.
• SLO wild-type, SLO derivatives such as , for example, rSL0.3, or SLO variants, such as,. " -for example, mSLO.3/6) is labelled with, for example, radioactive label, chemiluminescent label, bioluminescent label, fluorophore, enzyme, biotin, etc. using known labelling techniques.
• the SLO derivative is rSLO.3.
• a known amount of the labelled SLO derivative is admixed with insolubilized SLO monoclonal antibody and a sample suspected of containing SLO. Under .these parameters, the labelled SLO and sample SLO will compete with binding to the insolubilized SLO monoclonal antibody such that the amount of. insolubilized label is inversely proportional to the amount of SLO in the sample.
• Group A comprises insolubilized SLO polyclonal antibody, SLO antibody produced by recombinant DNA techniques, or SLO monoclonal antibody specific for a first SLO epitopic site
• Group B comprising a known quantity of labelled SLO polyclonal antibody, SLO monoclonal antibody specific for a different SLO epitopic site other than the first site, or SLO antibody produced by recombinant DNA techniques
• Group C comprises sample suspected of containing SLO.
• Insolubilized ternary complexes comprising insolubilized SLO antibody, SLO from the sample, and labelled SLO antibody are then formed such that the amount of insolubilized label is directly proportional to the amount of SLO in the sample.
• rSLO.3 In order to prepare antibodies to rSLO, 25 ⁇ g of rSLO.3 was administered intramuscularly to each of two legs of a rabbit. This was 'accomplished by mixing . equal volumes of rSLO.3, at an initial concentration of 0.2 g/dl in phosphate-buffered sailine ("PBS"), with FCA. This inoculum was then emulsified until a frothy- exture was achieved.
• PBS phosphate-buffered sailine
• One-half milliliter of the inoculum was administered as described.
• One-fifth milliliter of the inoculum was also administered intradermally to each of ten dorsal sites of the rabbit.
• the identical protocol was followed with one exception.
• the rSL0.3 was mixed with an equal volume of 50% FCA and 50% FICA.
• Booster injections of 25 ⁇ g of rSLO.3 were given monthly for several months.
• the booster injection protocol is identical to that described above, with two exceptions.
• rSLO.3 is combined in equal volumes of FICA, and one-fifth milliliter of the inoculum was administered intradermally to fifteen dorsal sites of the rabbit.
• Polyclonal antibodies to mSLO were also prepared, following the protocol described in Example 4 with the following exceptions: a) lOO ⁇ g of mSLO.3/6 was administered intramuscularly to each of two legs of two rabbits; and b) the initial concentration of mSLO.3/6 was 1 EXAMPLE 6
• the rabbit anti-rSLO 3 polyclonal antibody prepared by the method of Example 4 is' purified according to procedures well know in the art.
• a particularly preferred purification protocol is affinity purification.
• SLO wild- ype, an SLO derivative such as, for example, rSLO or an SLO variant, such as, for example, mSLO bound to affinity gel
• SLO is prepared by first solubilizing 100 mg of SLO in 30 ml of 0.1 M MOPS buffer, pH 7.5. Ten milliliters of unwashed Bio-Rad Affi-Gel 10TM affinity gel is then added to the SLO solution. The resulting slurry is allowed to react at room temperature for 2 hours. The reacted slurry is centrifuged at 1500 rpm; a preferred centrifuge is a Beckman TJ-6TM centrifuge. The supernatant is removed and the remaining gel-pellet is washed exhaustively with distilled water and then with PBS.
• the foregoing method provides purified polyclonal antibodies to rSLO.
• the polyclonal antibodies to rSLO should have substantially no cross reactivity with S. pyogenes antigens other than SLO, and most preferably, substantially no cross reactivity with antigens other-..than rSLO.
• the purification protocol of Example 6 is also applicable to pooled rabbit anti-mSLO.3/6 antisera obtained from the method of Example 5, such that purified polyclonal antibodies to mSLO are provided.
• the polyclonal antibodies to mSLO should have substantially no cross reactivity with S. pyogenes antigens other than SLO, and most preferably, substantially no cross reactivity with antigens other than mSLO.
• SLO antibodies can be generated using recombinant DNA techniques. Under this approach, wild- type SLO, SLO derivatives or SLO variants (as defined) can be utilized to obtain SLO antibodies .using standard immunization protocols such as those described above. Thereafter, the B-cell producing organ (e.g., spleen, PBL or lymph modes) is removed from the source of immunization. The lymphocytes are then isolated, followed by isolation of RNA. cDNA is then synthesized from the isolated mRNA such that cDNA libraries are obtained. Methodologies for cDNA cloning, such as those set forth in Sambrook, J. et al Molecular Clohincr: A Laboratory Manual, 2d Edition. Cold Spring Harbor
• an antibody expression library can be generated, using, for example, ImmunoZapTM cloning and expression systems (available from Stratacyte, La Jolla, CA. , USA). See also, Sastry, L.
• Antibodies including SLO antibody, have an approximate molecular weight of between about 100,000 to 130,000 kD. With an average molecular weight for each amino acid of about 110 kD, a 100,000 kD protein would be encoded by approximately 909 codons, which would be encoded by messages about 2727 bases long; a 130,000 kD protein would be encoded by approximately 1182 codons, which would be encoded by messages about 3545 bases long.
• purification of cDNA made from the B-cell producing organ, expressing SLO antibody and which are larger than about 3545.base pairs is preferred; purified fragments greater .than.about 2727 base pairs can be also be utilized.
• the isolated cDNA fragments are purified using standard techniques and ligated into an appropriate expression vector (such as, for example, ⁇ gtll) which has corresponding ends capable of annealing to such fragments.
• an appropriate expression vector such as, for example, ⁇ gtll
• the vector is then transfected into an appropriate cell, such as for example, ⁇ . coli, under conditions suitable for growth of an SLO antibody genomic library.
• the antibody proteins can be "lifted” from plaques using protein binding filters (e.g. PVDF membranes, Millipore) .
• the filter is then incubated with labelled SLO, or with SLO followed by washing and incubation with labelled anti-SLO antibody.
• Identification of the plaques expressing the SLO antibodies having affinity for SLO is possible. This method of screening is preferred in that thousands of plaques can be screened per membrane, such that millions of clones can be screened in a relatively short time ⁇ period.
• SLO antibodies as described can be labelled with enzymes, biotin and fluorochromes.
• Such labelled SLO monoclonal antibody can be used for, e.g., diagnostic purposes (such as those disclosed above) , screening __ . purposes, or therapeutic purposes.
• SLO monoclonal antibodies are preferably used, and most preferably, the IgG isotopes thereof. .-Prior to such labelling, the SLO antibodies are preferably purified. Purification can be by affinity chromatography with either protein A or SLO as the bonded ligand. Purification by high-performance ion-exchange chromatography can also be utilized. Crane, L.F. "Purification of Monoclonal Antibodies by High- Performance Ion-Exchange Chr ⁇ matography. " Chpt. 9, Monoclonal Antibody Production Techniques and Applications, L. B. Schook, Ed. Marcel Dekker, Inc. N.Y. , N.Y. (1987) (hereinafter "Monoclonal Antibody Production Techniques. ”) The foregoing' chapter is incorporated herein by reference. Purified IgG isotopes are digested with pepsin to yield F(ab') 2 , followed by reduction to yield Fab' .
• the Fab' fragments may then be labelled in accordance with the procedures outlined in e.g., Ishikawa, ⁇ . et al "Modification of Monoclonal Antibodies with Enzymes, Biotin, and fluorochromes and their
• particles functioning as reservoir or monolithic devices for important pharmacologic agents are typically biodegradable and nontoxic, bioadsorptive, retained by tissues, and exhibit sustained or controlled- release of pharmacologic agent (s).
• a monolithic device is one in which the agent (s) is dispersed in the particle matrix, while a reservoir device is one in which the agent(s) is encapsulated by the particle.
• Natural polymers used in the preparation of particles include polysaccharides, e.g., starch and cellulose-derivatives, and proteins, e.g., albumin, collagen and gelatin.
• Synthetic biodegradable polymers include polylactide, polyamino acids and copolymers of lactide-co-glycolide, lactide-co-E-caprolactore, N- (2-hydroxy-propyl) - methacrylamide, polyortho esters and polyanhydrides. Liposomes and lipoproteins are also available for delivery vehicles. Shaw, J. M. et al. "Drug Delivery Particles and Monoclonal Antibodies," Chpt. 15, Monoclonal Antibody Production Techniques'. The foregoing chapter is incorporated herein by reference.
• the particles can be coupled directly to the SLO antibody or to F(ab') 2 and : -Fab' fragments thereof.
• Such coupling methodologies are described in Shaw, J. M. , supra although after such coupling procedures will be readily apparent to the skilled artisan. Purification following such coupling is recommended in order to remove uncoupled SLO antibodies or F(ab') 2 and Fab' fragments.
• pharmaceutical agents can be entrapped in the particles.
• radioactive isotopes which are typically used for imaging ' processes in patients can be entrapped in the particles.
• the specific pharmaceutical agent(s) utilized depend principally on the target of the monoclonal antibody.
• SLO cardiotoxic effects thereof are well known, as well as the association with, e.g. rheumatic fever.
• one application of SLO monoclonal antibody coupled to particles comprising radioactive isotopes is the identification of specific compartmental and regional areas where SLO specifically interacts. This would then allow for the delivery of specific agents to such areas.
• pharmaceutical agents could be appropriately and readily.-. ' selected to be delivered to such tissue.
• the relationship between SLO and rheumatic fever is well documented, the specific interaction is not well understood.
• the Examples herein are not to be construed as limited to specific hybridoma cell lines which are preferred.
• the methodologies described for generating hybridoma cell lines capable of producing monoclonal antibody with an affinity to rSLO, mSLO and/or wild-type SLO are not to be construed as limited solely to preferred hybridoma cell lines.
• the preferred hybridoma cell lines disclosed above in no way constitute an admission, either actual or implied, that these are the only hybridoma cell lines to which Applicants are entitled. They are entitled to the full breadth of protection under applicable patent laws.
• the monoclonal antibodies generated from such cell lines are similarly not limited.
• hybridoma cell lines have been identified by Applicants as SLO ASH, SLO AS12, SLO AS16 and SLO AS21.
• the hybridoma cell lines were deposited on July 16, 1991 with ' the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852, under the provisions of the Budapest Treaty for the International-Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.
• the hybridoma cultures were tested by the ATCC on July 19, 1991, and determined to all be viable cultures.
• the ATCC has assigned the four hybridoma cell lines ATCC deposit numbers HB 10826, HB 10827, HB 10828 and HB 10829, respectively.

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