. US005403718A United States Patent [191 [11] Patent Number: 5,403,718 Durward et al. [451 Date of Patent: * Apr. 4, 1995

[54] METHODS AND ANTIBODIES FOR THE I OTHER PUBLICATIONS IMMUNE CAPTURE AND DETECHON 0F Nowinski et al, Science, 219:637-644 (11 Feb. 1983), BQRRELI-A BURGDORFERI - “Monoclonal Antibodies for Diagnosis of Infectious [76] Inventors: David W. Dorward, 401 N. 7th St; Diseases in Humans”. Tom G. Schwan, 601 S. 5th St.; . E . _ 1 E . Claude F. Garon, 904 Ponderosa Dr., Primary xammer Caro ' Bldwen all of Hamilton, Mont. 59840 [57] ABSTRACT [ * ] Notice; The portion of the term of this patent The invention relates to novel antigens associated with subsequent to Jun, 8, 2010 has been Borrelia burgdorferi which are exported (or shed) in disclaimed. vivo and whose detection is a means of diagnosing _ Lyme disease. The antigens are extracellular membrane [211 App 1' No" 929’172 vesicles and other bioproducts including the major ex [22] Filed: Aug. 11,1992 tracellular protein. The invention further provides anti bodies, monoclonal and/or polyclonal, labeled and/or Related US. Application Data unlabeled, that react with the antigens. The invention [63] Continuation-impart ofSer. No. 485 551 Feb. 27 1990 . relates to a math“ for immune capture of Speci?c mi‘ Pat No_ 5,217,871 ’ ’ ’ ’ , croorganisms for their subsequent cultivation. The in vention is also directed to a method of diagnosing Lyme clci? ------GolN 332940 disease by detecting the antigens in a biological sample ...... , . , taken from a host using the antibodies in conventional 530/3871; 530/388.4; 530/3895 immunoassay formats. The invention further relates to [58] Field of Search ...... 435/732, 7.33, 7.34, kits, for the diagnosis of Lyme disease, comprising the 435/735, 7.36, 7.37, 243, 261, 962, 7.94; antibodies and ancillary reagents. The advantage of the 530/388.4, 389.5, 39l.l antibodies used in the invention is that they react with [5 6] References Cited the antigens from geographically diverse strains of Bor- ‘ Us PATENT DOCUMENTS relia burgdorferi, but do not react with antigens from related Borrelia spirochetes. 5,008,186 4/ 1991 Grayston et al...... 435/736 FOREIGN PATENT DOCUMENTS 8901162 2/1989' WIPO ...... 435/755 2 Claims, 8 Drawing Sheets US. Patent Apr. 4, 1995 Sheet 1 of 8 5,403,718

I Ant? Ami- Anti 8. biaCk vesicie MEP OspA

wcvesicle Vesicle Ves'icie Vesicle Studs 1 W0 \ WE? ) WC \i

—OspaA US. Patent Apr. 4, 1995 Sheet 2 of 8 5,403,718

FIG. 2a US. Patent Apr. 4, 1995 Sheet 3 of 8 5,403,718

FIG. 31; FIG. 30 >

xi. US. Patent Apr. 4, 1995 Sheet 4 of 8 5,403,718

FIG. 4a US. Patent Apr. 4, 1995 Sheet 5 of 8 5,403,718

Human Seyum ARtFM E?

1 Human Mouse Urine Urine /~""—’¥-——\ /—’% Ur/zinfected Vesicle lnfeqted inieg?ed E 97 83 66

45

31 -OspA

22

14 US. Patent Apr. 4, 1995 Sheet 6 of 8 5,403,718

FIG. 6

W Anti-vesicle F(ab')2

Activated Surface

C: AntigenBorrelia Source

Antigen Capture

B. burgdorferi specific polyclonal lgG

immune Compiex

GD conjugateDetection

Visualization US. Patent Apr. 4, 1995 Sheet 7 of 8 5,403,718

FIG, 7

micropipet

micropipet lip

capillary pipet sleeve

Caraway capillary pipe:

5,403,718 1 2 against Borrelia burgdorferi in blood donors and in two METHODS AND ANTIBODIES FOR THE different groups of patients, p. 497-499. In J. L. Benach, IMMUNE CAPTURE AND DETECTION OF and E. M. Bosler (eds), Lyme disease and related disor BORRELIA BURGDORFERI ders. Annals of the New York Academy of Sciences 539; Stiernstedt, G., R. Gustafusson, M. Kaarlsson, B. This is a continuation-in-part of copending applica Svenungsson, and B. Skoldenberg. 1988. Clinical mani tion U.S. Ser. No. 07/485,551, ?led Feb. 27, 1990,‘ now festations and diagnosis of neuroborreliosis, p. 46-55. In US. Pat. No. 5,217,872. J. L. Benach, and E. M. Bosler (eds), Lyme disease and related disorders. Annals of the New York Academy of FIELD OF THE INVENTION 10 Sciences 539 and Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. The present invention relates to novel antigens asso 1988. Antigenic variability of Borrelia burgdorferi. p. ciated with Borrelia burgdorferi, antibodies that are 126-143. In. J. L. Benach, and E. M. Bosler (eds), raised against the antigens and the use of the antibodies Lyme disease and related disorders. Annals of the New to diagnose Lyme disease and for immune capture and York Academy of Sciences 539). Most mammalian cultivation of microorganisms. hosts mount an antibody response to the spirochete, however the antibodies are often serologically cross BACKGROUND OF THE INVENTION reactive with other species of Borrelia, and individuals with sero-negative infections have been encountered Since the demonstration of Borrelia burgdorferi as the using standard screening criteria (Dlesk, A., D. F. Bjar infectious agent of Lyme borreliosis (Burdorfer, W. et nason, P. Mitchell, and P. McCarty. 1988. Lyme disease at. 1982. Science 216:1317-1319), numerous studies presenting as seronegative rheumatoid arthritis, p. have documented the difficulty of culturing the spiro 454-455. In J. L. Benach, and E. M. Bos1er(eds.), Lyme chetes from or observing spirochetes in infected mam disease and related disorders. Annals of the New York malian hosts (Steere, A. C. 1989. New Engl. J. Med. Academy of Sciences 539; Fox, J. L. 1989. Interest in 321: 586-596; Szczepanski, A. et a1. 1991. Microbiol. Lyme disease grows. ASM News 55:65-66; Hyde, F. Rev. 55: 21-34). Factors such as the reportedly sparse W., R. C. Johnson, T. J. White, and C. E. Shelburn. distribution of B. burgdorfen' in hosts, the fastidious 1989. Detection of antigen in urine of mice and humans growth requirements, and the relatively slow growth infected with Borrelia burgdorferi, etiologic agent of rate of this spirochete compound the problems associ Lyme disease. J. Clin. Microbiol. 27:58-61; Magnarelli, ated with aseptic primary isolations. L. A. 1988. Serologic diagnosis of Lyme disease, p. The immunological interactions between the Lyme 154-161. In J. L. Benach, and E. M. Bosler (eds), Lyme disease spirochete, Borrelia burgdorferi, and its mamma disease and related disorders. Annals of the New York lian hosts are poorly understood (Bosler, E. M., D. P. Academy of Sciences 539; Sticht-Groh, V. R. Martin, Cohen, T. L. Schulze, C. Olsen, W. Bernard, and B. 35 and I. Schmidt-Wolf. 1988. Antibody titer determina Lissman. 1988. Host responses to Borrelia burgdorferi in tions againstBorreIia burgdorjferi in blood donors and in dogs and horses, p. 221-234. In J. L. Benach, and E. M. two different groups of patients, p. 497-499. In J. L. Bosler (eds), Lyme disease and related disorders. An Benach, and E. M. Bosler (eds), Lyme disease and nals of the New York Academy of Sciences 539; Dlesk, related disorders. Annals of the New York Academy of A., D. F. Bjarnason, P. Mitchell, and P. McCarty. 1988. 40 Sciences 539 and Wilske, B., V. Preac-Mursic, G. Lyme disease presenting as seronegative rheumatoid ' Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. arthritis, p. 454-455. In J. L. Benach, and E. M. Bosler 1988. Antigenic variability of Borrelia burgdorferi. p. (eds), Lyme disease and related disorders. Annals of 126-143. In. J. L. Benach, and E. M. Bosler (eds), the New York Academy of Sciences 539; Duray, P. H. Lyme disease and related disorders. Annals of the New and A. C. Steere. 1988. Clinical pathologic correlations 45 York Academy of Sciences 539:126-143). Furthermore, of Lyme disease by stage, p. 65-79. In J. L. Benach, and strain variation among B. burgdorferi isolates, and anti E. M. Bosler (eds), Lyme disease and related disorders. genic variation within populations render immunodiag Annals of the New York Academy of Sciences 539; nostics, based on monoclonal antibodies, potentially Fox, J. L. 1989. Interest in Lyme disease grows. ASM' insensitive and unreliable for detection of circulating News 55:65-66; Hyde, F. W., R. C. Johnson, T. J. 50 and excreted antigens in some hosts (Barbour, A. G., R. White, and C. E. Shelburn. 1989. Detection of antigen H. Heiland, and T. R. Howe. 1985. Heterogeneity of in urine of mice and humans infected with Borrelia burg major proteins in Lyme disease borreliae: a molecular dorferi, etiologic agent of Lyme disease. J. Clin. Mi analysis of North American and European isolates. J. crobiol. 27:58-61; Magnarelli, L. A. 1988. Serologic Infect. Dis. 152:478-484 and Wilske, B., V. Preac-Mur diagnosis of Lyme disease, p. 154-161. In J. L. Benach, 55 sic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. and E. M. Bosler (eds), Lyme disease and related disor Kramer. 1988. Antigenic variability of Borrelia burgdor ders. Annals of the New York Academy of Sciences feri. p. 126-143. In. J. L. Benach, and E. M. Bosler 539; Schwan, T. G., W. Burgdorfer, and C. F. Garon. _(eds.), Lyme disease and related disorders. Annals of 1988. Changes in infectivity and plasmid pro?le of the the New York Academy of Sciences 539:126-143). Lyme disease spirochete, Borrelia burgdarferi, as a result 60 Therefore, clinical symptoms, patient history and occa of in vitro cultivation. Infect. Immun. 56:1831-1836; sional primary isolations of the spirochete from blood Schwan, T. G., W. Burgdorfer, M. E. Schrumpf, and R. or tissue biopsies, provide the bases for most diagnoses H. Karstens. 1988. The urinary bladder, a consistent (Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Cole source of Borrelia burgdorferi in experimentally infected man, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. white-footed mice (Pemmyscus leucopus). J. Clin. Mi 65 Ziegler, A. G. Barbour, W. Burgdorfer, R. Edelman, crobiol. 26:893-895; Sticht-Groh, V., R. Martin, and I. and R. A. Kaslow. 1983. Spirochetes isolated from the Schmidt-Wolf. 1988. Antibody titer determinations blood of two patients with Lyme disease. N. Engl. J. 5,403,718 3 4 Med. 308:740-742; Dlesk, A., D. F. Bjarnason, P. burgdorferi- Lyme disease spirochete: silver staining Mitchell, and P. McCarty. 1988. Lyme disease present for nucleic acids. Scanning Microscopy Supplement 3, ing as seronegative rheumatoid arthritis, p. 454-455. In pages 109-115; Dorward, D. W., T. G. Schwan, and C. J. L. Benach, and E. M. Bosler (eds.), Lyme disease and F. Garon, 1991. J. Clin. Microbiol. 29:1162-1171). Indi related disorders. Annals of the New York Academy of rect evidence suggests these vesicles may be produced Sciences 539:454-455; Duray, P. H., and A. C. Steere. by spirochetes in vivo providing sustained antigenic 1988. Clinical pathologic correlations of Lyme disease challenge to hosts maintaining a limited population of by stage, p. 65-79. In J. L. Benach, and E. M. Bosler spirochetes (Fox, J. L. 1989. Interest in Lyme disease (eds, Lyme disease and related disorders. Annals of the grows. ASM News 55 :65-66). To determine whether B. New York Academy of Sciences 539:65-79 and Rawl burgdorferi vesicles occur in experimentally-infected ings, J. A., P. V. Fornier, and G. J. Teltow. 1987. Isola mice, polyclonal rabbit sera were generated against tion of Borrelia spirochetes from patients in Texas. J. vesicles and an 83 kilodalton (kDa), major extracellular Clin. Microbiol. 52:1148-1150.20). Such problems are protein (MEP). Using these reagents, a 2-stage immune often cited as factors in?uencing the reportedly poor electron-microscopic assay was developed for ?rst cap diagnostic acumen for Lyme disease (Fox, J. L. 1989. 15 turing then identifying extracellular B. burgdorferi infec Interest in Lyme disease grows. ASM News 55:65-66). tions. Considerable work is currently directed toward iden The closest known technology to the present inven tifying conserved, species-speci?c cell surface antigens tion was a diagnostic kit, initially produced by 3M for diagnostic use, and for epidemiological and pathoge Corp. (Fast Lyme, Cat. No. 700-500) that used mono netic studies. Expression of outer surface protein A 20 clonal antibodies to detect Lyme antigen in human (OspA) is considered universal among B. bulgdorfkri urine samples. The kit was limited to urine samples, isolates, but not among related spirochetes (Barbour, A. provided false-negative results with geographically G., R. H. Heiland, and T. R. Rowe. 1985. Heterogene diverse samples, and was marginally sensitive (Hyde F. ity of major proteins in Lyme disease borreliae: a molec W. et al, (1989), Detection of antigen in urine of mice ular analysis of North American and European isolates. 25 and humans infected with Borrelia burgdor?zn', etiologic J. Infect. Dis. 152:478-484; Barbour, A. G., S. L. Tes sier, and W. J. Todd. 1983. Lyme disease spirochetes agent of Lyme disease, J. Clin. Microbiol. 27:58-61). and ixodid tick spirochetes share a common surface Moreover, the kit technology, owned by BioWhittaker, is neither FDA approved for human testing nor cur antigenic determinant de?ned by a monoclonal anti rently marketed. body. Infect. Immun. 41:795-804; Bergstrom, S., V. G. 30 Bundoc, and A. G. Barbour. 1989. Molecular analysis of SUMMARY OF THE INVENTION linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia The present invention relates to a method for isola burgdorferi. Mol. Microbiol. 3:479-486; Hyde, F. W., R. tion and concentration of speci?c microorganisms from - C. Johnson, T. J. White, and C. E. Shelburn. 1989. 35 a biological source. The method allows for the speci?c Detection of antigen in urine of mice and humans in immune capture and subsequent cultivation of the iso fected with Borrelia burgdorferi, etiologic agent of lated microorganism. Lyme disease. J. Clin. Microbiol. 27:58-61; Magnarelli, The invention relates to novel antigens associated L. A. 1988. Serologic diagnosis of Lyme disease, p. with Borrelia burga’or?zri which are exported (or shed) 154-161. In J. L. Benach, and E. M. Bosler (eds.), Lyme 40 in vivo and whose detection is a means of diagnosing disease and related disorders. Annals of the New York Lyme disease. The antigens are extracellular membrane Academy of Sciences 539:154-161 and Wilsek, B., V. vesicles and other bioproducts including the major ex Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, tracellular protein. Another object of the invention is to and M. Kramer. 1988. Antigenic variability of Borrelia provide antibodies, monoclonal and/or polyclonal, la burgdorferi, etiologic agent of Lyme disease. J. Clin. 45 beled and/or unlabeled, that are raised against the anti Microbiol. 27:58-61 and Wilske, B., V. Preac-Mursic, gens. A further object of the invention is to provide a G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kra method of diagnosing Lyme disease by detecting the mer. 1988. Antigenic variability of Borrelia burgdorferi antigens in a biological sample taken from a host using p. 126-143. In J. L. Benach, and E. M. Bosler (eds.), the antibodies in conventional immunoassay formats. Lyme disease and related disorders. Annals of the New 50 Another object of the invention is to provide kits, for York Academy of Sciences 539:126-143). This protein the diagnosis of Lyme disease, comprising the antibod is immunogenic, however, surface-exposed regions ap ies and ancillary reagents. The advantage of the anti pear to be antigenically variable, since surface-reactive bodies used in the invention is that they react with the monoclonal antibodies to OspA fail to recognize some antigens form geographically diverse strains of Borrelia isolates (Barbour, A. G., R. H. Heiland, and T. R. 55 burgdarferi, but do not react with antigens from related Howe. 1985. Heterogeneity of major proteins in Lyme Borrelia spirochetes. disease borreliae: a molecular analysis of North Ameri BRIEF DESCRIPTION OF THE DRAWINGS can and European isolates. J. Infect. Dis. 152:478-484; Hyde, F. W., R. C. Johnson, T. J. White, and C. E. FIG. 1. Immunoblot analysis of antibodies used for Shelburn. 1989. Detection of antigen in urine of mice 60 the capture and detection of B. burgdorferi antigens. and humans infected with Borrelia burgdorferi. p. FIG. 2. 11-0 Localization of epitopes recognized by 126-143. In. J. L. Benach, and E. M. Bosler (eds.), IgG directed against the major extracellular protein Lyme disease and related disorders. Annals of the New (MEP). York Academy of Sciences 539:126-143). FIG. 3. a-f Immune electron microscopic detection Recent experiments have shown that OspA and sev 65 of B. burgdorferi antigens in mammalian urine and eral other proteins are exported from B. burgdorferi cells blood. in membrane vesicles (Garon, C. F., D. W. Dorward, FIG. 4. a-h Detection of B. burgdorferi antigens in and M. D. Corwin. 1989. Structural features of Borrelia macerated ixodid ticks and mouse tissues. 5,403,718 5 6 FIG. 5. Immunoblot analysis of B. burgdorferi anti coccus pyogenes, Bordetella pertussis, Haemophilus in?u gens, precipitated from infected mouse and human enzae, Pseudomonas aeruginosa, Mycobacterium tuber urine. culosum, Mycobacterium avium, Mycoplasma pneumonia, FIG. 6. Immune capture and detection of B. burgdor Legionella pneumophila, Listeria monocytogenes, Neisse feri antigens. ria meningitidis, Chlamydia psittaci, and the like. FIG. 7. Shows a micropipettor equipped with a 1-200 Sexually-transmitted pathogens, or potential patho ul polypropylene tip, which has been clipped off to gens that are isolated and cultivated from samples taken allow insertion of a capillary pipet sleeve. from the oral, rectal, genital, ocular regions of a host FIG. 8. A schematic diagram of the immune capture and secretions from these regions using the immune and cultivation procedure. capture and cultivation method of the present inven DETAILED DESCRIPTION OF THE tion, include, but are not limited to Neisseria gonor INVENTION rhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis and the like. The present invention is useful in capturing microor Vector-borne pathogens or potential pathogens are ganisms from a source material onto surfaces coated 15 isolated and cultivated from infected host tissues or host with antibodies directed against certain surface compo ?uids and from infected tissues or ?uids from vectors nents of a microorganism. The microorganisms that are such as ticks, mites, lice using the present invention of isolated by the present invention include bacteria, fungi immune capture and cultivation. Examples of such vec and parasites. The microorganisms include nonpatho tor-borne pathogens or potential pathogens include but genie, pathogenic and potentially pathogenic microor are not limited to Yersinia pestis, Rickettsia rickettsii, ganisms. The captured microorganism is placed into Rickettsia akari, Rickettsia prowazekii, Rickettsia tsutsu sterile medium for aseptic cultivation. The present in gamushi, Coxiella burnetti, Borrella burgdorferi, Borrelia vention of immune capture and cultivation is useful in hermsii, Borrelia duttoni, and the like. isolating a speci?c microorganism that is difficult to Disseminated pathogens or potential pathogens that obtain as a pure culture from a source containing a 25 mixture of different microorganisms especially if the are isolated from infected host tissues or host ?uids organism of interest is in low numbers as compared to using immune capture and cultivation include, but are the other microorganisms in the mixture. not limited to, Leptospira interrogans, Bacillus anthracis, The sources include but are not limited to environ Francisella tulerensis and the like. mental sources such as soil, water, air. The present Protozoan and/or parasite pathogens or potential invention allows microorganisms from water supplies, _ pathogens are also isolated and cultivated using the waste treatment sites, oil spills, mines, and agricultural present invention. Such protozoans and parasites in areas to be isolated by immune capture and cultivated. clude, but are not limited to, Toxoplasma gondii, Plas Also of interest as a source are tissues and ?uids from modiurn species, Trypanosoma species, Pneumocystis ticks, lice, reduviid bugs, mosquitoes, ?ies, and other 35 carinii, Schistosoma species, Leishmania species, and vectors known or suspected of being involved in the the like. transmission of infectious microorganisms to animals. The organisms are isolated from infected host tissue Of particular interest as a source are tissues and ?uids and host ?uids as well as from tissue and ?uid samples from a host such as an animal, preferably mammal, taken from the vectors. Vectors for the protozoans and containing or suspected to contain the microorganism parasites are generally known in the art and include, but of interest. The present invention is especially useful in are not limited to, mosquitoes, ?ies, reduviid bugs and isolating or immune capturing microorganisms that the like. occur at low density in tissues and ?uids of infected The present invention of immune capture and cultiva hosts or which may also contain potential contaminants. tion is also useful in isolating pathogenic or potentially The present invention is particularly useful in isolating 45 pathogenic fungi from the tissues or ?uids of an infected pathogenic or potentially pathogenic microorganisms host. An example of such a yeast includes Candida including but not limited to bacteria, fungi and para albicans and the like. sites. The immune capture method is particularly useful in Potentially pathogenic microorganisms that are im capturing and cultivating pathogenic microorganisms mune captured and cultivated using the present inven 50 with fastidious growth requirements and/or require tion include, but are not limited to, intestinal and/or anaerobiosis and microorganisms that have relatively food-borne pathogens such as: slow growth rates. Examples of microorganisms with Escherichia coli, Shigella dysenterae, Shigella ?exneri, relatively fastidious growth requirements, include but Salmonella typhi, Salmonella typhimurium, Yersinia en are not limited to, Campylobacter jejuni, Helicobacter terocolitica, Campylobacter jejuni, Helicobacter pylori, 55 pylori, Treponema denticola, Mycobacterium tuber Clostria'ium perfringens, Clostridium botulinum, Staphy culosum, Mycobacterium avium, Mycoplasma pneumonia, lococcus aureus, Bacteroidesfragilis, and the like. Legionella pneumophila, Listeria monocytogenes, Neisse Oral pathogens or potentially pathogenic microor ria meningitidtls, Chlamydia psittaci, Neisseria gonor ganisms that are isolated from the oral cavity of a host rhoeae, Chlamydia trachomatis, Treponema pallidum, using the immune capture and cultivation technique of 60 Rickettsia rickettsii, Rickettsia akari, Rickettsia prowaze the present invention include, but are not limited to, kii, Rickettsia tsutsugamushi, Coxiella burnetti, Borrelia Bacteroides gingivitis, Streptococcus mutants, Streptococ burgdorferi, Borrelia hermsii, Borrelia duttoni, Borrelia cus sanguis, Treponema denticola, and the like. turicatae, Leptospira interrogans, Francisella tulerensis, Respiratory and/or air-borne pathogens or potential and the like. pathogens that are isolated using the present invention 65 Examples of microorganism with anaerobic growth from samples such as bronchial secretions, oral-nasal requirements include, but are not limited to, Clostridium passages, pleural ?uid and lung biopsy samples include, perfringens, Clostridium botulinum, Bacteroides fragilis, but are not limited to, Streptococcus pneumonia, Strepto Bacteroides gingivalis, Bacillis anthracis, and the like. 5,403,718 7 8 Of particular interest is the spirochete, B. burgdorferi, logical Methods in Biomedical Sciences, 4th ed. (ed. D. which occurs at an apparently low density in tissues and M. Weir et al.), V014, p. 108, Blackwell Scienti?c Pub ?uids of infected hosts. This microorganism can be lications, Oxford; Harlow, E. et al. 1988, Antibodies: A captured using the present invention, and retained on Laboratory Manual, Cold Spring Harbor Laboratory, surfaces containing adsorbed antibodies directed Cold Spring Harbor, N.Y., all incorporated herein by toward cell-surface extracellular B. burgdor?zri anti reference). Basically, spleen cells from a hyperimmu gens. Relatively few contaminating, non-B. burgdorferi nized animal are fused with cells from a genetically organisms or host cells adhere to antibody-activated marked myeloma that has been adapted to grow in surfaces so that pure or relatively pure cultures of B. tissue culture. Mixed populations of the resulting hybrid burgdorferi are obtained upon culturing of the captured cells are tested for their ability to secrete antibody of the microorganisms. desired speci?city, and clones derived from individual Microorganisms are captured onto antibody cells are then established. The resulting monoclonal activated surfaces, and cultivated by transferring such antibodies are screened for their ability to speci?cally surfaces into culture medium. The culture media and immune capture the target microorganism of interest conditions for culturing the above identi?ed microor while not binding to non-target microorganisms. ganism, as well as other microorganisms, are known in The antibodies or antigen binding fragments of the the art and are detailed in Bergey’s Manual of Systemic antibodies may also be produced by genetic engineer Bacteriology (Eds. John G. Holt et al., Williams & ing. The technology for expression of both heavy and Wilkins, Baltimore-London, Vol. 1-1984, Vol. II-1986, light chains of antibody molecules in E. coli is the sub Vol. III & IV-l989), incorporated herein by reference. 20 ject of the PCT patent applications, publication num The immune capture system facilitates aseptic primary bers W0 901443, WO 901443, and W0 9014424 and in isolation of microorganisms from biological and/or Huse et al., 1989 Science 246: 1275-1281, incorporated environmental samples such as infected tissues soil, herein by reference. water, air, and the like. An antibody-activated, capillary tube-based selective Antibodies useful in the immune capture method for culture system was designed that allows the selective isolating speci?c microorganisms are antibodies that immobilization of B. burgdorfen‘ cells and cultivation of react with external components of the microorganisms. the spirochete. In this system, IgG antibodies, speci?c Such external components include but are not limited to for B. burgdorferi, generated against extracellular mem cell surfaces, surface coats, cell walls, slime layers, ex brane vesicle concentrates, or against an extracellular tracellular ?agella, pili and the like. The antibodies are 83 kDa multi-protein complex, are adsorbed onto the produced by immunizing mammals with the intact mi inner surface of capillary tubes. Suspensions of spiro croorganism or with puri?ed or partially puri?ed exter chetes containing or lacking contaminating bacteria, or nal components of the microorganism. The antibodies specimens from infected hosts are drawn within the are also produced by immunizing mammals with modi tube, and incubated. After expelling the contents of the ?ed external components of the microorganism or using 35 tubes, the tubes are washed with sterile buffer and synthetic peptides, carbohydrates, lipopolysaccharides, placed in culture medium containing or lacking antibi glycoproteins, glycolipids or recombinant peptides or otics. B. burgdorferi can be selectively and aseptically proteins, the structures of which one deduces from the cultured in vitro from pure and mixed cultures and in naturally occurring microorganism. These antibodies vivo from tissue or ?uid samples using this system. are capable of binding and retaining intact microorgan 40 The surface to which the antibodies are bound may isms onto antibody-activated surfaces for isolation and be glass, plastics, and the like. When a glass surface is subsequent cultivation. Preferred antibodies are anti used it may be pretreated with a basic solution such as bodies that are species-speci?c and thus are capable of 1N NaOH or other agent, to permit complete wetting of binding to a particular species of microorganism. The the surface, before the antibody containing solution is antibodies are polyclonal, or monoclonal, and may be a 45 added. Such pretreatment allows for an even layer of cocktail of antibodies with different reactivities for the antibody solution to coat the surface. microorganism of interest. Surfaces activated with antibodies dissolved in phos Mammalian lymphocytes are immunized in vivo or in phate-buffered saline are used to capture spirochetes vitro for production of antibodies. For in vivo immuni and successfully inoculate fresh growth medium. The zations of mammals, immunizations are repeated as 50 tubes are used immediately after coating with the anti necessary at intervals of up to a few weeks (e.g., 2-4 body solution, or stored wet, to avoid extensive salt weeks) so as to obtain a suf?cient titer of ‘antibodies. deposits, from the phosphate-buffered saline, that occur The microorganism, external component of the micro with drying. Tubes coated with antibody dissolved in organism, or the like is injected into the animal in appro phosphate-buffered saline were efficient in retention of priate solutions or adjuvants via an appropriate route 55 B. burgdorferi, and exclusion of contaminating E. coli. (i.e., intramuscular, intraperitoneal, subcutaneous, intra When long term storage of antibody coated tubes is venous, intradermal, and the like). needed, the tubes are coated with antibody in a solution If monoclonal antibodies are desired, the hybridoma of ammonium bicarbonate. Ammonium bicarbonate, a formation and monoclonal antibody production may be volatile buffer, is removed under vacuum, leaving an carried out using many different techniques which are 60 antibody ?lm on the surface of the glass. Antibody well known in the art. (Campbell, A. M. 1984. Mono activated tubes produced using ammonium bicarbonate clonal Antibody Technology. Laboratory Techniques allowed recovery of B. burgdor?zri from a mixture of B. in Biochemistry and Molecular Biology (ed. R. H. Bur burgdor?zri and E. coli, and provided long term storage don and P. H. van Knippenberg), Vol. 13. Elsevier, capabilities. Amsterdam; Goding, J. W. 1986. Monoclonal Antibod 65 The immune capture method of isolating microorgan ies: Principles and Practice, 2nd edition, Academic isms is capable of isolating speci?c microorganisms Press, London; Kipps, T. J. et a1. 1986, In: Handbook of from biological samples containing a variety of different Experimental Immunology: Applications of Immuno microorganisms with or without the use of supplemen 5,403,718 9 10 tal antibiotics. For example, antibody-activated capil results recon?rm that B. burgdorferi antigens persist in a lary tubes were capable of isolating B. burgdorferi from variety of host materials. Such antigens can be captured mixed cultures, containing S. aureus along with E. coli, and identi?ed with speci?c polyclonal antibodies, pro without the use of supplemental rifampin. Overnight viding a sensitive assay for diagnosing and studying growth of the serial dilutions of the mixed cultures lyme borreliosis. suggested nearly a l0-fold excess of contaminating bac Accordingly, the present invention relates to a sensi teria in the original mixture. Using the present invention tive immunoassay and diagnostic test kit for the detec of immune capture, B. burgdorferi was successfully re tion of B. burgdorferi-speci?c antigens in ticks, mice, covered from samples. Thus, both in systems supple dogs, and humans. When the assay involves the immune mented with or lacking rifampin, the capillary tubes capture of antigens with immobilized F(ab’)2 fragments, coated with antibodies enabled puri?cation of B. burg it is followed by speci?c antigen detection using poly dorferi from mixed cultures at nearly the same rate as clonal IgG. A reagent capable of reacting with poly from a diluted pure culture of B. burgdorferi. Using clonal IgG (e.g., labeled protein A, labeled protein G, control tubes that were not coated with antibodies, or labeled antipolyclonal IgG antibodies) may be used recovery rates from pure cultures of B. burgdorferi were during the assay. Using this assay, B. burgdorferz' anti reduced by 100-1000 fold, and B. burgdorferi could not gens and occasionally intact spirochetes were detected be recovered from mixed cultures. in ticks and mammalian urine, blood, and organs. Using tubes coated with the anti-83 (kilodalton) kda In one embodiment, a reagent capable of binding to antibodies, B. burgdorferi spirochetes were recovered immunoglobulin molecules is used in the detection from infected tick and murine samples. No isolates of B. 20 method. The reagent or immunoglobulin binding factor burgdorferi were obtained by direct inoculations of may bind speci?cally to a immunoglobulin class such as media with preparations from the infected tick and murine samples. IgG on IgM or may bind to any immunoglobulin class. Variables may be modi?ed to increase the probability Examples of such immunoglobulin binding factors in clude but are not limited to protein A and protein G, for successful isolation of this organism, or to target 25 other organisms for cultivation. The present immune and the like. capture system may be modi?ed for use with other When the detection method involves the use of pro substrates such as polystyrene, with enhanced antibody tein A or protein G, the capture antibodies must be, binding affinities, to increase the capacity for capturing F(ab’)2 or F(ab) fragments generated against extracellu organisms. Similarly, repeated contact of comparatively 30 lar membrane vesicle concentrates. Protein A binds to large volumes of source material to antibody activated intact IgG. Either intact antibodies or the antigert bind surfaces may allow for retention of additional targeted ing fragments can be used. When protein A, protein G, microorganisms, providing an increased chance of ef or other reagent which .binds to the Fc portion of anti~ fective isolation. Furthermore, since rinsing apparently bodies is used, the capture antibody must be a fragment. reduces the level of contaminants from immobilized 35 Antigens were captured with F(ab')2 fragments. Ini spirochetes, such immune capture methods facilitate tial characterizations of the anti-vesicle sera by im ampli?cation of B. burgdorferi DNA target sequences munoblot analysis and immune electron microscopy by polymerase chain reaction. The present immune demonstrated that serum IgG recognized multiple cell capture system is capable of detecting and obtaining and vesicle surface components including OspA and isolates of pathogenic or free-living microorganisms for 4-0 OspB, however, the sera also recognized some other diagnostic genetic, pathogenic, therapeutic, or other host antigens. Anti-vesicle F(ab’)2 effectively concen experimental purposes. trated and immobilized antigens from complex mix Current biological and serological techniques for tures, and appeared to block nonspeci?c adsorption of diagnosing Borrelia burgdorferi infections are often in host material and assay reagents to coated electron conclusive. In order to monitor Lyme disease, a rapid 45 microscopic grids. Because these antibodies cross and sensitive assay was developed for B. burgdorferi reacted with some host antigens, a second antibody was antigens infected hosts. Polyclonal rabbit antisera were used for speci?c detection of B. burgdorferi antigens. raised against membrane vesicles, and against an 83 kDa The detection antibody was generated against the 83 vesicle protein, puri?ed from in vitro B. burgdorferi kDa MEP. Surprisingly, immunoblots showed that the cultures. Immunoglobulin G (IgG) was recovered from 50 resulting polyclonal sera recognized OspA at 31 kDa these sera and tested for species-speci?c reaction with and OspB at 34 kDa, but had relatively weak activity several geographically diverse Borrelia isolates. Parlo with the original immunogen. Concurrent studies have dion-coated electron microscope grids were activated shown that OspA and the MEP appear to be glycosy with anti-vesicle F(ab')2 fragments, then incubated with lated with a variety of carbohydrates (Dorward, D. W., ?uids or macerated tissues from ticks, mice, dogs, and and C. F. Garon, 1990. Use of antibody and lectin-col humans. Captured antigens were assayed by immune loidal gold conjugates to detect surface and extracellu~ electron microscopy using anti-83 kDa antibodies (that lar glycoproteins produced in vivo by the Lyme disease is, antibodies raised against the 83 kDa MEP) and pro~ spirochete Borrelz'a burgdorferi in: Peachey, L. D., and tein A-colloidal gold conjugates. The results showed D. B. Williams (eds) Electron Microscopy, 1990, Pro that Lyme spirochetes shed surface antigens which ceedings of the XIIth International Congress for Elec were readily detectable in urine, blood, and several tron Microscopy, Vol. 3: Biological Sciences. San Fran organs from infected hosts. Positive mouse urine titers cisco Press, Inc., San Francisco, Calif. pp. 934-935). exceeded 1x106. Intact spirochetes were frequently Whether glycosylation or possible protein homology observed on grids incubated with blood, spleen, or blad~ accounts for these results remains unclear. The ?nding der preparations, and B. burgdog‘eri was re-isolated 65 that the anti-MEP IgG recognizes B. burgdorferi anti from all experimentally-infected mice. Immunoblot gens at 83 kDain mouse urine, and ll, 14, 22, 31, and 34 analysis con?rmed the presence of extracellular anti kDa in human urine, suggests that either common epi gens in positive mouse and human urine samples. These topes are expressed in several exported proteins, or the 5,403,718 11 12 MEP readily dissociates into subunits or degradative experimentation showed that continued immunization products. of rabbits with the 83 kDa band resulted in the produc As with previous characterizations of anti-OspA tion of IgG antibodies which also recognized OspB monoclonal antibodies (Barbour, A. G., R. H. Heiland, (Dorward, Schwan, & Garon, 1991. J. Clin. Microbiol. and T. R. Howe. 1985. Heterogeneity of major proteins 29:1162-1171). Previous work showed that B. burgdor in Lyme disease borrelia: a molecular analysis of North fen‘ sloughs OspA from cell surfaces (Barbour, A. G., S. American and European isolates. J. Infect. Dis. L. Tessier, and W. J. Todd 1983. Lyme disease spiro 152:478-484; Hyde, F. W., R. C. Johnson, T. J. White, chetes and ixodid tick spirochetes share a common sur and C. E. Shelburn. 1989. Detection of antigen in urine face antigenic determinant de?ned by a monoclonal of mice and humans infected with Borrelia burgdorferi, 10 antibody. Infect. Immun. 4l:795—804). Together these etiologic agent of Lyme disease. J. Clin. Microbiol. results suggest that the MEP, OspA and possibly OspB 27:58-61 and Wilske, B., V. Preac-Mursic, G. Schierz, share identity and may be components of a surface or R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. s-layer that can be released from cell surfaces. Antigenic variability of Borrelia burgdorfen'. p. 126-143. The detection of antigens in urine using monoclonal In. J. L. Benach, and E. M. Bosler (eds.), Lyme disease antibodies has recently been reported (Hyde, F. W., R. and related disorders. Annals of the New York‘ Acad C. Johnson, T. J. White, and C. E. Shelburn. 1989. emy of Sciences 539:126-143), the anti-MEP polyclonal Detection of antigen in urine of mice and humans in IgG was species-speci?c. Furthermore, whereas anti fected with Borrelia burgdorferi, etiologic agent of OspA monoclonal antibodies invariably fail to bind Lyme disease. J. Clin. Microbiol. 27:58-61). The fol some strains, this polyclonal antibody recognized all 20 lowing Example 1 con?rms that ?nding, and provides strains of B. burgdorferi tested, including several geo methods that may enhance the reported sensitivity of graphically diverse isolates. We presume that the poly detection (Hyde, F. W., R. C. Johnson, T. J. White, and clonal IgG binds to multiple epitopes on OspA and C. E. Shelburn. 1989. Detection of antigen in urine of MEP. Hence, divergence among B. burgdorferi strains, mice and humans infected with Borrelia burgdorferi, re?ected by amino acid sequence variation within these 25 etiologic agent of Lyme disease. J. Clin. Microbiol. proteins, could occur without complete loss of antibody 27:58-61). Example 1 also shows that extracellular B. recognition. burgdorferi antigens occur in tissues in which spiro When these reagents were used to examine experi chetes are infrequently reported (Duray, P. H., and A. mentally-infected mice by electron microscopy, B. D. Steere. 1988. Clinical pathologic correlations of burgdozferi-derived material was detected in urine, Lyme disease by stage, p. 65-79. In J. L. Benach, and E. blood, and macerated urinary bladder, spleen, liver, and M. Bosler (eds.), Lyme disease and related disorders. brain tissues. Aggregated antigens were not observed in Annals of the New York Academy of Sciences kidney tissue, however the relatively dense deposition 5 39:65-79). Such results suggest either that large quanti of gold in kidney preparations suggests B. burgdorferi ties of circulating antigert are deposited in these tissues, antigens were present. Minimal background labeling on 35 or that the antigen is secreted by a limited number of control grids lacking antigen and on grids incubated migrating spirochetes and the antigen persists in situ. with material from uninfected mice and humans, indi The possible pathological effects of this material are cated that gold deposition in this assay was speci?c for unknown, however determining its nature and the B. burgdor?zri antigens. mechanisms behind its deposition my lead to better a Intact spirochetes were observed on grids incubated 40 understanding of B. burgdorferi pathobiology. The rea with mouse blood, and with bladder and spleen tissue gents developed in Example 1 as discussed herein below specimens from mice. A spirochete was also observed in should facilitate such determinations. 1 pl of a human urine sample, suggesting this detection Both the F(ab’)2 and IgG preparations proved stable system may facilitate studies of tissue involvement com when stored as lyophilized powder. In these experi plicated by difficulty in demonstrating spirochetes in 45 ments the reagents were rehydrated just prior to use, infected hosts (Fox, J. L. 1989. Interest in Lyme disease indicating appropriate preparations could be stored and grows. ASM News 55:65-66). distributed in aliquots for use as needed. Combining Pre-incubation of the grids with anti-vesicle F(ab')2 immune capture with polyclonal antigen detection fragments dramatically increased the sensitivity of anti should reduce false-negative detection of B. burgdorferi, gen detection, particularly in complex samples such as resulting from minor antigenic variation, and enable blood and macerated tissues. Flocculent antigen was reliable demonstrations of this spirochete and its prod detectable in these samples without pre-incubations, ucts in vector, reservoir host, domestic animal, and however the grids contained considerable quantities of human urine, blood, or tissue samples. Although de unlabeled host material. Apparently, the F(ab’)2 frag signed initially for careful electron microscopic exami ments functioned both by concentrating Borrelia anti 55 nation of captured antigens, the system is readily adapt gens on the grids, and blocking non-speci?c adsorption able to more common clinical laboratory analysis proto of eukaryotic material. cols. Vesicles were resolved on the surfaces of spirochetes These clinical laboratory analysis protocols are well adhering to grids incubated with infected tissues, indi known in the art. The antibodies, particularly those cating these vesicles are produced by B. burgdorfen' in raised against the MEP, can be used in all the conven vivo. Gold-labeled, membranous vesicles were also tional immunoassay formats to detect the .antigens in observed in urine and blood. The majority of speci?c biological samples, and thus detect the presence of Bar gold labeling occurred on ?occulent material detected relia burgdor?eri. Both homogeneous and heterogeneous in B. burgdorjkri cultures and all infected animals. While immunoassays may be used. The anti-MEP antibody structurally-similar material was frequently on cell sur 65 can be directly labeled with conventional labels, i.e., faces, its exact nature is currently unknown. Western enzymes, radioisotopes, ?uorophores, colloidal metals blots showed that anti-MEP IgG and anti-OspA mono (gold), or these labels can be attached to antibody bind clonal antibodies react with the same bands. Further ing proteins (anti-anti-MEP antibodies, protein A, etc.) 5,403,718 13 14 to indirectly label the anti-MEP antibody. A preferred the resulting antigen/IgG complex may readily be de method of detecting the exported antigens is as follows. tected by radioimmunoassay (Gee, A. P. and J. J. Lan The anti-vesicle antibodies are bound to an inert solid gone (1983), Immunoassay using antigen-coated plastic substrate. The biological sample is brought into contact tube on radiolabeled or enzyme labeled protein A, with this substrate under conditions conducive for the Methods Enzymol. 922403-413; Umetsu, D. T., R. S. formation of immune complexes between the anti-vesi Geha (1987), In vitro production of antibody in cultures cle antibodies and any antigens associated with Borrelia of human peripheral blood lymphocytes. Methods En burgdorferi antigens in the sample. These antigens in zymol. 150:309-316). Alternatively, the polyclonal anti clude the intact Borrelia burgdorferi spirochete, the ex bodies may be contacted with a reagent (e.g., labeled) tracellular membrane vesicles, and the exported prote capable of reacting with polyclonal antibodies and the ins from the surface of the spirochete, including the 83 antibody/reagent complex is then detected. For in kDa MEP, and the 11, 14, 22, 31 and 34 kDa exported stance, the antibody/reagent complex may be visually proteins found in human urine as described in Example detected through a microscope. Preferably, the poly 1. The substrate is washed and then brought into clonal antibodies are of IgG class and a reagent capable contact with anti-MEP antibodies under condition con of reacting with polyclonal IgG is a protein A conju ducive to form a ternary sandwich immune complex gate, such as manufactured by Sigma Chemical Com consisting of an anti-vesicle (antigen) anti-MEP com pany, St. Louis, Mo. (e.g., protein A gold, Catalog No. plex. The anti-MEP antibody can be directly labeled P1039). Other suitable reagents capable of reacting with (see above) in which case the sandwich immune com IgG include commercially available protein G conju plex is directly detected. Alternatively, the substrate 20 gates or similar anti-IgG antibody conjugates. Protein with its ternary sandwich immune complex can be G gold conjugates are available from Sigma Chemical washed and then the anti-MEP antibody portion of the Co., St. Louis, Mo., Catalog No. P1671. complex is detected by means well known in the art. Although the polyclonal antibodies of the invention For example, a labeled anti-anti-MEP antibody can be are preferred, a system containing various monoclonal used; if the anti-MEP antibody was raised in species A 25 antibodies functioning as a polyclonal system is also (a rabbit for example), then a labeled anti-A antibody within the scope of the present invention. raised in a different species (i.e., mouse anti-rabbit) The use of the term “F(ab’)2 fragments” when de could be used to detect the anti-MEP in the ternary scribing reagents for the speci?c capture of Borrelia complex. Other labeled speci?c binding proteins that burgdorferi antigens encompasses the use of “F(ab) frag bind to the anti-MEP antibody can be used, such as 30 ments” or intact antibodies for this purpose. protein A. The most preferred method of detecting the Various samples can be tested for the presence of exported antigens uses F(ab'); fragments made from the bioproducts indicative of the presence of Lyme disease anti-vesicle antibodies bound to an inert solid substrate, spirochete in unknown biological samples by the an unlabeled anti-MEP IgG antibody and labeled pro method of the present invention. For diagnosis of Lyme tein A for detection. The results of Example 1 support 35 disease a body sample from a patient suspected of being the discussions and conclusions referred to hereinabove. infected will normally be diluted in an appropriate solu The present invention has proven effective for use tion such as physiological saline and this solution will with tears, urine, blood, and tissue biopsies from mam then be contacted with the diagnostic device containing mals, and with crushed ticks. The use of pooled poly the substrate and the immobilized F(ab’); fragments. clonal, instead of monoclonal, antibodies reduces the 40 Then, the antigen is detected with polyclonal IgG potential for loss of recognition caused by genetic and raised against the 83 kDa MEP and a reagent capable of antigenic variation among B. burgdorferi isolates. Fur reacting with polyclonal IgG syhasaprotein A con) - thermore, the sensitivity of this system for antigert de gate. When testing for the presence of bioproducts in tection in titered urine is at least 104 times greater than dicative of the presence of the Lyme disease spirochete reported by the 3M study. This conclusion is based 45 in, for instance, the blood of a patient to be tested, the upon the ?ndings of the present inventors when urine is blood is drawn from the patient in a routine manner and diluted 1:2 million and upon the report of a dilution of the blood is then optionally placed in a sterile solution. 1:64 (Hyde, F. W. et al (1989), Detection of antigen in This solution will then be tested for the presence of urine of mice and humans infected with Bo?elia burg bacteria. The biological sample may comprise mamma dorferi; etiologic agent of Lyme disease, J. Clin. Mi 50 lian urine, blood, tears, cerebral spinal ?uid, synovial crobiol. 27:58-61). ?uid and the like or organs. The organ may be selected Accordingly, the present invention relates to a from the group consisting of but not limited to macer method for detecting the presence of speci?ed microor ated urinary bladder, spleen, liver, lung, heart, kidney ganisms in samples, which comprises capturing Borrelia and brain tissue. The sample may also comprise an im burgdorferi antigens in said sample with F(ab’); frag 55 did tick. ments, F(ab) fragments or with intact or untreated anti Various insoluble substrates to which the F(ab')2 body molecules (e.g., IgG or IgM) raised against the fragments can be bound may be used. The substrate extracellular membrane vesicles exported from Borrelia should be capable of easily binding the F(ab’)2 frag burgdor?zri; and contacting the captured antigens with ments without interfering with the diagnostic test to be polyclonal antibodies raised against the 83 kDa MEP. conducted. Possible substrates include glass; thin layer The capture and contact of the antigens and antibodies chromatographic materials such as silica gel; synthetic may occur in physiological saline such as Dulbecco’s plastic material such as polyvinyl chloride, polystyrene, phosphate-buffered saline (dPBS). In addition, the Bor polypropylene and polyethylene. The substrates may be reIia burgdor?zri antigens in said sample may be captured in the form of ?at plates, glass beads, thin layers on with immobilized F(ab'); fragments, immobilized F(ab) 65 another substrate, microtiter plates, Petri dishes, latex fragments or immobilized intact antibody molecules beads, agarose or other types of beads, ?lter paper, capable of binding Borrelia burgdorferi antigens. The nylon ?lter membranes, bacterial or other types of cells, polyclonal antibodies may be radioactively labeled and glass slides, glass tubes, plastic tubes, etc. The substrate 5,403,718 15 16 may also be in the form of a membrane or ?lm of either lected to provide sufficient time for the bacterial anti a porous or nonporous nature. Preferably, the F(ab')2 gens to adsorb the F(ab’)2 fragments to a degree suffi fragments may be immobilized to an insoluble substrate cient to allow for accurate testing. The sample to be such as a solid surface selected from the group consist tested may be dissolved and/or diluted with various ing of Parlodion-coated electron microscopy grids, 5 liquids such as physiological saline, etc. nitrocellulose ?lter membranes, glass cover slips and After the solution has been contacted with the sub microtiter wells and the like. strate containing the F(ab’)2 fragments for a time suf? The F(ab’)2 fragments should be bound to the sub cient to allow the bacterial or antigens exported by the strate in an amount sufficient to and in a manner which bacteria to bind to the F(ab’)2 fragments, the substrate is allows binding of the antigens to be detected to the optionally washed to remove all unbound materials. F(ab’)2 fragments. From a practical point of view, the A test is then conducted to determine the presence of F(ab')2 fragments will usually be present in an amount the bacteria or antigens exported by the bacteria bound of at least 0.1 ng/mmZ, more preferably at least 1-1.7 to the F(ab')2 fragments on the substrate. Various tests ng/mm2 of surface area of the substrate. As far as the to accomplish this purpose are known in the art such as upper limit of the concentration of the F(ab)2 fragments the enzyme linked immunosorbent assay (ELISA), a on the substrate, the F(ab')2 fragments can be bound to radioimmune assay test, direct or indirect ?uorescent the saturation density of the substrate. For instance, the antibody test, etc. Basically, the substrate containing the saturation density of F(ab’); fragments on polystyrene is F(ab')2 fragments and suspected of containing bacterial established in a routine manner for a given manufactur antigen bound thereto is contacted with a material ers substrate (Engvall, E. and P. Perlmann (1972), En 20 which binds to the bacterial antigen to be tested. Such zyme-linked immunosorbent assay, ELISA III, Quanti materials include, for example, antibodies against the ?cation of speci?c antibodies by enzyme-labeled anti bacterial antigen (e.g., polyclonal IgG). The sub immunoglobulin in antigen coated tubes, J. Immunol. strate/F(ab)2 fragment/antigen/polyclonal IgG com 109:129-135; Voller A., D. Bidwell, G. Huldt, and E. plex is labeled with a reagent capable of reacting with Engvall (1974), A microplate method of enzyme-linked 25 polyclonal IgG such as protein A conjugates, protein G immunosorbent assay and its application to malaria, conjugates, etc. Bull. Wld. Hlth. Org. 51:209-211). The F(ab'); frag Reagents such as protein A and protein G may be ments can be bound as a molecular monolayer which “labeled” with a substance which may be easily de substantially completely covers the surface area of the tected. For example, the protein A may be conjugated substrate. Use of more than a molecular monolayer with an enzyme, radioactive material or element or F(ab')2 fragments bound to the substrate may result in a ?uorescent material. If the protein A is conjugated with waste of materials and may result in inef?cient binding an enzyme, the substrate is thereafter contacted with a of the F(ab’)2 fragments to the substrate. Preferably, the substrate for the enzyme which preferably turns color substrate is saturated with the F(ab')2 fragments after upon contact with the enzyme thereby indicating a experimentally determining the optimal quantity of 35 positive reaction. The protein A to be used should be F(ab'); fragments to use with a given substrate. one which reacts with the polyclonal IgG but which The F(ab’); fragments may be bound to the substrate does not react with the F(ab’)2 fragments bound to the in any suitable manner. Covalent or non-covalent (e.g., substrate thereby preventing a false positive reading. If hydrophobic) bonding may be used to bind the F(ab')2 the protein A is radioactively labeled, then the presence fragment to a substrate. Other forms of bonding such as 40 of radioactivity on the substrate should be measured. It ionic bonding may be used. Intact antibody molecules is also possible that the protein A may be ?uorescently or F(ab) fragments may be substituted for F(ab'); frag labeled. In this situation the treated substrate should be ments provided the substituted reagents can retain Bor exposed to ultraviolet light to determine the presence of relia burgdor?zn' antigens on the substrate, without caus the ?uorescent labeled material bound to the substrate. ing non-speci?c cross-reaction with the detection rea 45 Preferably, the protein A conjugates may be selected gents described herein. from the group consisting of colloidal gold, ?uorescent The F(ab’)2 fragments may also be bound to particles, materials such as ?uorescein isothiocyanate, rhodamine, such as latex particles, which are thereafter immobilized latex beads or other suitable beads, biotin, avidin, en by embedding in or binding to a porous membrane. The zymes such as horseradish peroxidase and alkaline phos latex particles may be of a size which can be embedded 50 phatase. Other suitable reagents capable of reacting by pressure into the pores of the porous membrane. with polyclonal IgG include protein G conjugates, Thus, for example, the average particle size of the latex similar anti-IgG antibody conjugates and direct conju particles may be about the same as, or slightly smaller gates with anti-MEP IgG, or F(ab) or F(ab'); fragments than, the average surface pore size of said porous mem made from anti-MEP IgG. brane. Alternatively, the particles may be bound to any 55 The detection system of the invention has proven porous or liquid permeable material such as a screen, effective at detecting antigert in mouse urine diluted net, etc. A material such as a binder may be used to bind l:2,000,000 in dPBS (physiological saline). the particles to the support as long as the binder does The present invention is further directed to a diagnos not interfere with the ability of the F(ab’)2 fragments to tic kit for detecting the presence of Borrelia burgdar?zri bind microorganisms. or exported antigens, comprising F(ab'); fragments, The samples suspected of containing Borrelia burg F(ab) fragments or intact or untreated antibody mole dor?zri or its exported antigens is contacted with the cules, capable of capturing and retaining Borrelia burg substrate containing the F(ab’)2 fragments described dorjferi antigens; polyclonal antibodies raised against the hereinabove. Preferably, the solution is contacted with 83 kDa MEP, these antibodies may be directly labeled the substrate until or before equilibrium is reached, e. g., 65 or a labeled speci?c binding protein that speci?cally 10 to 120 minutes, more preferably 30 to 60 minutes at binds the polyclonal antibodies is used with polyclonal a temperature of 20° to 37° C., preferably 25“ to 37° C. IgG (e.g., protein A conjugates) and capable of detect The precise time and temperature conditions are se ing the presence of antigen-antibody complex. For ex 5,403,718 17 18 ample, the F(ab‘)2 fragments, F(ab) fragments or intact dient, and removing banded membranes from the su antibody molecules are capable of being immobilized on crose gradient. a support optionally supplied with the test kit. A major extracellular protein (MEP) antigen was Accordingly, the kit for diagnosing Lyme disease puri?ed electrophoretically from extracellular vesicle may comprise (a) anti-MEP antibodies or (b) labelled extracts, separated by sodium dodecylsulfate-polya anti-MEP antibodies (0) anti-MEP antibodies and la crylamide gel electrophoresis (Judd, R. C. (1988). Puri beled anti-anti-MEP antibodies or labeled protein A or ?cation of outer membrane proteins of the Gram-nega (d) anti-vesicle antibodies and anti-MEP antibodies, etc. tive bacterium Neisseria gonorrhoea. Anal Biochem. The F(ab’)2, F(ab) fragments, intact antibody mole 1731307-316). The MEP is a protein with an apparent cules capable of binding Borrelia burgdorferi antigens, molecular mass of 83 kilodaltons (kDa), and it is shown IgG preparations and reagents capable of reacting with in FIG. 1. It constitutes the major protein component of polyclonal. IgG such as protein A conjugates may be in puri?ed extracellular vesicles (as described above). The the form of a powder. function of the MEP is unknown, however preliminary The present invention is also directed to a method of data suggested that the surface or s-layer of Borrelia diagnosing Lyme disease in a mammal (e,g., human and 15 burgdorferi was primarily made of the MEP. domestic animals such as dogs, cats, horses, goats, Recent data has elucidated the composition and ori sheep, cows, etc.) comprising the steps of taking a bio gin of the MEP. The 83 kDa MEP is composed of an logical sample suspected of containing Borrelia burgdor extracellular multiprotein complex. To characterize the feri antigens from a mammalian (e. g., human) host, cap 83 kDa band resolved by electrophoresis, the N-ter turing Borrelia burgdor?zri antigens in said sample with 20 minus of the predominant peptide in the band was se F(ab’)2 fragments, F(ab) fragments or with intact anti quenced. Peptide sequence and amino acid composition body molecules raised against the extracellular mem comparisons showed identity with the heavy chain of brane vesicles; and detecting the antigen with poly immunoglobulin M (IgM). Reduction sensitivity experi clonal antibodies raised against the 83 kDa MEP. The ments and the recognition of the band by antibodies 25 biological sample comprises, for instance, mammalian speci?c for rabbit p. chain indicated that the multi urine, blood, tears, cerebral spinal ?uid, synovial ?uid protein complex contained pentameric IgM. Immunoe or the like, or organ. The organ or biopsied portion lectron microscopy showed that anti-p. chain antibodies thereof is selected from the group consisting of macer and monoclonal antibodies to Osp A and Osp B bound ated urinary bladder, spleen, liver, lung, brain tissue, 30 to extracellular amorphous material surrounding cells. heart, kidney and skin. Furthermore, the Osps coprecipitated with either non In addition, the present invention can be used to de speci?c polyclonal rabbit IgM antibodies or with mu termine whether Ixodes or other ticks contain Borrelia rine monoclonal antihuman serum albumin IgM anti burgdorferi. For example, the tick may be crushed in bodies indicating that Osp A and Osp B are components Dulbecco’s phosphate-buffered saline pH 7.2 (dPBS) 35 of the 83 kDa MEP complex along with the predomi and used as the sample to be detected in the method of nant protein, nonspeci?c IgM. The origin of the IgM is the present invention. rabbit serum which was used as a culture medium sup Moreover, the present invention is directed to an plement (Dorward, D. W. et at. 1992, Infect. Immun. 60 antigen comprising puri?ed major extracellular protein (3): 838-844). isolated from Borrelia burgdorferi. Extracellular mem The puri?cation of the MEP is set forth in Example 1, brane vesicles were recovered from log phase cultures and it was used to immunize rabbits as described in of Borrelia burgdor?ari as described by Garon, et al. Example 1. Polyclonal IgG produced by rabbits, in (1989). Structural features of Borrelia burgdorferi— the response to this protein was used as a detection reagent Lyme disease spirochete: silver staining for nucleic for the diagnostic test. acids. Scanning Microscopy Supplement 3, pages 45 The present invention is further directed to an anti 109-115. Cells were removed from the cultures by cen body raised against the antigens described above. For trifugation at 20° C., for 20 min, at 10,000 x g. Cell example, the invention is directed to anti-vesicle F(ab')2 removal was assured by further centrifugation for 15 fragments which have a molecular mass of approxi min, at 20,000 x g, and by ?ltration through a 0.22 pm mately 95 kDa. They have an antigen binding valence ?lter. Vesicles were recovered from this ?ltrate by of 2, meaning that they can bind (iramobilize) two moles centrifugation for 1 hr at 257,000 x g, at 20° C. Vesicle of antigen per mole of F(ab’)2. For example, a l ug/ml pellets were resuspended in dPBS, then layered onto a solution of F(ab’)2 fragments would contain approxi 10—70% step sucrose gradient and dPBS, and centrifu mately 10.5 picomoles, capable of binding up to 21 gated for 2 hr at 259,000 x g at 4° C. Banded membranes picomoles of antigen. were then removed, diluted 1:1 in dPBS, and collected 55 The F(ab')2 fragments were made by digestion of by centrifugation for 15 rain at 435,000 x g at 4° C. anti-vesicle IgG with pepsin, followed by af?nity chro Such vesicles, when suspended at 1 mg per ml of matography, as described in Example 1. The IgG was dPBS, were used as immunogens in rabbits. Antibodies accordingly puri?ed from anti-vesicle serum by affmity produced by rabbits immunized with vesicles were used chromatography with protein A-agarose, as described to produce “anti-vesicle F(ab')2 fragments.” These vesi in Example 1. The F(ab')2 fragments can bind and im cles were also used as a source from which a “MEP” mobilize cell-surface and extracellular antigens pro antigen was puri?ed. duced by Borrelia burgdorferi, and they were used for Accordingly, the present invention is further directed such a purpose in this invention. to antigens of extracellular vesicles isolated by the steps Moreover, the present invention is further directed to of removing cells from Borrelia burgdorferi cultures by 65 polyclonal antibodies raised against the 83 kDa MEP. fractionation, for example, by centrifugation, ?ltering These antibodies were produced by rabbits in response the cells and collecting a ?ltrate, recovering the vesicles to immunization with puri?ed MEP, and were puri?ed from the ?ltrate, layering the vesicles on a sucrose gra by af?nity chromatography as described in Example 1. 5,403,718 19 20 The anti-MEP IgG binds to the MEP, and to a pro gen suspended in dPBS. Sera were collected over a tein at 31 kDa (Osp A), on immunoblots (FIG. 1), and it period of 10 weeks. binds to cell-surface, and extracellular antigens pro Immunoglobulin G (IgG) was puri?ed from the sera duced by Borrelia burgdorferi. The anti-MEP IgG also by affinity chromatography with protein A-agarose binds to a protein at 34 kDa (Osp B) as shown in Dor (Sigma Chemical Co., St. Louis, Mo.). Eluted IgG was ward, Schwan & Garon, 1991 (J. Clin. Microbiol. dialyzed overnight with water, and lyophilized for stor 24:1162-1171). It does not bind to antigens produced by age. related spirochetes such as other species of Borrelia, For some experiments, F(ab’); fragments were pro and Leplospira interrogans. Anti-MEP IgG is used in this duced from IgG, directed against vesicles, by passage invention to detect antigens produced by Borrelia burg 10 through pepsin-agarose (Sigma) as described in La dozferi, that have been captured and immobilized by the moyi, E., and A. Nisonoff, 1983, Preparation of F(ab’)2 anti-vesicle F(ab'); fragments. fragments from mouse IgG of various subclasses, J. EXAMPLE Immunol. Methods 56:235-243. Cleaved IgG was sub sequently passed through protein A-agarose and the MATERIALS AND METHODS 15 void volume was retained. F(ab) fragments can be pro Bacteria. The bacteria used in this study are described duced by passage through papain-agarose (Sigma) in in Table 1. stead of pepsin-agarose as described above. TABLE 1 Experimental mouse infections. White-footed mice (Peromyscus leucopus) were experimentally infected Bacteria used in Exam 1e 1. 20 with B. burgdorfen' by intra-peritoneal injection with 0.1 Anti Organism Strain Source MEP/GPA ml suspensions of spirochetes in dPBS at an OD600 "m of B. burgdad‘en' 19678 P. Ieucopus, N.Y. + 0.4 (Schwan, T. G., W. Burgdorfer, and C. F. Garon. B. burgdorferi 20004 I. ricinus, France + 1988. Changes in infectivity and plasmid pro?le of the B. burgdorfen' 26816 Microtus, R.I. + Lyme disease spirochete, Borrelia burgdorferi, as a result 25 B. burgdol?ri B31 1. dammini, N.Y. + of in vitro cultivation. Infect. Immun. 56:1831-1836 and B. burgdorferi G2 Human CSF, Germany + B. burgdor?zri HB19 Human blood, Conn. + Schwan, T. G., W. Burgdorfer, M. E. Schrumpf, and R. B. burgdaderi Sh-2-82 I. dammini, N.Y. + H. Karstens. 1988. The urinary bladder, a consistent B. anserina RML — source of Borrelia burgdorferi in experimentally infected B. coriaceae C053 Omithodorus coriaceus, — 30 white-footed mice (Peromyscus Ieucopus). J. Clin. Mi B. hermsii H81 0. hermsi, Wash. — crobiol. 262893-895). Urine, blood, and organs such as B. parkeri RML — bladder, spleen, liver, kidney, heart and brain were B. turicatae RML — collected from infected and uninfected animals. Infec I. inten'ogans ATCC 23581 — tion was con?rmed by culturing B. burgdorferi from All bacteria from Table 1 were maintained in BSK II 35 triturated urinary bladders, as described in Schwan, T. media described in (Barbour, A. G. 1984. Isolation and G., W. Burgdorfer, and C. F. Garon, 1988, Changes in cultivation of Lyme disease spirochetes. Yale J. Biol. infectivity and plasmid pro?le of the Lyme disease spi Med. 57:521-525). Whole cells (WC) and extracellular rochete, Borrelia burgdorferi, as a result of in vitro culti membrane vesicles were recovered by ?ltration and vation. Infect. Immun. 56:1831-1836 and Schwan, T. differential centrifugation as described in (Dorward, D. G., W. Burgdorfer, M. E. Schrumpf, and R. H. Kar W., and R. C. Judd. 1988. The isolation and partial stens. 1988. The urinary bladder, a consistent source of characterization of naturally-evolved outer membrane Borrelia burgdorferi in experimentally infected white blebs of Neisseria gonorrhoea. p. 349-356. In Gonococci footed mice (Peromyscus Ieucopus). J. Clin. Microbiol. and Meningococci, J. T. Poolman, H. C. Zanen, T. J. 26:893-895. Meyer, J. E. Heckels, P. R. H. Makela, H. Smith, and E. 45 Human, canine and tick materials. Clinical human C. Beuvery (eds), Kluwer Academic Publishers, Dor urine and Ixodes dammini ticks, collected from Juneau drecht, The Netherlands and Garon, C. F., D. W. Dor County, Wisconsin, were graciously provided by Dr. ward, and M. D. Corwin. 1989 Structural features of Paul Duray. Human urine samples were also provided Borrelia burgd0rfen'- the Lyme disease spirochete: by laboratory volunteers. Acute human serum from silver staining for nucleic acids. Scanning Microscopy 50 Southampton, N.Y., was supplied by Dr. Alan Mac Supplement 3, pages 109-115). Donald. Whenever possible, assays on human speci Antibodies. Polyclonal rabbit sera were raised against mens were compared with serological data and/or pa membrane vesicles and the 83 kDa MEP, electrophoret tient histories. Urine and blood from a dog, naturally ically puri?ed from vesicles (Dorward, D. W., and R. infected in Bridgewater, N.J., were provided by Sara C. Judd. 1988. The isolation and partial characterization 55 Stephens, D. V. M., Missoula, Mont. of naturally-evolved outer membrane blebs of Neisseria Immunoblot analysis. Antigens, precipitated from gonorrhoea. p. 349-356. In Gonococci and Meningo urine with antiMEP antibodies, WC, and vesicles, were cocci, J. T. Poolman, H. C. Zanen, T. J. Meyer, J. E. solubilized and subjected to sodium dodecylsulfate Heckels, P. R. H. Makela, H. Smith, and E. C. Beuvery polyacrylamide gel electrophoresis using the discontin (eds.), Kluwer Academic Publishers, Dordrecht, The uous buffer system of Laemmli (Laemmli, U. K. 1970. Netherlands and Judd, R. C. 1988. Puri?cation of outer Cleavage of structural proteins during the assembly of membrane proteins of the Gram-negative bacterium the head of bacteriophage T4. Nature (London) Neisseria gonorrhoea. Anal. Biochem. 173:307-316). 227:680-685), with the modi?cations described in Judd, Emulsions of antigen, and monophosphorylated lipid A R. C. 1982, 125I-peptide mapping of protein III isolated and trehalose dimycolate (Ribi ImmunoChem, Inc., 65 from four strains of Neisseria gonorrhoea, Infect. Immun. Hamilton, Mont.) were prepared according to manufac 37:622-631. Separated promins were electroblotted turer’s instructions and used as primary immunogen. onto nitrocellulose, blocked with 0.05% Tween 20 in Immunized rabbits were periodically boosted with anti dPBS or 5% nonfat dry milk in dPBS, and probed with 5,403,718 21 22 serum or IgG, as described in Batteiger, R., W. J. Ne and sera taken from rabbits after prolonged immuniza whall, and R. B. Jones, 1982, The use of Tween 20 as a tion with MEP gave similar results in the immune cap blocking agent in the immunological detection of prote' ture assay for detection of B. burgdorferz' antigens. ins transferred to nitrocellulose membranes, J. To determine whether these polyclonal sera could be Immunol. Methods 55:297-307. Resulting immune com used to concentrate and speci?cally detect intact WC plexes were labeled with protein A-horseradish peroxi and elaborated vesicles, Parlodion-coated electron mi dase, and detected by chromogenic assay. croscope grids were absorbed with F(ab')2 fragments Electron microscopy. Copper grids were coated with made from anti-vesicle IgG. Such “activated” grids Parlodion, then incubated for 10 rain, at room tempera were incubated either in vitro cultures of B. burgdorferi ture, on a 6 pl droplet of anti-vesicle F(ab’)2 fragments or with media alone. Antigens adhering to the grids dissolved at 1 pg per ml of dPBS. The grids were were detected with anti-MEP IgG and protein A-col~ ' washed twice for 10 min with dPBS, then incubated for loidal gold conjugates (FIG. 2). FIGS 2a-2c describes 10 min on a 6 pl droplet of antigen prepared as follows: the parlodion-coated grids were activated with anti urine, diluted 1:10 or as speci?ed in dPBS; blood, di vesicle F(ab’)2 fragments, then incubated with cultured luted 1:10 in dPBS; and ticks or mouse organs such as 15 B. burgdorferi cells (20, b) or with culture media (20). bladder, spleen, liver, kidney, heart and brain, macer The grids were then probed with anti-MEP IgG (2a, 0) ated in an equal volume of dPBS in glass tissue grinders. or pre-immune serum (2b), labeled with protein A-col Following two, 10 min washings in dPBS, the grids loidal gold conjugates, and examined by electron mi were transferred onto 6 pl droplets of anti-MEP IgG croscopy. Gold particles adhered to cell and vesicle and incubated at room temperature for 20 min. Two 20 surfaces and to ?occulent material surrounding cells. more dPBS washings were done, then antigen~antibody Only background levels of gold were observed on grids complexes were labeled for 20 min with 6 pl of protein incubated with media, or probed with pre-immune A-colloidal gold conjugates, prepared by the methods serum. Bars, 200 nm. Both WC and vesicles adhered to of Robinson and co-workers (Robinson, E. N., Z. A. activated grids. Heavy labeling was evident on the sur McGee, ‘J. Kaplan, M. E. Hammond, J. K. Larson, T. 25 faces of cells and vesicles, and on ?occulent material M. Buchanan, G. K. Schoolnik. 1984. Ultrastructural surrounding these structures. Sparse deposition of gold localization of speci?c gonococcal macromolecules on the control grids lacking antigen, or incubated with with antibody-gold sphere immunological probes. In pre-immune serum, was considered non-speci?c back fect. Immun. 46:361-366). The grids were washed twice ground. for 5 min each in dPBS, twice for ?ve min each in To determine the speci?city of the anti-MEP poly 0.25M ammonium acetate, then negatively stained with clonal IgG seven strains of B. burgdory‘éri, ?ve other 0.5% ammonium molybdate, pH 6.5. The grids were Borrelia species, and Leptospira interrogans (Table l) dried in air, and observed at 75 kV with a model HU were assayed. All seven B. burgdor?zri isolates from the llE-l transmission electron microscope (Hitachi, Ltd., U.S. and Europe were speci?cally labeled, whereas the Tokyo, Japan). 35 other spirochetes retained only background levels of gold. RESULTS Activated grids were then incubated with possible in To address whether membrane vesicles are released vivo sources of B. burgdorferi antigens to assess the by B. burgdorferi in vivo, polyclonal rabbit antibodies possibility of adapting this system to clinical samples. were raised against vesicles and the 83 kDa MEP. After The samples tested included macerated Ixodes sp. ticks, puri?cation from collected sera, IgG from the rabbits specimens of urine, blood, or macerated tissues from was screened for reactivity with WC and vesicles by mice, dogs, and humans (FIGS. 3, 3a-3f, 4a-4h). immunoblot analysis (FIG. 1). In FIG. 1, electroblotted FIG. 3 discloses the immune electron microscopic whole cell (WC) and vesicle proteins were stained with detection of B. burgdorferi antigens in mammalian urine buffalo black (B. Black), probed with polyclonal rabbit 45 and blood. Parlodion-coated grids were activated with IgG generated against extracellular vesicles or the 83 anti-vesicle F(ab’)2 fragments, incubated with urine or kilodalton major extracellular protein (MEP), or blood samples, probed with anti-MEP IgG, and labeled probed with the anti-OspA monoclonal antibody, 5332 - with protein A-colloidal gold conjugates. Heavily (Rocky Mountain Laboratories Collection). Anti-vesi— labeled ?occulent antigens were aggregated on grids cle IgG recognized several WC and vesicle proteins, incubated with urine from infected mice, dogs, and including OspA and OspB. The anti-MEP IgG recog humans (3a, c, d, respectively), and with blood from nized a 31 kilodalton protein, corresponding to OspA in infected mice (3b). Labeled membrane vesicles were WC and vesicle lanes. Accordingly, blotted proteins occasionally observed (30, insert). No speci?c labeling were either stained with buffalo black, or probed with was observed in urine and blood from uninfected mice anti-vesicle, polyclonal anti-MEP, or monoclonal anti 55 (3e, j). Bars, 200 nm. OspA antibodies, then labeled and detected as described Capture and labeling of ?occulent antigen was evi above. Numerous WC and vesicle proteins were recog dent in urine and blood of ?ve experimentally-infected nized by anti-vesicle IgG. Polyclonal IgG from rabbits mice (3a, b). Some ?elds contained labeled membranous immunized with the MEP, reacted primarily with a 31 structures resembling vesicles (FIG. 3a, insert). Similar kDa protein corresponding to OspA by electrophoretic ?occulent material was evident in urine from a natural migration. Only minimal labeling of the 83 kDa immu ly-infected dog (3c) and a clinical human patient (3d). nogen occurred. Reaction with monoclonal antibody Labeled ?occulent antigen was detected in mouse urine 5332 con?rmed the presence of OspA in WC and vesi diluted up to 2X 10~6in dPBS. No evidence of material, cles. With prolonged immunization of rabbits with the speci?cally labeled with gold, was detected in samples 83 kDa MEP, the sera showed reactivity with both the 65 from uninfected mice or humans (3e, t). A total of 39 Osp A and Osp B proteins. (Dorward, Schwan, & human urine samples were tested. These included two Garon, 1991, J. Clin. Microbiol. 29:1162-1171). Sera negative samples provided by laboratory personnel, and taken from rabbits after a few immunization with MEP 37 clinical samples from patients suspected of having