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United States Patent [191 [11] Patent Number: 5,403,718 Durward Et Al

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United States Patent [191 [11] Patent Number: 5,403,718 Durward Et Al . 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.
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