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. 1984 Nov;46(2):519–525. doi: 10.1128/iai.46.2.519-525.1984

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

P Minden, P J Kelleher, J H Freed, L D Nielsen, P J Brennan, L McPheron, J K McClatchy
PMCID: PMC261565  PMID: 6389346

Abstract

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.

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Selected References

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  1. Baker L. A., Sharpton T., Minden P., Campbell P. A. Adjuvant and mitogenic properties of a supernatant fraction of sonically treated Myobacterium bovis (BCG). Infect Immun. 1976 Jul;14(1):83–87. doi: 10.1128/iai.14.1.83-87.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bardana E. J., Jr, McClatchy J. K., Farr R. S., Minden P. Universal occurrence of antibodies to tubercle bacilli in sera from non-tuberculous and tuberculous individuals. Clin Exp Immunol. 1973 Jan;13(1):65–77. [PMC free article] [PubMed] [Google Scholar]
  3. Brunda M. J., Minden P., Sharpton T. R., McClatchy J. K., Farr R. S. Precipitation of radiolabeled antigen-antibody complexes with protein A-containing Staphylococcus aureus. J Immunol. 1977 Jul;119(1):193–198. [PubMed] [Google Scholar]
  4. Chang J. Y., Knecht R., Braun D. G. Amino acid analysis in the picomole range by precolumn derivatization and high-performance liquid chromatography. Methods Enzymol. 1983;91:41–48. doi: 10.1016/s0076-6879(83)91009-1. [DOI] [PubMed] [Google Scholar]
  5. Coates A. R., Hewitt J., Allen B. W., Ivanyi J., Mitchison D. A. Antigenic diversity of Mycobacterium tuberculosis and Mycobacterium bovis detected by means of monoclonal antibodies. Lancet. 1981 Jul 25;2(8239):167–169. doi: 10.1016/s0140-6736(81)90355-x. [DOI] [PubMed] [Google Scholar]
  6. Daniel T. M., Anderson P. A. The use of immunoabsorbents for the purification of mycobacterial antigens. J Lab Clin Med. 1977 Aug;90(2):354–360. [PubMed] [Google Scholar]
  7. Daniel T. M., Balestrino E. A., Balestrino O. C., Davidson P. T., Debanne S. M., Kataria S., Kataria Y. P., Scocozza J. B. The tuberculin specificity in humans of Mycobacterium tuberculosis antigen 5. Am Rev Respir Dis. 1982 Oct;126(4):600–606. doi: 10.1164/arrd.1982.126.4.600. [DOI] [PubMed] [Google Scholar]
  8. Daniel T. M., Janicki B. W. Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties. Microbiol Rev. 1978 Mar;42(1):84–113. doi: 10.1128/mr.42.1.84-113.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferguson H. R., McClatchy J. K., Sharpton T. R., Minden P. Immunological method to differentiate between antigens of tubercle bacilli, other mycobacterial species, and non-acid-fast bacteria. Infect Immun. 1978 Oct;22(1):101–106. doi: 10.1128/iai.22.1.101-106.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilead Z., Jeng Y. H., Wold W. S., Sugawara K., Rho H. M., Harter M. L., Green M. Immunological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation. Nature. 1976 Nov 18;264(5583):263–266. doi: 10.1038/264263a0. [DOI] [PubMed] [Google Scholar]
  11. Goding J. W. Antibody production by hybridomas. J Immunol Methods. 1980;39(4):285–308. doi: 10.1016/0022-1759(80)90230-6. [DOI] [PubMed] [Google Scholar]
  12. Goren M. B. Immunoreactive substances of mycobacteria. Am Rev Respir Dis. 1982 Mar;125(3 Pt 2):50–69. doi: 10.1164/arrd.1982.125.3P2.50. [DOI] [PubMed] [Google Scholar]
  13. Hansen E. J., Robertson S. M., Gulig P. A., Frisch C. F., Haanes E. J. Immunoprotection of rats against Haemophilus influenzae type B disease mediated by monoclonal antibody against a haemophilus outer-membrane protein. Lancet. 1982 Feb 13;1(8268):366–368. doi: 10.1016/s0140-6736(82)91394-0. [DOI] [PubMed] [Google Scholar]
  14. Hasty D. L., Beachey E. H., Simpson W. A., Dale J. B. Hybridoma antibodies against protective and nonprotective antigenic determinants of a structurally defined polypeptide fragment of streptococcal M protein. J Exp Med. 1982 Apr 1;155(4):1010–1018. doi: 10.1084/jem.155.4.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunkapiller M. W., Hewick R. M., Dreyer W. J., Hood L. E. High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 1983;91:399–413. doi: 10.1016/s0076-6879(83)91038-8. [DOI] [PubMed] [Google Scholar]
  16. Imai M., Nomura M., Gotanda T., Sano T., Tachibana K., Miyamoto H., Takahashi K., Toyama S., Miyakawa Y., Mayumi M. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies. J Immunol. 1982 Jan;128(1):69–72. [PubMed] [Google Scholar]
  17. Kelleher P. J., Mathews H. L., Woods L. K., Farr R. S., Minden P. A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells. Cancer Immunol Immunother. 1983;14(3):185–190. doi: 10.1007/BF00205358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kessler S. W. Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol. 1976 Nov;117(5 Pt 1):1482–1490. [PubMed] [Google Scholar]
  19. Klotz S. A., Drutz D. J., Tam M. R., Reed K. H. Hemorrhagic proctitis due to lymphogranuloma venereum serogroup L2. Diagnosis by fluorescent monoclonal antibody. N Engl J Med. 1983 Jun 30;308(26):1563–1565. doi: 10.1056/NEJM198306303082604. [DOI] [PubMed] [Google Scholar]
  20. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  21. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  25. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  26. Meyer T. J., Ribi E. E., Azuma I., Zbar B. Biologically active components from mycobacterial cell walls. II. Suppression and regression of strain-2 guinea pig hepatoma. J Natl Cancer Inst. 1974 Jan;52(1):103–111. doi: 10.1093/jnci/52.1.103. [DOI] [PubMed] [Google Scholar]
  27. Minden P., Farr R. S. Binding between components of the tubercle bacillus and humoral antibodies. J Exp Med. 1969 Nov 1;130(5):931–954. doi: 10.1084/jem.130.5.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Minden P., Mathews H. L. Suppression and immunotherapy of the guinea pig line 10 hepatocarcinoma mediated by heat-killed disrupted Mycobacterium bovis strain Bacillus Calmette-Guérin. Cancer Res. 1980 Sep;40(9):3214–3217. [PubMed] [Google Scholar]
  29. Minden P., McClatchy J. K., Cooper R., Bardana E. J., Jr, Farr R. S. Shared antigens between Mycobacterium bovis (BCG) and other bacterial species. Science. 1972 Apr 7;176(4030):57–58. doi: 10.1126/science.176.4030.57. [DOI] [PubMed] [Google Scholar]
  30. Minden P., McClatchy J. K., Farr R. S. Shared antigens between heterologous bacterial species. Infect Immun. 1972 Oct;6(4):574–582. doi: 10.1128/iai.6.4.574-582.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minden P., McClatchy J. K., Wainberg M., Weiss D. W. Shared antigens between Mycobacterium bovis (BCG) and neoplastic cells. J Natl Cancer Inst. 1974 Nov;53(5):1325–1331. doi: 10.1093/jnci/53.5.1325. [DOI] [PubMed] [Google Scholar]
  32. Nowinski R. C., Tam M. R., Goldstein L. C., Stong L., Kuo C. C., Corey L., Stamm W. E., Handsfield H. H., Knapp J. S., Holmes K. K. Monoclonal antibodies for diagnosis of infectious diseases in humans. Science. 1983 Feb 11;219(4585):637–644. doi: 10.1126/science.6297006. [DOI] [PubMed] [Google Scholar]
  33. YOUMANS A. S., YOUMANS G. P. IMMUNOGENIC ACTIVITY OF A RIBOSOMAL FRACTION OBTAINED FROM MYCOBACTERIUM TUBERCULOSIS. J Bacteriol. 1965 May;89:1291–1298. doi: 10.1128/jb.89.5.1291-1298.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yoshida N., Nussenzweig R. S., Potocnjak P., Nussenzweig V., Aikawa M. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science. 1980 Jan 4;207(4426):71–73. doi: 10.1126/science.6985745. [DOI] [PubMed] [Google Scholar]
  35. Yoshida N., Potocnjak P., Nussenzweig V., Nussenzweig R. S. Biosynthesis of Pb44, the protective antigen of sporozoites of Plasmodium berghei. J Exp Med. 1981 Oct 1;154(4):1225–1236. doi: 10.1084/jem.154.4.1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zavala F., Cochrane A. H., Nardin E. H., Nussenzweig R. S., Nussenzweig V. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J Exp Med. 1983 Jun 1;157(6):1947–1957. doi: 10.1084/jem.157.6.1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de StGroth S. F., Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immunol Methods. 1980;35(1-2):1–21. doi: 10.1016/0022-1759(80)90146-5. [DOI] [PubMed] [Google Scholar]

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