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Production of potent polyclonal antibodies to bacterial membrane amphiphiles

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Abstract

Lipid A (LA), ketodeoxyoctonate (KDO) and lipoteichoic acids (LTA) were used to produce homologous polyclonal antibodies. These haptens were administered to rabbits in differing immunogenic forms, using multiple intradermal and intraperitoneal injections with complete Freund adjuvant. Booster injections were either made intradermally with incomplete Freund adjuvant or intravenously in saline. The immune-response was monitored regularly with an enzyme-immunoassay. Lipid A and KDO covalently linked to bovine serum albumin (BSA), with hapten densities per BSA molecule of 17 and 9, respectively, produced nondetectable immune-response. Acid-hydrolysed and intact cells of Salmonella minnesota Re 595 used as LA and KDO immunogens, respectively, produced significant immune-response when administered intradermally or intraperitoneally. Good immune-response was obtained with LTA covalently linked to BAS. However, a better result was obtained with crude LTA, containing 21.5% proteins. Generally, the lengthy immunization schedules used produced IgG antibodies to the antigens and the highest reciprocal titres attained were 75,000, 55,000 and 150,000 for LA, KDO and LTA, respectively. Meaningful expression of antisera titres by enzyme-immunoassay is discussed. We defined titre as the reciprocal antiserum dilution of the intercept of the mid-point on the linear section ending at 0.2 absorbance on the antiserum dilution curve.

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References

  1. Abraham GE, Grover PK (1971) Covalent linkage of steroid hormones to protein carriers for use in radioimmunoassay. In: Odell WD, Daughaday WH (eds) Principles of competitive protein-binding assays. JB Lippincott Company, Philadelphia Toronto, pp 140–152

  2. Barclay GR, Yap PL, McClelland DBL, Jones RJ, Roe EA, McCann MC, Micklem LR, James K (1986) Characterisation of mouse monoclonal antibodies produced by immunisation with a single serotype component of a polyvalent Pseudomonas aeruginosa vaccine. J Med Microbiol 21:87–90

  3. Barrett DJ, Ammann AJ, Stenmark S, Wara DW (1980) Immunoglobulin G and M antibodies to pneumococcal polysaccharides detected by enzyme-linked immunosorbent assay. Infect Immun 27:411–417

  4. Brade H (1985) Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba. J Bacteriol 161:795–798

  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248–254

  6. Colwell DE, Michalek SM, Briles DE, Jirillo E, McGhee JR (1984) Monoclonal antibodies to Salmonella lipopolysaccharide: antigen O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium. J Immunol 133:950–957

  7. Erlanger BF (1980) The preparation of antigenic hapten-carrier conjugates: A survey. In: Van Vunakis H, Langone JJ (eds) Methods in enzymology. Academic Press, New York, pp 85–104

  8. Fiedel BA, Jackson RW (1976) Immunogenicity of a purified and carrier-complexed streptococcal lipoteichoic acid. Infect Immun 13:1585–1590

  9. Fink PC, Galanos C (1981) Determination of anti-lipid A and lipid A by enzyme immunoassay. Immunobiol 158:380–390

  10. Fischer W, Koch HW, Haas R (1983) Improved preparation of lipoteichoic acids. Eur J Biochem 133:523–530

  11. Galanos C, Lüderitz O (1984) Lipopolysaccharide: properties of an amphipathic molecule. In: Rietschel ET (ed) Handbook of endotoxin, vol I: Chemistry of endotoxin. Elsevier Science Publishers. The Netherlands, pp 46–58

  12. Galanos C, Lüderitz O, Westphal O (1971) Preparation and properties of antisera against the lipid-A component of bacterial lipopolysaccharides. Eur J Biochem 24:116–122

  13. Galanos C, Lüderitz O, Rietschel ET, Westphal O (1977) Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component. In: Goodwin TW (ed) International review of biochemistry, vol 14: Biochemistry of lipids II. University Park Press, Baltimore, pp 239–335

  14. Habeeb AFSA (1966) Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem 14:328–336

  15. Ibrahim GF (1986) A review of immunoassays and their application to salmonellae detection in food. J Food Protect 49:299–310

  16. Ibrahim GF, Fleet GH, Lyons MJ, Walker RA (1985) Production of potent Salmonella H antisera by immunization with polymeric flagellins. J Clin Microbiol 22:347–351

  17. Johns MA, Bruins SC, McCabe WR (1977) Immunization with R mutants of Salmonella minnesota. II. Serological response to lipid A and the lipopolysaccharide of Re mutants. Infect Immun 17:9–15

  18. Knox KW, Wicken AJ (1973) Immunological properties of teichoic acids. Bacteriol Rev 37:215–257

  19. Knox KW, Hewett MJ, Wicken AJ (1970) Studies on the group F antigen of lactobacilli: antigenicity and serological specificity of teichoic acid preparations. J Gen Microbiol 60:303–313

  20. Lambert PA, Hancock IC, Baddiley J (1977) Occurrence and function of membrane teichoic acids. Biochim Biophys Acta 472:1–12

  21. Lüderitz O, Staub AM, Westphal O (1966) Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae. Bacteriol Rev 30:192–255

  22. Lüderitz O, Freudenberg MA, Galanos C, Lehmann V, Rietschel ET, Shaw DH (1982) Lipopolysaccharides of gram-negative bacteria. Curr Top Memb Trans 17:79–151

  23. Mattsby-Baltzer I, Kaijser B (1979) Lipid A and anti-lipid A. Infect Immun 23:758–763

  24. Mineo JR, Camargo ME, Ferreira AW (1980) Enzyme-linked immunosorbent assay for antibodies to Toxoplasma-gondii polysaccharides in human toxplasmosis. Infect Immun 27:283–287

  25. Müller G, Sjöberg O, Andersson J (1973) Immunogenicity, tolerogenicity, and mitogenicity of lipopolysaccharides. J Infect Dis 128:S52-S56

  26. Mullan NA, Newsome PM, Cunnington PG, Palmer GH, Wilson ME (1974) Protection against Gram-negative infections with antiserum to lipid A from Salmonella minnesota R595. Infect Immun 10:1195–1201

  27. Nikaido H, Nakae T (1979) The outer membrane of Gram-negative bacteria. Adv Microbiol Physiol 20:163–250

  28. Nossal GJV, Ada GL, Austin CM (1964) Antigens in immunity. II. Immunogenic properties of flagella, polymerized flagellin and flagellin in the primary response. Aust J Exp Biol Med Sci 42:283–294

  29. Vaitukaitis J, Robbins JB, Nieschlag E, Ross GT (1971) A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol 33:988–991

  30. Wicken AJ, Knox KW (1977) Immunological properties of lipoteichoic acids. Prog Immunol 3:135–143

  31. Wicken AJ, Knox KW (1980) Bacterial cell surface amphiphiles. Biochim Biophys Acta 604:1–26

  32. Wicken AJ, Gibbens JW, Knox KW (1973) Comparative studies on the isolation of membrane lipoteichoic acid from Lactobacillus fermenti. J Bacteriol 113:365–372

  33. Yolken RH, Wyatt RG, Kim HW, Kapikian AZ, Chanock RM (1978) Immunological response to infection with human reovirus-like agent: measurement of anti-human reovirus-like agent immunoglobulin G and M levels by the method of enzyme-linked immunosorbent assay. Infect Immun 19:540–546

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Correspondence to G. F. Ibrahim.

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Ibrahim, G.F., Lyons, M.J. Production of potent polyclonal antibodies to bacterial membrane amphiphiles. Med Microbiol Immunol 176, 305–313 (1987). https://doi.org/10.1007/BF00194890

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Keywords

  • Lipid
  • Bovine Serum Albumin
  • Polyclonal Antibody
  • Intact Cell
  • Bacterial Membrane