Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Apr;78(4):2493–2496. doi: 10.1073/pnas.78.4.2493

Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.

Y H Jou, R B Bankert
PMCID: PMC319373  PMID: 7017733

Abstract

An efficient technique has been developed for coupling protein antigens to erythrocyte membranes. The procedure involves three steps. First, 3-(2-pyridyldithio)propionyl residues are introduced into the protein by reaction with a heterobifunctional reagent, N-succinimidyl 3-(pyridyldithio) propionate. Second, the addition of disulfide groups to sheep erythrocytes (SRBC) is achieved by coupling dithiodiglycolic acid to SRBC with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The disulfide bonds of the dithiodiglycolyl-SRBC conjugate are then reduced with dithiothreitol. Finally, the 3-(2-pyridyldithio)propionyl-protein conjugate is covalently coupled to the thiolated SRBC through thiol/disulfide exchange to form the disulfide-linked antigen-SRBC conjugate. The procedure requires only 10-500 microgram of protein antigen for the preparation of 50 microliter of packed protein-coupled SRBC. Antibodies binding to antigen on the erythrocyte initiate a complement-dependent immune lysis of the target cells. Target cells prepared by this method are stable for at least 4 wk at 4 degrees C in phosphate buffer (pH 7.2) and are capable of detecting as little as 40 pg of antibody in a hemolytic assay without noticeable nonspecific lysis.

Full text

PDF
2496

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BOYDEN S. V. The adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination by antiprotein sera. J Exp Med. 1951 Feb;93(2):107–120. doi: 10.1084/jem.93.2.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bankert R. B., DesSoye D., Powers L. Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay. J Immunol Methods. 1980;35(1-2):23–32. doi: 10.1016/0022-1759(80)90147-7. [DOI] [PubMed] [Google Scholar]
  3. Bankert R. B., Mayers G. L., Pressman D. Mechanisms of B cell tolerance. I. Tolerance to dextran B1355 induced with the oxidized dextran. J Immunol. 1977 Apr;118(4):1265–1270. [PubMed] [Google Scholar]
  4. Bankert R. B., Mayers G. L., Pressman D. Synthesis of a lipopolysaccharide designed to conjugate haptens and proteins to erythrocyte for use as target cells in passive hemagglutination and hemolytic assays. J Immunol. 1979 Dec;123(6):2466–2474. [PubMed] [Google Scholar]
  5. Carlsson J., Drevin H., Axén R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem J. 1978 Sep 1;173(3):723–737. doi: 10.1042/bj1730723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Citkowitz E. The hyaline layer: its isolation and role in echinoderm development. Dev Biol. 1971 Mar;24(3):348–362. doi: 10.1016/0012-1606(71)90085-6. [DOI] [PubMed] [Google Scholar]
  7. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  8. GYENES L., SEHON A. H. THE USE OF TOLYLENE-2,4-DIISOCYANATE AS A COUPLING REAGENT IN THE PASSIVE HEMAGGLUTINATION REACTION. Immunochemistry. 1964 Apr;1:43–48. doi: 10.1016/0019-2791(64)90055-2. [DOI] [PubMed] [Google Scholar]
  9. Gold E. R., Fudenberg H. H. Chromic chloride: a coupling reagent for passive hemagglutination reactions. J Immunol. 1967 Nov;99(5):859–866. [PubMed] [Google Scholar]
  10. Golub E. S., Mishell R. I., Weigle W. O., Dutton R. W. A modification of the hemolytic plaque assay for use with protein antigens. J Immunol. 1968 Jan;100(1):133–137. [PubMed] [Google Scholar]
  11. Jerne N. K., Henry C., Nordin A. A., Fuji H., Koros A. M., Lefkovits I. Plaque forming cells: methodology and theory. Transplant Rev. 1974;18:130–191. doi: 10.1111/j.1600-065x.1974.tb01588.x. [DOI] [PubMed] [Google Scholar]
  12. Johnson H. M., Brenner K., Hall H. E. The use of a wate-soluble carbodiimide as a coupling reagent in the passive hemagglutination test. J Immunol. 1966 Dec;97(6):791–796. [PubMed] [Google Scholar]
  13. LING N. R. The coupling of protein antigens to erythrocytes with difluorodinitrobenzene. Immunology. 1961 Jan;4:49–54. [PMC free article] [PubMed] [Google Scholar]
  14. Lee C. I., Wang M. C., Murphy G. P., Chu T. M. A solid-phase fluorescent immunoassay for human prostatic acid phosphatase. Cancer Res. 1978 Sep;38(9):2871–2878. [PubMed] [Google Scholar]
  15. Mayers G. L., Bankert R. B. Immunochemistry of monoclonal antibodies. Transplant Proc. 1980 Sep;12(3):413–416. [PubMed] [Google Scholar]
  16. ROWLEY D. A., FITCH F. W. HOMEOSTASIS OF ANTIBODY FORMATION IN THE ADULT RAT. J Exp Med. 1964 Dec 1;120:987–1005. doi: 10.1084/jem.120.6.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stuchbury T., Shipton M., Norris R., Malthouse J. P., Brocklehurst K., Herbert J. A., Suschitzky H. A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem J. 1975 Nov;151(2):417–432. doi: 10.1042/bj1510417. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES