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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.

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Selected References

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

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