PMID: 7017733Apr 1, 1981Paper

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

Proceedings of the National Academy of Sciences of the United States of America
Y H Jou, Richard B Bankert

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.

References

Jan 1, 1974·Transplantation Reviews·N K JerneI Lefkovits
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Dec 1, 1964·The Journal of Experimental Medicine·D A ROWLEY, F W FITCH

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Citations

May 9, 1986·Biochimica Et Biophysica Acta·D M Wojchowski, A J Sytkowski
Oct 1, 1982·Developmental Biology·B L Hylander, R G Summers
Jun 11, 1982·Journal of Immunological Methods·C S OwenP H Maurer
Apr 21, 1989·Journal of Immunological Methods·D KokkinopoulosM Papamichail
Feb 1, 1983·The Journal of Experimental Medicine·S S Chen, D H Katz
Jan 1, 1982·Annals of the New York Academy of Sciences·C L LeeT M Chu
Sep 28, 2005·Clinical Immunology : the Official Journal of the Clinical Immunology Society·Qiang LouRichard B Bankert
Dec 16, 1983·Biochemical and Biophysical Research Communications·D CantenysM Massol
Apr 1, 1990·Perceptual and Motor Skills·F C SeitzR Quam
Apr 18, 1985·Nature·U D StaerzM J Bevan
Jul 20, 2013·Biomacromolecules·Jason KerrAndrea M Kasko

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