Some methodologic aspects of the chromium chloride method for coupling antigen to erythrocytes.

The influence of various methodological variables on the CrCl3 technique for coupling antigen to the surface of erythrocytes has been investigated. Tests should be performed in protein- and phosphate-free medium. The CrCl3 stock solution should be stored at a pH of 5.0 and working dilultions prepared in acetate buffer (pH 5.5). The coupling procedure itself was performed as described by Goding (1976) with slight modifications. The relation between antigen and CrCl3 concentrations was found to be of crucial importance as excess of antigen inactivates CrCl3 whereas lack of antigen or excess of CrCl3, leads to spontaneous agglutination. A preliminary test based on determination of the minimum concentration of CrCl3 which produces spontaneous agglutination in the absence of antigen, and coupling if antigen is available, can be employed to predict the optimal concentration with sheep, human or chicken red blood cells. The pH-dependency of the coupling process is emphasized.