The chromic chloride method of coupling antigens to erythrocytes: definition of some important parameters

Factors affecting the efficiency of the chronic cloride method of coupling antigen to erythrocytes were studied using a radioactive tracer. The amount of chromic chloride required for optimal coupling was related to the concentration of protein in the reaction mixture. Coupling was considerably more efficient when the chromic chloride solution was allowed to 'age' prior to use. In the system studied, the addition of piperazine buffer made little difference to the degree of coupling. The reaction was shown to proceed very rapidly. 50% of the final uptake of antigen took place within seconds, and the reaction was essentially complete 5 min after initiation. The importance of adequate mixing during the addition of the chromic chloride solution was emphasised.