The use of homocarnosine as a spacer in indirect haemagglutination

Homocarnosine (gamma-butyryl-L-histidine) was bound to erythrocytes through the action of glutaraldehyde in order to increase the number of available groups for diazotization on the surface of the cells. Diazotization was performed with tetrazotizized di-o-anisidine (TOD). TOD decomposed rapidly at an alkaline pH. The reagent was stabilized by the use of borate buffers. The presence of homocarnosine on erythrocytes enhanced the coupling of histidine by diazotization. TOD formed bonds of varying lability with alifatic amino and imino groups. This inhibited completely the formation of stable bonds with histidine and tyrosine. The relative amounts of stable bonds formed by TOD and human serum albumin and TOD and gamma-globulin varied inversely with the concentration of the proteins. The two proteins were used as model antigens and the presence of homocarnosine on the surface of the erythrocytes increased the sensitivity of the haemagglutination reaction by a factor of 4-8.