Comparison of methyl-p-hydroxybenzimidate and 1,3,5-trichlorotriazine in producing sensitive target cells for use in the hemolytic spot assay

Two bifunctional reagents were shown to be useful in the coupling of immunogens to the surface of either nucleated or non-nucleated cells. The heterobifunctional reagent methyl-p-hydroxybenzimidate was used to couple aromatic amino-haptens to the surface of SRBC which resulted in a stable and sensitive target cell capable of detecting as little as 20 pg of purified anti-hapten antibody in the hemolytic spot assay. The multifunctional reagent 1,3,5-trichlorotriazine yielded similar results when coupling amino-haptens to the surface of SRBC for use in the hemolytic spot assay. This reagent was also used to couple protein to the surface of SRBC which were able to detect as low as 1 ng of purified anti-protein antibody in the hemolytic spot assay. The sensitized SRBC produced using either of these coupling reagents were shown to remain stable for several months giving reproducible results from one test to another. Lastly, 1,3,5-trichlorotriazine was used to couple the hapten 4-aminophthalate to the surface of nucleated cells with retention of >90% cell viability with continued growth and cellular division in culture.