Antitrinitrophenyl (TNP) Plaque Assay. Primary Response of Balb/c Mice to Soluble and Particulate Immunogen

TLDR

The study developed a hemolytic plaque assay to detect individual anti‑TNP antibody‑producing cells. TNP was conjugated to erythrocytes via TNBS to create a stable reagent for assessing anti‑hapten responses to TNP‑KLH. Soluble TNP‑KLH elicited a primary anti‑TNP response that was stronger when the immunogen was particulate on bentonite; TNP‑BSA inhibited both primary and secondary antibody‑producing cells, but only secondary cells were fully suppressed at equivalent hapten levels, indicating higher affinity of secondary antibodies.

Abstract

A technique was developed for the detection of individual cells producing anti-TNP antibody by the hemolytic plaque technique. Conjugation of TNP directly to the erythrocyte surface by use of TNBS resulted in a stable reagent that permitted a study of the antihapten response to TNP-KLH. It was possible to induce a primary anti-TNP response with soluble TNP-KLH but the response was greater when the immunogen was made particulate by coating it onto bentonite. Both primary and secondary responding cells (those brought out by antiglobulin serum) were inhibited by TNP-BSA added to the plating medium but at an equivalent concentration of hapten only the secondary cells were completely inhibited. This was interpreted to indicate the higher binding affinity of secondary antibody.