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

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. Author's note. As we were in process of submitting this manuscript, we became aware of an abstract which indicated that responses of similar magnitude to those reported here with TNP could be obtained in Balb/c mice to DNP using a DNP-BSA-SRBC plaquing reagent. [H. Yamada and A. Yamada, Federation Proc. 28, 428, (1969)]. We wish to express our appreciation to Dr. B. Merchant and B. Mackler for helpful advice at the inception of this project and to W. W. Bullock for valuable discussion. We also appreciate the excellent assistance of L. P. Elwell.