Abstract
A combination rosette-plaque assay was used to demonstrate that most (92%) of the plaque-forming cells (PFC) bound hapten to their surface 5 to 8 days after one injection of 3-nitro-4-hydroxy-5-iodophenyl acetic acid (NIP) coupled to diphtheria toxoid, (NIP-TOX). There was a significant decrease in hapten binding by these cells later in the primary response. Rosette formation was observed when NIP-TOX-primed lymph node cells bound NIP-coupled sheep red blood cells stabilized with pyruvic aldehyde (NIP/PASRBC). This suspension of cells was then plaqued on fresh NIP-coupled sheep red blood cells (NIP/SRBC). A rosetted lymph node cell in the center of a plaque clearly demonstrated simultaneous hapten binding and anti-hapten antibody secretion. The specificity of hapten binding and antibody secretion was established by rosette and plaque inhibition with NIP coupled to bovine serum albumin (NIP-BSA). The specificity of each was further characterized by comparative inhibition with analogues of NIP (also coupled to BSA): 3,5-dinitro-4-hydroxyphenyl acetic acid (NNP-BSA), 3,5-diiodo-4-hydroxyphenyl acetic acid (DIP-BSA) and 3-nitro-4-hydroxyphenyl acetic acid (NP-BSA). The homologous hapten NIP was always the best inhibitor of plaques (97.8% inhibition) and rosettes (96.2% inhibition). NNP was always a better plaque and rosette inhibitor than either DIP or NP. This hierarchy of inhibition, and the correlation between rosette and plaque inhibition demonstrated the similarity of receptor and antibody specificity.
