Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues. Quantitation and specificity of binding.

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from . Cytophilic antibody as the cause of antigen binding was excluded. The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood. Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.