To gauge the proximity between cooperating T and B cells required for effective triggering of antibody production, guinea pigs were immunized with bifunctional antigens in which the haptenic and carrier determinants were separated by rigid spacers of varied dimension. These took the form 2,4-dinitrophenol-(proline)n-L-tyrosine-p-azobenzenearsonate, where n varied from 1 to 40 proline residues. Animals immunized with n = 10 and n = 22 compounds made strong anti-DNP antibody responses, whereas animals immunized with bifunctional compounds containing longer spacers did not make antibody detectable by precipitation. It can be calculated on the basis of very strong physicochemical evidence for the rigidity and axial translation of poly-L-proline chains in solution that the cut-off point for effective interaction between T and B cells lies between 69 and 97 A U.

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