The titer of high affinity anti-trinitrophenyl antibodies can be determined by two different spectroscopic methods. A fluorescence approach, based on the approx 10-fold increase in fluorescence of the probe, 1,1-dihydro, 2,4,6-trinitrocyclohexadienate, on binding to homologous antibody has been used to titrate both specifically purified antibodies and an IgG subfraction. We have simplified a previously published method, based on the observation that these antibodies cause the breakdown of complexes formed between trinitrophenyl amino acids and sulfite. A study of the kinetic aspects of this latter reaction indicates the rate-determining step in the reaction is the dissociation of the hapten-sulfite complex. This suggests the antibody can bind to free hapten, but not to the hapten-sulfite complex.
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