The Kinetics of the Reactions Between Antibodies to the 2, 4 Dinitrophenyl Group and Specific Haptens.*

It has been realized for some time that the bjmolecular reaction of an antibody molecule with its specific antigen or hapten occurs at a very rapid rate.Until recently, only estimates of absolute rate constants have been published.If rate constants could be determined with an accuracy of a few percent, an accuracy hitherto unattained with such systems, new insight might result into the mechanism of the antigen-antibody reaction and the nature of biochemical specificity generally.To obtain kinetic data of such accuracy, three essential requirements must be met: ( a ) The antigen-antibody system must be as well-defined as possible.The concentrations of the reacting species must be accurately known.The bimolecular combination reaction must be isolated and distinguished from aggregation and disaggregation reactions; ( b ) some property of the system must undergo a kinetically-correlated and accurately measurable change upon combination of the antigen and antibody; ( c ) some means of following this change over very short time intervals must be available.The first requirement can best be met by utilizing pure antihapten antibodies with their specific univalent haptens.Protein antigens, with their unknown valences, the heterogeneity of their antigenic sites, and the multitude of aggregation and disaggregation reactions they undergo with their specific antibodies, are generally unsatisfactory for such investigations.The second requirement would most conveniently be met if some substantial spectral change were to accompany a given hapten-antibody reaction.In most cases so far studied, however, the absorption spectrum of a colored hapten is only very little altered on combination with its specific antibody.One exception to this was discovered by Wofsy et nZ.1.2These authors found that the dye hapten, DNPNS, undergoes a marked spectral change at pH-7 on binding to antibodies specific to the DNP hapten, and that this change is associated with a pK shift of the naphtholic OH group of the dye from -6.5 in the free state to -9.0 when bound to a n t i b ~d y .~ This finding permitted preliminary spectrophotometric rate measure-ments2$ to be made with this hapten-antibody system.By suitable molecular design, it may be possible to duplicate this pK and spectral shift quite * Abbreviations : DNP-2,4 dinitrophenyl; DNPNS-2 ( 2,4 dinitrophenylazo ) -1- naphthol-3,6 disulfonic acid.f Contribution No. 1723. 4The assuniption is made in such a case that the rate-limiting step is the haptenantibody combination, and not the protonation of the hapten.