Specific Excitation Energy Transfer from Antibodies to Dansyl‐Labeled Antigen

Abstract

The "loop') peptide region of hen egg-white lysozyme was labeled with the dansyl group. The binding of the labeled peptide to the anti-"loop" antibodies led to a specific excitation energy transfer from the antibody to the dansyl group on the hapten. This sensitized fluorescence was used for quantitative evaluation of the binding and homogeneity parameters of the "loop"-anti-"loop" interaction. The association constant observed was 3.2 x lo6 M-l and the homogeneity index interpreted as a result of excitation energy transfer to these haptens, and was used for quantitative studies of the affinity and heterogeneity of the antibodies.More recently, antibodies to the dansyl group have also been prepared [13], and the fluorescence behaviour of the antibody-bound hapten yielded information about the nature of the environment of the binding site [14]. I n these studies the antibody specificity was directed to determinants in which the dansyl group played an immunodominant role.Unusual Abbreviations. Dansyl, l-dimethylaminonaphthalene-5-sulfonyl; "loop", amino acid sequence 60 to 83 of lysozyme, containing a disulfide bridge between half-cystine residues 64 and 80.Enzyme. Lysozyme or mucopeptide N-acetylneuraminylhydrolase (EC 3.2.1.17).

Antibodies Reactive with Native Lysozyme Elicited by a Completely Synthetic Antigen

“…In the transfer reaction of the excitation energy from the antibodies to the dansylated loop, antibodies to the synthetic loop bring about the same enhancing effect as the antibodies to the natural loop. When the data obtained by this technique were quantitatively analyzed, the homogeneity index and the association constant of the antibodies to the synthetic loop were found to be quite similar to the values reported (20) for antibodies to the natural loop. Likewise, the specificity of the reactions with the two types of antibodies is identical (Fig.…”

“…The association constants of two preparations of the antisynthetic loop antibodies were 1.1 X 106 and 0.6 X 106 M-1, the homogeneity indices 0.88 and 1.05, respectively. A value of 3.2 X 106 M-l was previously (20) The increase in fluorescence induced by the interaction with the antibodies could be specifically inhibited by either lysozyme or the loop peptide, as shown in Fig. 6, but not by the open-chain loop peptide.…”

“…5. From the data of this fluorometric titration, the binding parameters of these antibodies were calculated (20). The association constants of two preparations of the antisynthetic loop antibodies were 1.1 X 106 and 0.6 X 106 M-1, the homogeneity indices 0.88 and 1.05, respectively.…”

“…In a previous publication (20) we have reported that, by interaction of anti-loop antibodies with a loop preparation dansylated at its amino terminus, an excitation energy transfer from the antibodies to the dansyl group is observed, which is manifested in enhanced fluorescence. The same effect is brought about by the antibodies to the synthetic loop, as shown in Fig.…”

“…Each of the modified bacteriophage preparations (about 1000 plaqueforming units) was incubated for 2 hr with different dilutions of the antiserum, and was subsequently plated with bacteria for monitoring of the surviving phages. Fluorescence measurements and fluorometric titrations were performed (20) with a dansylated loop preparation with the aid of the Turner Spectrofluorometer, model 210.…”