Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Abstract

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.

Use of a Biomimetic Peptide in the Design of a Competitive Binding Assay for Biotin and Biotin Analogues

“…Because of this similarity, one could propose that biocytin, like biotin, would bind much more strongly to streptactin than Strep-tag II-BAP and that the dose-response curves for biocytin and biotin would be similar on the basis of theoretical predictions outlined in Ref. (8). This is clearly demonstrated in Fig.…”

“…For a weakly binding ligand, one would expect a less steep slope of the doseresponse curve compared to biotin, since the ratio K*/K is now higher (8). As can be seen in the figure, the desthiobiotin dose-response curve has a broader dynamic range, a less steep slope, and a higher detection limit (2 ϫ 10 Ϫ6 M) than biotin and biocytin.…”

“…The K d for biotin and streptavidin is 10 Ϫ15 M, whereas that for Strep-tag II is 7.24 ϫ 10 Ϫ5 M. Even if the K d for biotin and streptactin were on the order of 10 Ϫ9 M, one would expect that a binding assay for biotin using the Strep-tag II-BAP conjugate would have a small dynamic range, with a steep slope. Bachas and Meyerhoff have demonstrated theoretically that the difference between the binding constants of the free ligand (K) and the labeled ligand (K*) can have a significant effect on the shape of the dose-response curve, provided that this difference is large enough (8). The steepness of the slope of the dose-response curve was shown to increase with a decrease in the ratio K*/K.…”

“…A more desirable scenario would be one in which the enzyme-ligand conjugate contains one attached ligand per enzyme subunit at a predetermined position. The fact that oligosubstituted enzyme-ligand conjugates result in better sensitivity and detection limits than multiply substituted conjugates has been demonstrated both theoretically (8) and experimentally (9,10).…”

Enzyme-Linked Competitive Binding Assays Based on the Biotin/Avidin Interaction

“…The dose response behavior of the competitive binding assay is also affected by the ratio of binder/enzyme conjugate employed in the assay mixture, as demonstrated both theoretically 10 and experimentally. 11,12 In the present work, three different ratios of the ALPbiotin and avidin concentrations were selected; these yield a similar extent of analytical signal for each dose-response curve (i.e., curves C, D and E in Fig.…”