Figure 5
Dose–response curves of competitive ELISAs obtained with the scFvs shown in Fig. 4. The vertical bars indicate SD for intra-assay variance (n = 4). The midpoints of the curves (pg/assay) were as follows: scFv#4mut, 12.6 ± 0.12 [mean ± SD (n = 4)]; #M3rd(amb), 15.0 ± 0.76; #M3rd(Q), 16.8 ± 1.57; #R3-4, 14.3; #R4-2, 15.6; #R4-1, 19.0; #R5-1, 27.6; and #WT, 220 (average of determinations in duplicate). In these assays, the scFv concentrations were adjusted to give bound enzyme activities at B0 (the reaction without E2 standard) of approximately 1.0–1.5 absorbance after a 30-min enzyme reaction. The background absorbance (observed without addition of scFvs) was lower than 5.0% of the B0 absorbance. We should note that, in the original article where scFv#M3rd(amb) (denoted as scFv#m3-a18 therein) was generated14, we reported its Ka value as 1.3 × 1010 M−1, and the midpoint value in the ELISA using this scFv was determined to be 10.0 ± 1.2 pg/assay. In this study, we re-determined the Ka in triplicate. The midpoint values were also re-determined to perform equal and strict comparisons between the scFvs, because we had to use a newly prepared E2–BSA conjugate to coat the ELISA microplates. Difference in the quality of these conjugates (mainly in the hapten/protein molar ratio) influences on the ELISA sensitivity and often makes it difficult to strictly reproduce previous experimental data.
