Effects of the competitor on antibody-hapten binding in immunoassays.
The effects of competitors on antibody (Ab)-hapten binding in an immunoassay were investigated using a goat antimethamphetamine (MA) antibody (Ab). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with keyhole limpet hemocyanine (KLH) and used as an immunogen. The antiserum was purified by affinity chromatography with various ligands, including 4-ABMA-protein conjugates, free haptens, and protein G. Direct and indirect competitive enzyme-linked immunosorbent assays (ELISA) were conducted with a competitor of 4-ABMA-fluorescein isothiocyanate (4-ABMA-FITC). The results were compared to those of ELISA with a different competing antigen, 4-ABMA-ovalbumin (4-ABMA-OVA), in terms of sensitivity and specificity. In both direct and indirect assay formats, the sensitivity was much improved with 4-ABMA-FITC, compared to that with 4-ABMA-OVA, suggesting that different labels on the same haptenic moiety for competitors considerably influence the assay performance. All the purified Abs also showed a distinct feature of strong affinity for benzphetamine with 4-ABMA-FITC, whereas they had their respective binding specificities with 4-ABMA-OVA. Comparing the results to those from other assay systems, we determined that the assay sensitivity was dependent on both the system and the competitor employed, and that the specificity was primarily dependent on the competitor used.
Choi
Jeongeun
J
Bioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology, Seoul. jechoi@physiology.wisc.edu
Kim
Choonmi
C
Choi
Myung Ja
MJ
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006241:Haptens / Q000276:immunology*; D012680:Sensitivity and Specificity
D000906:Antibodies; D006241:Haptens