Influence of the antibody purification method on immunoassay performance: hapten-antibody binding in accordance with the structure of the affinity column ligand.
The effects of ligands for immunoaffinity chromatography on the immunoassay were investigated with three goat anti-methamphetamine (anti-MA) antibodies (Abs). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with proteins and used as immunogens. All the antisera produced were purified by affinity chromatography with various ligands of 4-ABMA-proteins and of haptens as well as protein G: 4-ABMA-bovine serum albumin (4-ABMA-BSA), 4-ABMA-keyhole limpet hemocyanine (4-ABMA-KLH), 4-ABMA-ovalbumin (4-ABMA-OVA), MA, 4-ABMA, and amphetamine were used as ligands. Enzyme-linked immunosorbent assay (ELISA) was conducted to examine characteristics of the purified Abs with the 4-ABMA-OVA competitor coated. The results obtained revealed that characters of the purified Abs were closely related with chemical structures of ligands used. The Abs from the MA and the amphetamine columns showed better sensitivities than those from the others in each antiserum. Particularly, the Ab from the amphetamine column gave the best results in terms of sensitivity and specificity. The recognition or the affinity of the Ab selected was considered to be affected by the structure of the ligand concerned. These results suggest that the Ab purification method should be considered as an important parameter which has great influence on the performance of immunoassays with polyclonal Abs.
Choi
J
J
Bioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology, Seoul, 130-650, Korea. jechoi@kistmail.kist.re.kr
Kim
C
C
Choi
M J
MJ
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006041:Goats; D006241:Haptens; D006433:Hemocyanins / Q000032:analysis / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D008694:Methamphetamine / Q000031:analogs & derivatives / Q000032:analysis* / Q000276:immunology; D010047:Ovalbumin / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D006241:Haptens; D012710:Serum Albumin, Bovine; D008694:Methamphetamine; C006508:N-(4-aminobutyl)methamphetamine; D010047:Ovalbumin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin