A single-step method for the purification of anti-FITC antibodies by use of a coumarin immunosorbent.
Monoclonal anti-fluorescein isothiocyanate (FITC) antibody cross-reacts with 7-hydroxy coumarin derivatives conjugated to BSA. This property permitted the affinity purification of monoclonal anti-FITC antibodies from ascitic fluid using an-immunosorbent consisting of a 7-hydroxy coumarin derivative linked to Sepharose 4B. Ascitic fluid was applied to the immunosorbent column and, after washing, the bound antibody was eluted under extremely mild conditions using 3 M MgCl2. Antibody eluted in this manner was greater than 96% pure as assessed by SDS-PAGE. A polyclonal sheep anti-FITC antibody was also purified from serum on the same immunosorbent to greater than 94% purity. This simple and rapid method for the purification of anti-FITC antibodies will find applications in both immunodiagnostic procedures and in studies of hapten-antibody interactions. The affinity constant of the purified monoclonal anti-FITC antibody conjugated to horseradish peroxidase was assessed by ELISA and was found to be 1.5 x 19(9) M-1.
Samuel
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Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, U.K.
Abuknesha
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RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins; D012710:Serum Albumin, Bovine; D012756:Sheep; D013861:Thiocyanates; D014468:Umbelliferones / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D013861:Thiocyanates; D014468:Umbelliferones; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate