一种使用香豆素免疫吸附剂纯化抗异硫氰酸荧光素抗体的单步方法。

A single-step method for the purification of anti-FITC antibodies by use of a coumarin immunosorbent.

作者信息

Samuel D, Abuknesha R A

机构信息

Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, U.K.

出版信息

J Immunol Methods. 1990 Oct 4;133(1):133-9. doi: 10.1016/0022-1759(90)90327-r.

DOI:10.1016/0022-1759(90)90327-r

PMID:2120348

Abstract

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.

摘要

单克隆抗异硫氰酸荧光素（FITC）抗体与偶联到牛血清白蛋白（BSA）上的7-羟基香豆素衍生物发生交叉反应。这一特性使得可以使用由连接到琼脂糖4B上的7-羟基香豆素衍生物组成的免疫吸附剂从腹水液中亲和纯化单克隆抗FITC抗体。将腹水液应用于免疫吸附柱，洗涤后，使用3M氯化镁在极其温和的条件下洗脱结合的抗体。通过SDS-PAGE评估，以这种方式洗脱的抗体纯度大于96%。还在相同的免疫吸附剂上从血清中纯化了多克隆羊抗FITC抗体，纯度大于94%。这种简单快速的抗FITC抗体纯化方法将在免疫诊断程序和半抗原-抗体相互作用研究中得到应用。通过酶联免疫吸附测定（ELISA）评估了与辣根过氧化物酶偶联的纯化单克隆抗FITC抗体的亲和常数，发现其为1.5×10⁹M⁻¹。