We developed a two-step ELISA to determine the immunoreactive fraction of monoclonal antibodies in conditions of antigen excess. An antibody aliquot at limiting dilution was incubated in wells coated with increasing amounts of antigen up to concentrations that bind 100% of antibody. At equilibrium, a supernatant aliquot was transferred to a second plate coated with excess of antiglobulin, and the captured antibody was incubated with peroxidase-conjugated anti-IgG. Antibody was quantitated from the enzyme velocity gradient in a kinetic ELISA, and the immunoreactive fraction calculated as (1 - gradienti/gradientT) x 100, where i and T are the gradients for the free and total antibody fractions. For four distinct monoclonal antibodies (anti-diphtheria toxoid, -cholera toxin, -bovine serum albumin (BSA), and -trinitrophenyl-BSA), measurement of inter-assay variability yielded values ranging from 3.1 to 7.4 (% coefficient of variation), which supports method repeatability. This ELISA is simple, precise, and applicable to mono- and polyclonal antibodies.

中文翻译：

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用于确定单克隆抗体免疫反应部分的动力学ELISA。

我们开发了两步ELISA法来确定在抗原过量的条件下单克隆抗体的免疫反应性分数。将有限稀释的抗体等分试样在用逐渐增加量的抗原包被的孔中孵育，直至达到可结合100％抗体的浓度。在平衡时，将上清液等分试样转移至用过量的抗球蛋白包被的第二板中，并将捕获的抗体与过氧化物酶偶联的抗IgG一起孵育。在动力学ELISA中根据酶速度梯度对抗体进行定量，并且将免疫反应性分数计算为（1-gradienti / gradientT）×100，其中i和T是游离和总抗体部分的梯度。对于四种不同的单克隆抗体（抗白喉类毒素，-霍乱毒素，-牛血清白蛋白（BSA）和-三硝基苯基-BSA），测定批间变异性得出的值介于3.1到7.4（变异系数百分比）之间，支持方法的可重复性。此ELISA简单，精确，适用于单克隆和多克隆抗体。