Gupta R K, Siber G R
Massachusetts Public Health Biologic Laboratories, State Laboratory Institute, Boston, MA 02130, USA.
J Immunol Methods. 1995 Apr 12;181(1):75-81. doi: 10.1016/0022-1759(94)00331-p.
One of the methods for calibration of antigen specific antibodies in a serum involves parallel titration of known concentrations of purified normal immunoglobulins (Igs) and a specific antiserum. We evaluated the effect of three methods for capturing known concentrations of purified normal mouse Igs (IgG, IgG subclasses and IgM) on the anti-tetanus toxoid antibody concentrations assigned to a reference mouse serum: (1) direct coating, (2) capture by pre-coated anti-mouse IgG (Fc) specific antibodies, and (3) capture by pre-coated anti-mouse IgG (Fab) specific antibodies. Direct coating of purified Igs onto plastic was the least efficient and would greatly overestimate antigen specific antibody concentrations. Equivalent concentrations of purified IgG subclasses and IgM produced higher absorbances when captured by anti-Fab antibodies pre-coated on plates than by anti-Fc antibodies. As a consequence, the anti-Fc capture method resulted in the assignment of IgG1, IgG2a and IgG2b tetanus specific antibody concentrations which exceeded the total IgG1, IgG2a and IgG2b concentrations. Additionally, the sum of tetanus specific IgG subclass antibodies exceeded the tetanus specific IgG antibody concentration by > 2-fold. In contrast, the anti-Fab capture method resulted in 2-9-fold lower assigned tetanus antibody concentrations which did not exceed the total Ig concentrations. We conclude that when using enzyme-linked immunosorbent assays, parallel titration of purified normal Ig standards captured by anti-Fab antibodies is superior to anti-Fc capture or direct coating for calibrating antigen specific antibodies in a reference serum.
血清中抗原特异性抗体校准的方法之一是对已知浓度的纯化正常免疫球蛋白（Ig）和特异性抗血清进行平行滴定。我们评估了三种捕获已知浓度纯化正常小鼠Ig（IgG、IgG亚类和IgM）的方法对参考小鼠血清中抗破伤风类毒素抗体浓度的影响：（1）直接包被，（2）用预包被的抗小鼠IgG（Fc）特异性抗体捕获，以及（3）用预包被的抗小鼠IgG（Fab）特异性抗体捕获。将纯化的Ig直接包被在塑料上效率最低，会极大地高估抗原特异性抗体浓度。当用预包被在板上的抗Fab抗体捕获时，纯化的IgG亚类和IgM的等效浓度产生的吸光度高于用抗Fc抗体捕获时。因此，抗Fc捕获法导致指定的IgG1、IgG2a和IgG2b破伤风特异性抗体浓度超过了总的IgG1、IgG2a和IgG2b浓度。此外，破伤风特异性IgG亚类抗体的总和超过破伤风特异性IgG抗体浓度2倍以上。相比之下，抗Fab捕获法导致指定的破伤风抗体浓度低2至9倍，且未超过总Ig浓度。我们得出结论，在使用酶联免疫吸附测定时，用抗Fab抗体捕获的纯化正常Ig标准品进行平行滴定，在校准参考血清中的抗原特异性抗体方面优于抗Fc捕获或直接包被。
