MEASUREMENT OF MONOCLONAL-ANTIBODY AFFINITY BY NONCOMPETITIVE ENZYME-IMMUNOASSAY
MEASUREMENT OF MONOCLONAL-ANTIBODY AFFINITY BY NONCOMPETITIVE ENZYME-IMMUNOASSAY
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DOI：
10.1016/0022-1759(87)90187-6
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发表时间：
1987-06-26
影响因子：
2.2
通讯作者：
VLAHOS, WG
中科院分区：
文献类型：
--
作者：
BEATTY, JD;BEATTY, BG;VLAHOS, WG
酶联免疫吸附试验(EIA)广泛应用于抗体浓度的测定。抗体的亲和常数(Kaff)对EIA法定量有影响。因此，能够用固相环评测定卡夫)是很重要的。根据质量作用定律，使用抗原(包被平板)和抗体的系列稀释液，用EIA法测定了Kaff。用抗原(Ag)包被微量滴度板，然后与单抗(Ab)孵育。将平板依次与第二种酶-抗体结合物(EAC)和酶底物孵育。用OD法测得的酶产物反映了抗体在平板上[Ag-Ab]和[Ag2-Ab]上的粘附量。使用连续稀释的抗体导致OD与井中加入的总抗体的对数之间的S型曲线。不同抗体在上高原的OD(OD-100)的比较反映了不同抗体识别的抗原表位的相对数量，前提是过量使用EAC。[AB]t和[Ab‘]t分别是涂有[Ag]和[Ag’‘]的平板在OD-50和OD-50’‘时井中可测的总抗体浓度。[AG]和[Ag‘’]不是真正的抗原浓度，而是平板上抗原密度的测量。对于[Ag‘’]=[Ag]/2，Kaff=1/2(2[AB‘’]t-[AB]t)。用5种不同的抗CEA抗体和包被液中不同比例的CEA测定Kaff。EIA法测定的Kaff与可溶性相抑制法测定的Kaff具有良好的相关性。该方法具有简便、快速、可靠的特点。
Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (Kaff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure Kaff) by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigen (coating the plate) and antibody, Kaff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag2 Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab]t and [Ab'']t were the measurable total antibody concentrations in the wells at OD-50 and OD-50'' for plates coated with [Ag] and [Ag''], respectively. [Ag] and [Ag''] were not true antigen concentrations, but were a measure of antigen density on the plate. For [Ag'']=[Ag]/2, Kaff=1/2(2[Ab'']t-[Ab]t). Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, Kaff was measured. Kaff determined by EIA correlated well with Kaff measured by soluble phase inhibition assay. This EIA method of estimation of Kaff is simple, rapid, and reliable.