Conrad D H, Studer E, Gervasoni J, Mohanakumar T
Int Arch Allergy Appl Immunol. 1983;70(4):352-60. doi: 10.1159/000233347.
Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.
通过用大鼠IgE免疫BALB/c小鼠并将脾细胞与非分泌性骨髓瘤P3/X63Ag8.653融合，制备了针对大鼠IgE的Fc和Fab部分的杂交瘤抗体。其中两种抗体，命名为A2和B5，得到了广泛的表征。使用来自IR 162和IR2免疫细胞瘤的大鼠IgE以及大鼠IgG进行的竞争性结合实验表明，A2和B5都是ε链特异性的，而不是抗独特型的。A2还与小鼠IgE表现出一些交叉反应性。当用胰凝乳蛋白酶处理IgE以产生F(ab')2和Fab片段时，经酶处理的IgE与B5保持反应性，但与A2的反应性丧失。在56℃下对IgE进行热变性导致IgE对A2和大鼠嗜碱性白血病细胞上的Fc受体的反应性逐渐丧失；B5的反应性保持不变。A2显然不与IgE的Fc上与大鼠嗜碱性白血病细胞Fc受体结合所涉及的相同位点相互作用；A2对IgE与大鼠嗜碱性白血病细胞的结合影响很小。因此，数据表明B5的抗原位点在IgE分子的Fab区域，而A2与IgE Fc反应。在双标记实验中也评估了使用这些抗体测量细胞结合IgE的情况，并讨论了使用二价抗体定量细胞表面抗原时的潜在问题。
