Chen P P, Nonaka M, O'Hair C H, Cohen P A, Zuraw B L, Katz D H
J Immunol. 1984 Oct;133(4):1909-13.
This study was performed to address the controversy concerning human IgE biosynthesis in vitro induced by stimulation with pokeweed mitogen (PWM) or other agents. The controversy has focused on the specificity of reagents employed for quantitatively determining human IgE in culture supernatant fluids. Specifically, questions have been raised as to whether certain anti-human IgE antibody reagents possess anti-idiotypic reactivities, thereby resulting in reactions with Fab determinants of polyclonal immunoglobulins which would yield false-positive readings of IgE protein levels. We present a detailed analysis confirming that the goat anti-human IgE antibody designated GAHE(PS), which was initially isolated by affinity chromatography with the same IgE(PS) myeloma protein used for immunization, binds poorly, if at all, with IgG, IgA, or IgM immunoglobulins, even at excessive concentrations (100 micrograms/ml). Moreover, GAHE(PS) displayed no reactivity with Fab fragments of IgG or free L-chains prepared from pooled polyclonal IgG isolated from Cohn fraction II. A second GAHE reagent was prepared by purification by affinity chromatography on a second, completely unrelated IgE myeloma protein (DZA), which differed from IgE(PS) in light chain class, thereby resulting in a reagent, designated GAHE(DZA), which was completely devoid of any possible reactivity with L-chain or idiotypic determinants affiliated with IgE(PS). By utilizing both reagents, the studies presented here confirmed that PWM-stimulated human lymphoid cell cultures synthesize increased quantities of IgE, which can be detected in comparable amounts by both GAHE(DZA) and GAHE(PS) in supernatant fluids from such cultures. Because incorporation of the reversible protein synthesis inhibitor, cycloheximide, totally abolished the PWM-induced increases in IgE levels in such cultures, these results verify that such increases reflect de novo synthesis of human IgE as a result of PWM stimulation in vitro.
本研究旨在解决关于体外用人商陆丝裂原（PWM）或其他试剂刺激诱导人IgE生物合成的争议。该争议聚焦于用于定量测定培养上清液中人IgE的试剂的特异性。具体而言，有人质疑某些抗人IgE抗体试剂是否具有抗独特型反应性，从而导致与多克隆免疫球蛋白的Fab决定簇发生反应，这会产生IgE蛋白水平的假阳性读数。我们进行了详细分析，证实最初通过与用于免疫的相同IgE(PS)骨髓瘤蛋白进行亲和层析分离得到的山羊抗人IgE抗体GAHE(PS)，即使在过高浓度（100微克/毫升）下，与IgG、IgA或IgM免疫球蛋白的结合也很差，甚至根本不结合。此外，GAHE(PS)与从Cohn组分II中分离的混合多克隆IgG制备的IgG Fab片段或游离轻链没有反应性。通过在第二种完全不相关的IgE骨髓瘤蛋白（DZA）上进行亲和层析纯化制备了第二种GAHE试剂，该蛋白在轻链类别上与IgE(PS)不同，从而产生了一种名为GAHE(DZA)的试剂，它与与IgE(PS)相关的轻链或独特型决定簇完全没有任何可能的反应性。通过使用这两种试剂，本研究证实PWM刺激的人淋巴细胞培养物合成了增加量的IgE，在这种培养物的上清液中，GAHE(DZA)和GAHE(PS)都能检测到相当数量的IgE。由于加入可逆性蛋白质合成抑制剂环己酰亚胺完全消除了这种培养物中PWM诱导的IgE水平升高，这些结果证明这种升高反映了体外PWM刺激下人IgE的从头合成。
