Allauzen S, Joly S, Granier C, Molina F, Bouix O, Pau B, Bouanani M
CNRS UMR 9921, Faculté de Pharmacie, Montpellier, France.
Mol Immunol. 1995 Jan;32(1):27-36. doi: 10.1016/0161-5890(94)00133-l.
Seven anti-human insulin monoclonal antibodies (mAb) were produced according to an efficient immunization protocol elaborated in our laboratory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M-1 to 1.0 x 10(10) M-1 when insulin was in solution and from 6.0 x 10(6) M-1 to 2.5 x 10(8) M-1 when insulin was adsorbed onto the microtiter plate. The antigenic sites on the insulin molecule recognized by these mAbs were mapped using two approaches. MAb pairs capable of binding simultaneously to human insulin in solution (using a two-site ELISA) or adsorbed onto a microtiter plate (using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on soluble insulin. Two distinct antigenic regions common to both the adsorbed and the soluble forms of insulin were defined by our mAbs. In a second approach, the immunological cross-reactivities of these mAbs with species variants of insulin, chemically modified insulin of known structure and a panel of 78 overlapping nonapeptides covering the entire sequence of human proinsulin were assessed. Evidence was obtained that the epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one group of mAbs (group I) was precisely defined. This group recognized a highly conserved region of the insulin molecule including residues 10-17 of the A chain.
按照我们实验室精心制定的高效免疫方案，制备了7种抗人胰岛素单克隆抗体（mAb）。当胰岛素处于溶液中时，它们与胰岛素分子结合的亲和常数范围为5.0×10⁸ M⁻¹至1.0×10¹⁰ M⁻¹；当胰岛素吸附在微量滴定板上时，亲和常数范围为6.0×10⁶ M⁻¹至2.5×10⁸ M⁻¹。使用两种方法绘制了这些单克隆抗体识别的胰岛素分子上的抗原位点。首先寻找能够同时结合溶液中的人胰岛素（使用双位点ELISA）或吸附在微量滴定板上的人胰岛素（使用竞争性ELISA）的单克隆抗体对。在吸附的人胰岛素表面定义了三个抗原区域，在可溶性胰岛素表面定义了四个抗原区域。我们的单克隆抗体定义了胰岛素吸附形式和可溶性形式共有的两个不同抗原区域。在第二种方法中，评估了这些单克隆抗体与胰岛素物种变体、已知结构的化学修饰胰岛素以及覆盖人胰岛素原整个序列的一组78个重叠九肽的免疫交叉反应性。获得的证据表明，单克隆抗体识别的表位包括胰岛素分子在进化过程中保守的残基。精确确定了一组单克隆抗体（I组）的表位特异性。该组识别胰岛素分子的一个高度保守区域，包括A链的第10 - 17位残基。
