Papouchado M L, Ermácora M R, Poskus E
Cátedra de Immunología-Instituto de Estudios de la inmunidad Humoral, Universidad de Buenos Aires-CONICET, Argentina.
J Autoimmun. 1996 Oct;9(5):689-97. doi: 10.1006/jaut.1996.0090.
The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.
采用间接免疫荧光法（IIF）研究了在CHO细胞中表达的人谷氨酸脱羧酶（GAD65）与GAD抗体之间的反应。单克隆抗体GAD1和GAD6分别识别构象性和连续性GAD表位，产生了不同的染色模式。26例新诊断的胰岛素依赖型糖尿病（IDDM）患者的血清中有12例呈现出多种抗GAD特异性IIF图像，涵盖了用单克隆抗体观察到的两种极端情况。21例正常血清在该检测中均未呈阳性。作为对照，使用体外产生的、折叠的35S-GAD65通过参考免疫沉淀（IP）检测对血清进行检测。只有1例患者血清在用热和去污剂变性的35S-GAD65进行IP检测时发生反应，这表明大多数自身抗体仅识别折叠抗原。11例患者血清的IIF和IP检测均为抗GAD阳性。这些血清的IIF反应性被脑提取物中的可溶性GAD阻断。1例血清仅IIF呈阳性，其反应性未被可溶性GAD阻断。8例血清仅IP呈阳性。我们的结果表明，与传统的IP检测相比，抗GAD抗体在转化的CHO细胞环境中识别人类GAD65的能力存在差异。在未来改进现有IDDM自身抗体检测方法的尝试中应考虑这些差异。
