Schlosser M, Witt S, Ziegler B, Ziegler M
Institute of Diabetes, Gerhardt Katsch, Karlsburg, F.R.G.
J Immunol Methods. 1991 Jun 24;140(1):101-9. doi: 10.1016/0022-1759(91)90131-x.
A rapid, effective and sensitive CELISA for the detection of monoclonal islet cell reactive antibodies (mc-ICRA) using the insulin-producing rat insulinoma cell line (RIN) is described. RIN cells are a suitable target for this monoclonal antibody assay as shown by a comparative study with normal rat islet cells. We tested the influence of the target cell preparation and obtained the best sensitivity and reliability with the CELISA using desiccated cells or desiccated cell homogenate with a cell number of 5 x 10(4) cells per well rather than an adsorbed cell homogenate. Furthermore, ethanol fixation of RIN cells resulted in a loss of antigenicity as shown particularly by the detection of islet cell surface antibodies. We also compared the binding of mc-ICRA in RIN-CELISA with data obtained by indirect immunofluorescence using viable RIN cells as targets. By permeabilization of the cell membrane by desiccation or sonication, more antibodies are detected in CELISA (surface and cytoplasmic antibodies), whereas in immunofluorescence on viable RIN cells, only surface reactive antibodies are detected.
本文描述了一种使用产胰岛素大鼠胰岛素瘤细胞系（RIN）快速、有效且灵敏地检测单克隆胰岛细胞反应性抗体（mc-ICRA）的捕获酶联免疫吸附测定（CELISA）。与正常大鼠胰岛细胞的比较研究表明，RIN细胞是该单克隆抗体检测的合适靶标。我们测试了靶细胞制备的影响，发现使用干燥细胞或干燥细胞匀浆（每孔5×10⁴个细胞）的CELISA具有最佳的灵敏度和可靠性，而非吸附的细胞匀浆。此外，RIN细胞的乙醇固定导致抗原性丧失，尤其是在检测胰岛细胞表面抗体时表现明显。我们还将RIN-CELISA中mc-ICRA的结合情况与以活RIN细胞为靶标的间接免疫荧光法获得的数据进行了比较。通过干燥或超声处理使细胞膜通透化后，在CELISA中可检测到更多抗体（表面和细胞质抗体），而在活RIN细胞的免疫荧光检测中，仅能检测到表面反应性抗体。
