Andersson G, Ekre H P, Alm G, Perlmann P
Research and Development Immunobiology, Kabi Biopharma, Stockholm, Sweden.
J Immunol Methods. 1989 Dec 20;125(1-2):89-96. doi: 10.1016/0022-1759(89)90081-1.
Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.
用重组干扰素-γ免疫后制备了抗人干扰素-γ（IFN-γ）的小鼠单克隆抗体（mAb）。选择了两种识别天然IFN-γ上不同表位的单克隆抗体（1-D1K和7-B6-1）用于开发双位点酶联免疫吸附测定（ELISA）。在含1%牛血清白蛋白的磷酸盐缓冲盐水（PBS）、用过的培养基或胎牛血清中稀释的IFN-γ，其检测灵敏度相似，但在正常人血清中稀释时，灵敏度降低至约50%。对个体正常人血清进行了检测，14份中有3份在ELISA中出现假反应性。一种对个体差异和灵敏度降低有主要影响的血清因子可被吸附到蛋白A-琼脂糖上并从其上洗脱下来。基于这些观察结果，我们建立了一种新的ELISA方法，该方法能够检测人血清和血浆样本中的低水平IFN-γ。该方法中的修改易于使用基本实验室设备进行应用。
