Pereira H A, Martin L E, Spitznagel J K
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.
J Immunol Methods. 1989 Feb 8;117(1):115-20. doi: 10.1016/0022-1759(89)90125-7.
The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.
利用酶联免疫吸附测定法（ELISA）已实现对人多形核白细胞的一种高度疏水的天然阳离子抗原CAP57的定量分析。决定ELISA灵敏度和精密度的一个重要特征是减少非特异性蛋白质-蛋白质结合，尤其是在抑制试验中，从而消除了现有方法所获得的高背景值。使用含肝素的磷酸钾缓冲液洗涤固相结合抗原并封闭非特异性结合位点，在很大程度上提高了这种灵敏度。抑制试验使用的抗原浓度范围为0.9至120纳克。该测定法具有高度特异性，可使用单克隆抗体和多克隆抗体进行。仅当抑制混合物中存在高浓度抗原（大于100纳克）时才观察到非特异性反应。所述技术极其简单，具有高度可重复性，未来在临床环境中检测阳离子抗菌蛋白方面可能具有价值。
