Kala M, Bajaj K, Sinha S
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
Anal Biochem. 1997 Dec 15;254(2):263-6. doi: 10.1006/abio.1997.2378.
An efficient means for the detection of antigen-specific binding by phage-displayed antibodies would facilitate the selection of such phage, especially from libraries with large repertoires of V-genes. We report the development and characterization of a magnetic bead phage ELISA which detects antigen binding phage which could not be detected by conventional ELISA. We were attempting to select phage binding to the oncodevelopmental antigen, heat-stable alkaline phosphatase (HSAP). Although there was an obvious enrichment in the phage titers after successive rounds of selection, we were unable to detect antigen-binding phage by ELISA on a plastic surface. However, ELISA with a suspension of superparamagnetic particles covalently conjugated to HSAP effectively identified antigen-binding phage after the fourth round of selection. This method could also detect antigen-specific binding of individual phage clones. Some of the phage clones bound to either amino- or carboxy-terminal-conjugated HSAP, perhaps reflecting the differences in the exposed epitopes. It is suggested that a sensitive method such as magnetic bead phage ELISA be tried before declaring a phage selection as unsuccessful or concluding that a phage clone does not bind antigen.
一种用于检测噬菌体展示抗体的抗原特异性结合的有效方法，将有助于此类噬菌体的筛选，尤其是从具有大量V基因库的文库中筛选。我们报告了一种磁珠噬菌体酶联免疫吸附测定法（ELISA）的开发和特性，该方法可检测常规ELISA无法检测到的抗原结合噬菌体。我们试图筛选与肿瘤发生相关抗原热稳定碱性磷酸酶（HSAP）结合的噬菌体。尽管经过连续几轮筛选后噬菌体滴度有明显富集，但我们无法通过塑料表面的ELISA检测到抗原结合噬菌体。然而，用与HSAP共价偶联的超顺磁性颗粒悬浮液进行的ELISA在第四轮筛选后有效地鉴定出了抗原结合噬菌体。该方法还可以检测单个噬菌体克隆的抗原特异性结合。一些噬菌体克隆与氨基末端或羧基末端偶联的HSAP结合，这可能反映了暴露表位的差异。建议在宣布噬菌体筛选失败或得出噬菌体克隆不结合抗原的结论之前，尝试使用磁珠噬菌体ELISA等灵敏方法。
