Chames P, Coulon S, Baty D
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, Marseille, France.
J Immunol. 1998 Nov 15;161(10):5421-9.
Immunoassays are widely used to determine steroid concentrations. However, they are limited by the specificity of antisteroid mAbs. We used the phage display system combined with molecular modeling and site-specific randomization to improve the affinity and the fine specificity of an anti-cortisol mAb. Using parsimonious mutagenesis, we have generated a library of mutant Ab fragments (scFv) derived from this Ab by randomizing five amino acids chosen by molecular modeling and Ab-hapten contact structural analysis. Anti-cortisol Ab fragments were selected from the library in the presence of steroid analogues to block cross-reacting binders. Specific elution with free cortisol allowed the recovery of clones with up to eightfold better affinity and fivefold less cross-reactivity than the wild-type scFv. This approach can be applied to any anti-hapten Ab and represents a useful approach for obtaining highly specific Abs for use in steroid immunoassays.
免疫测定法被广泛用于测定类固醇浓度。然而，它们受到抗类固醇单克隆抗体特异性的限制。我们使用噬菌体展示系统结合分子建模和位点特异性随机化来提高抗皮质醇单克隆抗体的亲和力和精细特异性。通过简约诱变，我们通过对分子建模和抗体-半抗原接触结构分析所选择的五个氨基酸进行随机化，从该抗体产生了一个突变抗体片段（单链抗体）文库。在类固醇类似物存在的情况下从文库中筛选抗皮质醇抗体片段，以阻断交叉反应性结合物。用游离皮质醇进行特异性洗脱，使得能够回收与野生型单链抗体相比亲和力提高多达八倍且交叉反应性降低五倍的克隆。这种方法可应用于任何抗半抗原抗体，是获得用于类固醇免疫测定的高特异性抗体的一种有用方法。
