Generation of mutant antibodies with ultrahigh affinity based on a novel and universal selection method and their application to trace analysis

与传统抗体相比，具有更高亲和力的半抗原特异性抗体工程有望使半抗原免疫测定具有亚飞摩尔范围的灵敏度。从这些观点出发，我们开发了一种新的通用方法来选择具有高亲和力的抗半抗原抗体片段的噬菌体颗粒。选择11-脱氧皮质醇（11-DC）、17α-羟孕酮和雌二醇作为模型靶半抗原，并通过包含还原可切割二硫键的间隔物与生物素共价偶联。这些生物素（CB）偶联类固醇具有良好的生化和化学性质：偶联物与相关的抗类固醇抗体和链霉亲和素同时结合，并且在50 mM二硫苏糖醇（DTT）的作用下，二硫键很容易断裂。对11-DC （Ka=1.3×10^<10> M^<-1）具有高亲和力的单链Fv片段（scFvs）的噬菌体颗粒与cb偶联的11-DC孵育，所得复合物在固定化NeutrAvidin上捕获。DTT处理可使目标噬菌体恢复；这个过程完全独立于抗原-抗体相互作用的解离。5轮连续筛选使抗11- dc噬菌体从超过10万倍的非特异性噬菌体颗粒中分离和富集。该方法将普遍适用于选择针对多种半抗原的超高亲和力噬菌体抗体。

The engineering of hapten-specific antibodies with a much higher affinity than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity. From these points of view, we developed a novel and universal method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), 17α-hydroxyprogesterone, and estradiol were selected as model target haptens, and were covalently conjugated to biotin via a spacer including a reductively cleavable disulfide bond. These cleavable biotin(CB)-conjugated steroids exhibited desirable biochemical and chemical properties : the conjugates were bound simultaneously with relevant anti-steroid antibody and streptavidin, and the disulfide bond was easily cleaved by the treatment with 50 mM dithiothreitol(DTT). Phage particles displaying high affinity single-chain Fv fragments(scFvs) specific for 11-DC (Ka=1.3×10^<10> M^<-1) were incubated with the CB-conjugated 11-DC and the resulting complexes was captured on immobilized NeutrAvidin. The DTT treatment allowed the recovery of target phages ; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC-phage from among a 100,000-fold excess of nonspecific phage particles. This method will be universally applicable for selection of extra-high affinity phage antibodies against a wide variety of haptens.