Cacalano N A, Aggarwal R, Quesniaux V F, Cleveland W L, Erlanger B F
Department of Microbiology, Columbia University, New York, NY 10032.
Mol Immunol. 1992 Jan;29(1):107-18. doi: 10.1016/0161-5890(92)90162-q.
Monoclonal antibodies to cyclosporine A (Cs), a potent immunosuppressant, were generated in BALB/c mice using a novel antigen prepared by linking Cs to a protein carrier via a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa). Twenty-two monoclonal anti-Cs antibodies were generated, using Cs-BBa-bovine serum albumin (Cs-BBa-BSA) as the immunogen. They were characterized with respect to affinity by Scatchard analysis of a radioimmunoassay (RIA), and with respect to specificity by an ELISA in which a series of singly substituted Cs derivatives were examined as inhibitors. McAb affinities ranged from 5 x 10(-8) M to 2 x 10(-10) M. Based on ELISA inhibition data with Cs analogs, and on the binding to two Cs-BSA conjugates in which opposite sides of the Cs molecule are exposed, the antibodies fell into five epitope recognition groups. Binding to Cs was also studied by ELISA in competition with cyclophilin (CyP), a Cs-binding protein whose epitope specificity has been well characterized. Competition by CyP was found to correlate with antibody specificity, not with affinity, i.e. CyP competed best with antibodies having specificities most similar to that of CyP. Epitope mapping can, therefore, be accomplished in a system in which two different species of binding proteins compete for the same antigen. This type of characterization may be useful in identifying antibodies whose combining sites mimic those of a receptor.
通过使用一种新型抗原，即通过光活性交联剂4-苯甲酰苯甲酸（BBa）将环孢素A（Cs）与蛋白质载体相连而制备的抗原，在BALB/c小鼠中产生了针对强效免疫抑制剂环孢素A的单克隆抗体。以Cs-BBa-牛血清白蛋白（Cs-BBa-BSA）作为免疫原，产生了22种单克隆抗Cs抗体。通过放射免疫分析（RIA）的Scatchard分析对它们的亲和力进行了表征，并通过ELISA对其特异性进行了表征，在ELISA中检测了一系列单取代的Cs衍生物作为抑制剂。单克隆抗体的亲和力范围为5×10^(-8) M至2×10^(-10) M。基于Cs类似物的ELISA抑制数据，以及基于与两种Cs-BSA缀合物的结合情况（其中Cs分子的相对两侧暴露），这些抗体分为五个表位识别组。还通过ELISA研究了与亲环蛋白（CyP）竞争结合Cs的情况，亲环蛋白是一种Cs结合蛋白，其表位特异性已得到充分表征。发现CyP的竞争与抗体特异性相关，而不是与亲和力相关，即CyP与特异性最类似于CyP的抗体竞争最激烈。因此，表位作图可以在两种不同种类的结合蛋白竞争同一抗原的系统中完成。这种类型的表征可能有助于鉴定其结合位点模拟受体结合位点的抗体。
