de Haard H J, Kazemier B, Koolen M J, Nijholt L J, Meloen R H, van Gemen B, Hoogenboom H R, Arends J W
Biosciences Research Unit, Organon Teknika, Boxtel, Maastricht, The Netherlands.
Clin Diagn Lab Immunol. 1998 Sep;5(5):636-44. doi: 10.1128/CDLI.5.5.636-644.1998.
By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.
通过应用组合文库技术，我们生成了首个针对人类免疫缺陷病毒1型（HIV-1）主要衣壳蛋白p24的重组抗体片段。利用来自病毒裂解物免疫小鼠脾脏的B细胞抗体可变区（V）基因构建了单链Fv片段（scFvs）文库。通过淘选或用生物素化抗原富集来筛选抗体，得到了四个不同的抗体片段家族。通过亲和力测定、Western印迹上的抗原识别以及肽扫描分析对不同类型的scFvs进行了表征。其中一种scFvs的表位位于与亲环素A（CypA）结合相关的残基附近，因此使其成为治疗应用的有吸引力的候选物。将该scFV的V基因序列与先前描述的针对这一免疫显性表位的单克隆抗体的V基因序列进行比较，发现VH和Vκ区域的相同组合被使用。因此，这是从文库衍生的抗体片段中检索到原始组合的罕见例子之一。经过适当的亲和力和形式改进后，我们最好的重组scFvs可能构成一种灵敏的p24检测方法的基础，用于测量病毒载量。此外，抗p24 scFvs可表达为细胞内抗体（intrabodies）以辅助治疗HIV感染。
