A single chain Fv specific against Western equine encephalitis virus

A recombinant single chain Fv (scFv) specific against Western equine enceph alitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to El component of viral envelope glycoprotein. V-L and V-H gene segments of 11D2 scFv were g enerated and joined together with a (gly(4)ser)(3) linker by polymerase cha in reaction (PCR), The resulting scFv was successfully expressed in P. past oris expression system, Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same V-L and V-H gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that V-H of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while V-L domain did not belong to any known subgr oup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antib ody for detection showed different band pattern among clones derived from d ifferent plasmids, This was thought to be due to the different glycosylatio n where amino acid substitution occurred. Successful purification of 11D2 s cFv could be done by immobilized metal affinity chromatography with an unop timized yield of 700 mu g/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysi s and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11 D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclona l antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.