Molecular cloning, expression and mutagenesis of an anti-insulin single chain Fv (scFv)

The immunoglobulin variable region genes of a murine anti-insulin IgG-producing hybridoma were rescued and cloned into a bacterial expression vector. The variable regions of the gamma heavy chain and the kappa light chain were expressed independently and together as a single chain antibody (scFv). The variable heavy chain alone demonstrated the ability to bind to insulin. The kappa light chain did not show any binding activity towards insulin. The scFv was constructed by PCR assembly using a (Gly₄Ser)₃ linker between the carboxyl end of the variable heavy chain and the amino terminus of the kappa light chain. The scFv bound insulin at an IC₅₀ of 3.5 × 10^-8 M whereas the parent antibody bound insulin at 1.0 × 10^-8 M. Mutagenesis of the variable heavy chain complementarity determining regions (CDR) indicated that CDR1 and CDR3 were important for binding to insulin. Position 99 in CDR3 of the heavy chain was found to be a critical position for the ability of the scFv to bind to insulin.