THERMAL STABILIZATION OF A SINGLE-CHAIN FV ANTIBODY FRAGMENT BY INTRODUCTION OF A DISULFIDE BOND

A disulphide bond was introduced into a single-chain Fv form of the an ticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light cha in and Asp-H106 of the heavy chain with cysteines, by site-directed mu tagenesis. To maintain the salt-bridge from the latter residue to Arg- H98, Tyr-107 was also altered to Asp, The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and sur face plasmon resonance analysis of binding kinetics, Compared with the parent scFv, the disulphide bonded form was shown to have enhanced th ermal stability, by Fourier transform IR spectroscopy, The T-m was rai sed from 60 degrees C to 69 degrees C, The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression a dvantages of the scFv.