DETERMINATION OF ACTIVE SINGLE-CHAIN ANTIBODY CONCENTRATIONS IN CRUDEPERIPLASMIC FRACTIONS

We have measured active single chain antibody (scFv) concentrations un der mass transfer limitation conditions using surface plasmon resonanc e on the BIAcore. For the creation of a standard curve scFv 4Dwt, deri ved from monoclonal antibody (mAb) 4D, directed against human chorioni c gonadotropin (hCG), was purified by positive affinity chromatography . Determination of the active antibody fraction after purification was performed using anti-FLAG, reactive against a tag sequence C-terminal ly fused to the scFv. Two independent experiments showed that the acti vity remaining represented over 75% of the total amount of purified pr otein. Calibration curves on high density antigen surfaces showed a li near relationship between antibody concentration and binding rates. Pe riplasmic fractions of six mutant scFvs, also derived from mAb 4D, rev ealed a clear difference in the amount of soluble active scFv expresse d in the periplasm of E. coli compared with the total amount of antibo dy present, indicating the necessity of measuring active antibody conc entrations. This rapid concentration determination method will be part icularly useful for accurately comparing affinity constants, using ant ibody concentrations determined with the BIAcore, of the many differen t scFv fragments routinely isolated from phage display libraries.