Quantitative measurement of bitagged recombinant proteins using an immunometric assay: Application to an anti-substance P recombinant antibody

We have developed two different immunometric assays to directly quantify bo th the total and the active fractions of a recombinant antibody (single cha in fragment variable, or ScFv) as obtained in a crude extract from an Esche richia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha- Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recomb inant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directe d against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), a cts as tracer. In parallel, for the determination of the active fraction, t he capture is performed using microtiter plates coated with the antigen, wh ile solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb , A synthetic peptide in which the two Tag sequences were joined was used a s a standard, thus avoiding the laborious purification of a recombinant pro tein as reference. The method was applied to the direct measurement, in per iplasmic extracts, of the total and active; fractions of an ScFv produced a t different induction temperatures. (C) 2000 Academic Press.