AGGLUTINATION-INHIBITION ASSAY FOR THE DETECTION OF RECOMBINANT PROTEINS TAGGED WITH PEPTIDE EPITOPES

We have demonstrated that the expression of recombinant proteins label ed with an immunoreactive epitope can be rapidly assessed and quantita ted using a modified haemagglutination inhibition assay in enzyme-link ed immunosorbent assay (ELISA) trays. The agglutination of erythrocyte s from a droplet of whole blood provided a simple visual assay. The ad ditional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent an tibody with specificity to the same epitope. In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG(R) in conjunction with the M2 anti-FLAG antibody, which had affinity to FLA G incorporated either at the C-terminus or N-terminus of the recombina nt protein. The agglutination-inhibition (AI) assay was configured to detect as little as 1 mg/L of soluble recombinant protein in a 30-min assay. Since the AI assay was substantially more rapid and convenient than dot-blot or Western blot analyses, our laboratory now uses this m ethod routinely for the assay of FLAG-labeled recombinant products fol lowing protein expression and subsequent small- and large-scale purifi cation procedures.