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Updated: Jul 17, 2026
Peptide-based Identification of Functional Motifs and their Binding Partners
Published on: July 1, 2013
Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes
O Dolezal1, G Coia, P J Hudson
1CSIRO Division of Biomolecular Engineering, Parkville, VIC, Australia.
We have demonstrated that the expression of recombinant proteins labeled with an immunoreactive epitope can be rapidly assessed and quantitated using a modified haemagglutination inhibition assay in enzyme-linked immunosorbent assay (ELISA) trays. The agglutination of erythrocytes from a droplet of whole blood provided a simple visual assay. The additional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent antibody with specificity to the same epitope. In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG in conjunction with the M2 anti-FLAG antibody, which had affinity to FLAG incorporated either at the C-terminus or N-terminus of the recombinant 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 method routinely for the assay of FLAG-labeled recombinant products following protein expression and subsequent small- and large-scale purification procedures.
We have demonstrated that the expression of recombinant proteins labeled with an immunoreactive epitope can be rapidly assessed and quantitated using a modified haemagglutination inhibition assay in enzyme-linked immunosorbent assay (ELISA) trays. The agglutination of erythrocytes from a droplet of whole blood provided a simple visual assay. The additional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent antibody with specificity to the same epitope. In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG in conjunction with the M2 anti-FLAG antibody, which had affinity to FLAG incorporated either at the C-terminus or N-terminus of the recombinant 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 method routinely for the assay of FLAG-labeled recombinant products following protein expression and subsequent small- and large-scale purification procedures.
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