title: Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes.
abstract: 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.
authors.affiliation: CSIRO Division of Biomolecular Engineering, Parkville, VIC, Australia.
authors.forename: O
authors.initials: O
authors.lastname: Dolezal
authors.forename: G
authors.initials: G
authors.lastname: Coia
authors.forename: P J
authors.initials: PJ
authors.lastname: Hudson
mesh_terms: D000469:Alkaline Phosphatase; D018033:Antibodies, Bispecific; D015153:Blotting, Western; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D008156:Luciferases / Q000032:analysis; D008163:Luminescent Measurements; D009842:Oligopeptides; D010455:Peptides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis*; D001616:beta-Galactosidase / Q000032:analysis
chemical_list: D018033:Antibodies, Bispecific; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D008156:Luciferases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase