Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes.
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.
Dolezal
O
O
CSIRO Division of Biomolecular Engineering, Parkville, VIC, Australia.
Coia
G
G
Hudson
P J
PJ
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
D018033:Antibodies, Bispecific; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D008156:Luciferases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase