Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes

Biotechniques. 1997 May;22(5):897-904. doi: 10.2144/97225st02.

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.

Publication types

  • Technical Report

MeSH terms

  • Alkaline Phosphatase
  • Antibodies, Bispecific*
  • Blotting, Western
  • Epitopes*
  • Escherichia coli / metabolism
  • Genes, Reporter
  • Hemagglutination Inhibition Tests*
  • Humans
  • Luciferases / analysis
  • Luminescent Measurements
  • Oligopeptides
  • Peptides*
  • Plasmids
  • Polymerase Chain Reaction
  • Recombinant Proteins / analysis*
  • beta-Galactosidase / analysis

Substances

  • Antibodies, Bispecific
  • Epitopes
  • Oligopeptides
  • Peptides
  • Recombinant Proteins
  • FLAG peptide
  • Luciferases
  • Alkaline Phosphatase
  • beta-Galactosidase