A Method for Checking Recombinant Protein Quality to Troubleshoot for Discordant Immunoassays
Tran-Nguyen, T. K.
Show abstract
Discordant results of recombinant protein-based enzyme-linked immunosorbent assays (ELISA) and other antigen detection tests are a common and vexing problem in scientific research and clinical laboratories. The reproducibility of immunoassays based on antibody specificity can be adversely affected by cryptic changes in the composition of the analyte (e.g., the protein antigen). By use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry analyses, we found varying levels of purity and the extent of post-translational modifications (PTMs), particularly of N-linked glycosylation and phosphorylation, among varied lots of recombinant glucose-regulated protein 78 (GRP78) expressed in Escherichia coli. We expect these lotto-lot variabilities of both purity and PTMs led to inconsistent results stemming from ELISA assays that measured GRP78 autoantibody levels in patient plasma specimens. We present these analyses to draw readers attention to a potentially common, yet seldom appreciated problem, in laboratory assays using recombinant proteins as antigens for antibody detection, and propose a workflow to detect and troubleshoot for this typical problem.
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