ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST–protein immune sera¹

Abstract

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.

Diagnostic and Prognostic Utility of the Synaptic Marker Neurogranin in Alzheimer Disease

“…The CSF neurogranin levels were measured using a 2-site immunoassay that uses an affinity-efficient trapping and purification technique for polyclonal antibodies developed in the Laboratory of Jack H. Ladenson, PhD, Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri (eMethods in the Supplement) 42 …”

Development of an Immunoassay for the Kidney-Specific Protein myo-Inositol Oxygenase, a Potential Biomarker of Acute Kidney Injury

“…Polyclonal antibodies were purified by first removing cross-reacting anti-GST with a GST column (#20205, Pierce) according to manufacturer's instructions. Antibodies were then affinity purified as described previously (34). Mouse monoclonal antibodies were produced at Maine Biotechnology Services, Inc (Portland, ME) using recombinant GST-MIOX as the immunogen.…”