Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase

• Published in: Protein expression and purification 2017-07, Vol.135, p.16-23
• Main Authors: Abud, J.E., Luque, E.H., Ramos, J.G., Rodriguez, H.A.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - isolation & purification; Avidin - chemistry; Biotin - chemistry; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Glutathione S-transferase; Glutathione Transferase - biosynthesis; Glutathione Transferase - genetics; Glutathione Transferase - isolation & purification; Helminth Proteins - biosynthesis; Helminth Proteins - genetics; Helminth Proteins - isolation & purification; Hybridomas - immunology; Hybridomas - pathology; Immuno-polymerase chain reaction; Limit of Detection; Mice; Mice, Inbred BALB C; Monoclonal antibodies; Polymerase Chain Reaction - methods; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Schistosoma japonicum - chemistry; Schistosoma japonicum - enzymology; Spleen - cytology; Spleen - immunology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2017.04.014

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected “Universal-IPCR” format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays. •We developed an IPCR assay to measure rGST and compared its analytical performance with that of an rGST-ELISA.•rGST-IPCR assay provided a 100-fold increase in the LOD in comparison with rGST-ELISA.•rGST-IPCR assay might be very useful when measurement of very low levels of GST is necessary.