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

ABUD, J.E.; LUQUE, E.H.; RAMOS G.; RODRIGUEZ, H.A.

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2017 vol. 135 p. 16 - 27

1046-5928

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tagfrom its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is oftenrequired. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determinethe remnants of GST during purification process. In the present study, we developed both a conventionalenzyme-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, usinga pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridomatechnology. Two of these were rationally selected as capture and detection antibodies, allowing thedevelopment of a sandwich ELISA with a limit of detection (LOD) of 0.01 mg/ml. To develop the rGST-IPCRassay, 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 antibodieson PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentrationeffect were studied and determined. Under optimized assay conditions, the rGST-IPCR assayprovided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGSTELISA.The proposed method exhibited great potentiality for application in several fields in whichmeasurement of very low levels of GST is necessary, and might provide a model for other IPCR assays