Efficient detection of pre-proinsulin by double antibody sandwich ELISA

Abstract ObjectiveTo generate monoclonal antibodies against pre-proinsulin (PPI), and establish sandwich ELISA method to provide a basis for PPI detection in recombinant human insulin production. MethodsThe Balb /c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained by conventional cell fusion technique and ELISA screening.The antibody was purified using a Protein G gel column and identified for purity by SDS-PAGE. Pairing effect was found by the sandwich ELISA, and the specificity of the paired antibody was determined. A paired antibody with better specificity was selected to establish sandwich ELISA, and construct a quantitative curve, the accuracy and sensitivity of the method were evaluated. ResultsSix anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer. The sandwich ELISA method was established with P5 for plating and P2 for detection antibodie. The linear range of the quantitative curve of PPI by sandwich ELISA was 0. 645-82.5 pg/mL, the recovery was 89%-95%, and the limit of detection was 3.06 pg/mL. ConclusionSix monoclonal antibodies against PPI were generated and the sandwich ELISA method was established to detect PPI in process control and product release control for recombinant human insulin production.