Development and Validation of an Electrochemiluminescent ELISA for Quantitation of Oral Insulin Tregopil in Diabetes Mellitus Serum

To detect residual pre-proinsulin (PPI) in recombinant human insulin production, an analytical method based on double-antibody sandwich ELISA was developed in this study. The BALB/c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained using the conventional cell fusion technique and ELISA screening. We purified the antibody using a Protein G gel column and identified its purity by SDS-PAGE. The sandwich ELISA was used to explore the pairing effect, and the specificity of the paired antibody was determined. We selected a paired antibody with relatively good specificity to establish sandwich ELISA, constructed a quantitative curve, and evaluated the accuracy and sensitivity of the method. Six anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer value. The sandwich ELISA method was established with P5 for plating and P2 as detection antibodies. The linear range of the quantitative curve of PPI by sandwich ELISA was 0.645 to 82.5 pg/mL, the recovery was 95% and the detection limit was 3.06 pg/mL. In this study, we prepared six anti-PPI monoclonal antibodies and established the sandwich ELISA method to detect PPI in process control and product release control for recombinant human insulin production.

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Development and Validation of an Electrochemiluminescent ELISA for Quantitation of Oral Insulin Tregopil in Diabetes Mellitus Serum

Abstract: The ELISA method is sensitive and can be used to support accurate measurement of Tregopil with no cross-reactivity to Glargine and its metabolites in clinical studies.

Cited by 4 publications

References 19 publications

Controlling insulin release from reverse hexagonal (HII) liquid crystalline mesophase by enzymatic lipolysis

Controlling insulin release from reverse hexagonal (HII) liquid crystalline mesophase by enzymatic lipolysis

A sustained zero-order release carrier for long-acting, peakless basal insulin therapy

Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA

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