Abstract

Soybean peroxidase (SBP) is characterized by relatively high heat resistance, wide substrate scope, good stability and broad pH applicability. These advantages led SBP to be used as labeledantibody for immunoassays. In this work, monoclonal antibody was linked with SBP to beta-HCG (Mab II-SBP) for enzyme-linked immunosorbent assay (ELISA) or chemiluminescence assay to explore its potential as an alternative to the widely used horseradish peroxidase (HRP) based ELISA. The Mab II-SBP was prepared by the sodium periodate oxidation method. The effects of enzyme coupling rate and molar ratio of Mab II to enzyme on assay performance were investigated. The optimum mass concentration ratio of Mab II and SBP was determined to be 1:3. The best coat concentration of the monoclonal antibody to alfa-HCG antibody (Mab I) was 4 μg/mL. Under optimum conditions, the HCG concentration was determined by the proposed ELISA assay with linear response range from 0.18 to 18 ng/mL and detection limit of 0.07 ng/mL at a signal-to-noise ratio of 3. Both enhancers, 3-(10’-phenothiazinyl) propane- 1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH), amplified chemiluminescence intensity nearly 200-fold in comparison to the SBP-luminol-H₂O₂ system. The present work demonstrates the feasibility and rationale of SBP-labeling antibodies for ELISA and chemiluminescence assays.

Keywords: Chemiluminescence, ELISA, enhancers, enzyme-labeled antibody, immunoassay, soybean peroxidase.

Graphical Abstract