Heterologous immunoassay strategy for enhancing detection sensitivity of banned dye rhodamine B in fraudulent food

Abstract

Abstract Background The high quality of antibody (Ab) is critical for an immunoassay; usually, an Ab with low affinity is often regarded as a “bad” one in the immunoassay development. How to use a “bad” Ab to develop a highly sensitive immunoassay is still a huge challenge. Methods In this study, a heterologous immunoassay strategy was designed to enhance the sensitivity for the detection of banned dye, rhodamine B (RB), in fraudulent food. The RB Ab could not recognize RB by pairing with homologous coating antigen (Ag). However, the Ab showed unexpected high specificity and sensitivity recognition after being replaced by heterologous coating Ag. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed based on the heterologous strategy. Results The detection limit of icELISA for chilli powder, Chinese prickly ash, hot-pot seasoning, and chilli sauce was 0.002 μg/kg, and the recoveries of the four samples ranged from 76.0 to 102.0%, with the coefficient of variation between 3.9 and 18.8%. Parallel experiment for 20 market samples with high-performance liquid chromatography (HPLC) was performed on to confirm the performance of the practical application of the developed icELISA, and the results of the two methods had good correlation. Molecular modeling inferred that the carboxyl group of hapten and its exposure level played an important role in the hapten-Ab recognition. Conclusions The proposed icELISA can be used for the surveillance screening of RB in a range of seasoning foods, and the heterologous strategy is an effective approach to enhance the sensitivity in an immunoassay.

Keywords

• Rhodamine B
• ELISA
• Immunoassay
• Antibody
• Hapten
• Heterologous