Abstract
BackgroundThe 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.MethodsIn 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.ResultsThe 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.ConclusionsThe 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.
Highlights
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
Molecular modeling inferred that the carboxyl group of hapten and its exposure level played an important role in the hapten-Ab recognition
The proposed Indirect competitive enzyme-linked immunosorbent assay (icELISA) can be used for the surveillance screening of Rhodamine B (RB) in a range of seasoning foods, and the heterologous strategy is an effective approach to enhance the sensitivity in an immunoassay
Summary
Materials and reagents RB (99.0%) was obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). 15 mg of BSA was added to 2 mL of phosphate buffer (PBS, pH 7.4, 10 mM) containing RB (10 mg), followed by the addition of EDC (20 mg) under continuous stirring for 30 min in a dark chamber. Immunogen 2 RBITC–BSA conjugate was prepared by direct reaction of RBITC and BSA [24]. Two kinds of immunogens, RB–BSA and RBITC-BSA, were injected to the New Zealand rabbits, respectively. Goat anti-rabbit IgG-HRP (200 ng/mL, 100 μL/well) was added to each well After another 30 min of incubation at 37 °C, the plates were washed five times again. The cartridge was preconditioned with 5 mL of methanol followed by 5 mL of N-hexane:acetone (8:2, v/v) prior to the addition of the extracted supernatant. Analysis of market samples Twenty natural samples (chilli powder: No 1–5; hotpot seasoning: No 6–10; Chinese prickly ash: No 11–15; chilli sauce: No 16–20) were extracted by the above method and detected by both icELISA and HPLC–FLD
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