Pilot production of a sensitive ELISA kit and an immunochromatographic strip for rapid detecting citrinin in fermented rice

Abstract

Citrinin (CTN) is a mycotoxin primarily produced by Monascus species. Excess consumption of CTN may lead to nephrotoxicity and hepatotoxicity. A pilot study for commercial production of competitive direct enzyme-linked immunosorbent assay (cdELISA) kit and an immunochromatographic strip (immunostrip) for screening CTN in red yeast rice is established in this study. The coating antibody and the CTN-horse radish peroxidase (HRP) concentrations were optimized to increase the sensitivity and specificity of cdELISA kit. The conjugation methods/ratios of CTN to HRP as well as the long-term stability of kit components were also evaluated. The IC₅₀ and detection limit of the ELISA kit were determined to be 4.1 and 0.2 ng mL^-1, respectively. Analysis of 20 red yeast rice samples using ELISA kits revealed the contamination levels of CTN from 64 to 29 404 ng g^-1. The on-site rapid detection of CTN with the immunostrip showed that CTN levels in seven samples exceeded the regulatory limit of 5 ppm. Additionally, the coefficient correlation between the results of HPLC and ELISA kits of 20 samples was 0.96. Sensitive and convenient tools at commercial levels for detection of CTN contamination in food are established herein to protect the health of the public.

Fig. 1. Characterization of the ELISA kit for CTN. (A) Comparison of the different conjugation method for CTN–HRP. (B) Comparison of the different conjugation ratio between CTN and HRP. (C) Comparison of CTN Ab concentration for coating onto the plate (D) cross-reactivities of CTN, 1-hydroxy-2-naphthoic acid and ochrartoxin A in the ELISA kit. (E) Comparison of the blocking buffer and 0.1% BSA using in the blocking step (F) stability analysis of the ELISA kit stability storage at 4 °C. A was the absorbance value of the D day; A₀ was the absorbance value of the first day; the absorbance of the A₀, CTN-free present was 2.5; each analysis was duplicated.

Fig. 2. Optimization of the immunostrip for CTN. (A) Comparison of the particle size of the gold nanoparticle. (B) Comparison of the quantity of antibodies on the surfaces of gold nanoparticles. (C) Comparison of the test line reagents CTN–OVA and CTN–BSA. (D) Comparison of the pore size of nitrocellulose.

Fig. 3. Characterization of immunostrip for CTN standard. (A) Visual detection limit of the immunostrip for CTN (B) the standard curve of the test line intensity on the immunostrip. (C) Long-term stability analysis of the immunostrip.

Fig. 4. Analysis of CTN contamination levels in 20 red yeast rice samples with immunostrip.

Fig. 5. Analysis of CTN with high performance liquid chromatography (A) HPLC chromato-grams of 5 (), 10 (), 20 (), and 40 () ng of CTN standard. The inlet figure is the calibration curve of CTN. (B) HPLC chromatograms of 30 ng of the CTN standard () and one red yeast rice sample with CTN contamination () (C) the correlation coefficients of HPLC and ELISA kit for analyzing the CTN levels in red yeast rice samples.