Carboxyl-Functionalized, Europium Nanoparticle-Based Fluorescent Immunochromatographic Assay for Sensitive Detection of Citrinin in Monascus Fermented Food

A fluorescent immunochromatographic test strip (FICTS) based on the use of europium nanoparticles (EuNPs) was developed and applied to detect citrinin (CIT) in Monascus fermented food. The sensitivity of the immunoassay to detect CIT was greatly improved by the use of a specific monoclonal antibody to attach EuNPs to form a probe. Under optimum conditions, the visual detection limit was 2.5 ng/mL, and the detection limit of the instrument was 0.05 ng/mL. According to the results, the IC50 was 0.4 ng/mL. Matrix interference from various Monascus fermented foods was investigated in food sample detection. The immunosensor also demonstrated high recoveries (86.8–113.0%) and low relative standard deviations (RSDs) (1.8–15.3%) when testing spiked Monascus fermented food. The detection results of this method showed a good correlation (R2 > 0.98) with high-performance liquid chromatography (HPLC). The results showed that the FICTS method could be used as a rapid, sensitive method to detect CIT in Monascus fermented food.

. UV spectrum of the CIT artificial antigens. (a) UV spectrum scan of CIT-BSA. (b) UV spectrum scan of CIT-HSA. Figure S2a indicates that the CIT-BSA band was found after the appearance of the BSA band, reflecting a significant hysteresis. Therefore, CIT successfully binds to the residues of BSA to form a conjugate. Significant hysteresis indicates that more CIT was attached to the BSA. This identification result was consistent with the UV-vis spectra identification results. In addition, the CIT-HSA strip also reflected this characteristic, indicating that CIT was successfully coupled to HSA ( Figure S2b). However, the migration difference between CIT-HSA and HSA was much smaller than between BSA and CIT-BSA. This suggested that the CIT was less attached to the HSA than does CIT-BSA.

S2. Production of Monoclonal Antibody
The "Administrative Rules for experimental animals in Zhejiang Province" (2009) were strictly complied with in this study to minimize animals' suffering. Three Balb/c female mice were purchased from the laboratory animal center of Hangzhou Normal University (Hangzhou, China).
Referring to other previous studies, antibodies were produced in Balb / c mice [2]. Specifically, three Balb/c mice were immunized by subcutaneous injection of CIT-HSA (200 μg in 100 μL sterile PBS) and emulsified with an equal volume of Freund's complete adjuvant. For the subsequent immunizations (three times, at two-weekly intervals) 100 μg of CIT-HSA conjugate (in 100 μL sterile PBS) in the same volume Freunds incomplete adjuvant was used. Blood was obtained via retro-orbital venous plexus and stored at 4 °C overnight. Then, the segregated sera were examined for titer and inhibition ratio against free CIT by using indirect competition enzyme-linked immunosorbent assay, in which CIT-BSA was used as the coating antigen. When the antiserum titer no longer changed, the mice whose antiserum showed the strongest inhibition were further fused. Three days prior to spleen harvest, 100 μg of CIT-HSA was injected into the selected mice by spleen in 0.9% NaCl solution.
Murine myeloma cells Sp2/0 were maintained in an exponential growth stage in RPMI 1640 cell culture medium supplemented with 10% fetal bovine serum (FBS). The splenocytes from the immunized mice were fused with the myeloma cells. Hybridomas were selected in a HAT medium (DMEM medium containing 15% FBS). The cultures in 96-well plates were maintained in a 5% CO2 incubator at 37 °C. The hybridoma that secreted antibodies specific to CIT were subcloned by the serial dilution method. Then the stable cells were expanded and cryopreserved in liquid nitrogen. The stable cells were produced for ascites and purified by octanoic acid-sulfur ammonium method to obtain monoclonal antibodies.

S3. Indirect Competitive ELISA
After checkerboard optimization, the indirect competitive ELISA was processed as the standard procedure for detection of antisera or identification of antibodies [3]. The detailed procedure was as follows. The 96-well plates coated with CIT-BSA were blocked at 4 °C for 24 h. After the reaction was completed, the 96-well plates were dried at 37 °C for 5 h and stored at 4 °C until use. PBS and diluted CIT standard were added to the closed 96-well plates at 50 μL/well. Then, anti-CIT monoclonal antibody and goat anti-mouse-horseradish peroxidase were diluted and added to the closed 96-well plates at 50 μL/well. The reaction was carried out at 25 °C for 30 min. After the reaction was completed, the plates were washed with PBST for 3 times. Then, the TMB chromogenic reagent was added to the 96-well plates at 100 μL/well. The reaction was performed at 25 °C for 10 min. The stop solution was added to the plate at 50 μL/well, and the OD value was measured at 450 nm with a microplate reader.

S4. Identification of Anti-CIT Monoclonal Antibody
After the stable cells were produced for ascites and purified by octanoic acid-sulfur ammonium method to obtain monoclonal antibodies, the antibody affinities were determined using an indirect competitive ELISA ( Figure S3). On account of the greater binding rates, the monoclonal antibody of clone 4B9 was selected for further use.  Table S1 was showed that the IC50 was 5.53 ± 0.76 ng/mL. According to calculation, the anti-CIT mAb had a CR of less than 0.1% for the other six different mycotoxins. The results showed that the anti-CIT mAb had almost no cross-reactivity to other six different mycotoxins. It could be seen that the anti-CIT mAb showed good specificity.

S5. Supplementary Note to Table S2
For the detection of small molecular substances, the analysis characteristics of the whole method are reflected in the calibration cruves range, detection time and 50% inhibition (IC50), limit of detection (LOD) and so on. Then the comparison of the analytical characteristics of this method and other methods for CIT is summarized in Table S2, which showed that the high sensitivity of the present study detects CIT. is usually defined as the minimum concentration of the component that can be detected with appropriate confidence. 50% inhibition (IC50), the half maximal inhibitory concentration, represents the concentration of an inhibitor that is required for 50% inhibition of things like an enzyme, a cell, a cell receptor or a microorganism. NR: Not reveal.