Immunochromatographic strip for ultrasensitive detection of fumonisin B1

ABSTRACT A sensitive monoclonal antibody (mAb) (1H1) against fumonisin B1 (FB1) was produced in our laboratory. A rapid, simple, and an immunochromatographic strip has been developed for the ultrasensitive detection of FB1 in corn samples. Under the optimized condition, the cut-off limits of test strips for FB1 were found to be 50 ng/mL in 0.01 M PBS and 25 ng/mL in corn samples. Results could be obtained within 5 min. The results revealed that the developed method is a rapid, sensitive, and simple tool for the detection of FB1.

The effect on human health has been proved, the regulations for fumonisins only in America and Swiss that have recommended levels. The America advisory level for FB 1 , FB 2 , and FB 3 in corn and corn products for human consumption ranges from 2 to 4 ppm; meanwhile in Swiss fumonisins in corn and corn products for human consumption is 1 ppm (Wang, Quan, Lee, & Kennedy, 2006;Zheng, Richard, & Binder, 2006).
Immunochemical methods for fumonisins based on direct and indirect competitive enzyme-linked immunosorbent assays (ELISA) have been developed (Rodriguez-Cervantes et al., 2013;Wang, Bao, et al., 2016;Zou et al., 2014). ELISA is very useful in cases when high amounts of samples have to be tested. It has high specificity, requires simple sample preparation steps, and is cost effective (Ding, Liu, Song, Kuang, & Xu, 2017;Kong et al., 2017;Wang et al., 2017;. However, a long analysis time is needed because of the required incubation time for enzyme-substrate reactions, which is not suitable for detection on-site. The lateral flow immunochromatographic assay is very popular in the detection of residues because it is simple, rapid (can be performed in 5-10 min), specific, and sensitive   Peng, Kuang, & Xu, 2015). The aim of this study was to develop a lateral flow immunochromatographic test strip for the detection of FB 1 in corn. Nitrocellulose (NC) high-flow plus membranes (Pura-bind RP) were obtained from Whatman-Xinhua Filter Paper Co., Ltd. (Hangzhou, China). Glass fibre membrane (CB-SB08) used for the sample pad, polyvinylchloride (PVC) backing material, and absorbance pad (SX18) were supplied by Goldbio Tech Co., Ltd. (Shanghai, China). A BioDot TSR 3000 Membrane Strip Reader (Gene Company Limited, Shanghai Branch, Shanghai, China) was used to test the colour intensities of colloidal gold on the test zone. The complete Freund's adjuvant (FCA), incomplete Freund's adjuvant (FIA), and enzyme immunoassay-grade HRP-labelled goat anti-mouse immunoglobulin were obtained from Sigma (St. Louis, MO, USA) and specific monoclonal antibodies (anti-FB 1 monoclonal antibody (mAb), 1H1) were generated in our laboratory.
Preparation and characterization of anti-FB 1 monoclonal antibody FB 1 was conjugated to KLH or BSA ( Figure 2) according to previous reports (Christensen, Yu, & Chu, 2000;Wang et al., 2014). KLH or BSA was dissolved in phosphate-buffered saline (PBS; 0.01 M, pH 7.4) into 10 mg/mL and dialysed at 4°C for 24 h in 0.01 M PBS (pH 7.4) containing 0.2% glutaraldehyde (GA) (v/v). The dialysis bag was then transferred to 0.01 M PBS overnight to remove the free GA. FB 1 was added to the dialysis, stirred at 4°C overnight. Tris was then added to the mixture and stirred again for 2 h. In the end, the mixture was dialysed against 0.01 M PBS at 4°C for 3 days, and stored at −20°C. All the products were characterized by SDS-PAGE.
Female BALB/c mice (6-8 weeks old) were used to produce the monoclonal antibody (mAb). The mice were immunized subcutaneously with the FB 1 -KLH conjugate. The first immunization using FCA and FIA was used in the subsequent boost injection. The mice were immunized every three weeks using 100 µg for the first immunization and 50 µg for the remaining (2 to 5 immunizations). We analysed the antibodies in blood samples of mice analyzed by ELISA and the mouse with the highest titre was scarified and its splenocytes were fused with Sp 2/0 murine myeloma cells, and the hybridomas were then screened with an indirect ELISA. The selected hybridoma cells were expanded and injected into BALB/c mice to produce the mAb (Deng et al., 2012;. The ascites was harvested and purified with the caprylic acidammonium sulfate precipitation method (Kuang et al., 2013). The purified antibody solution was stored at −20°C until further use.

Development of the immunochromatographic strip
Preparation of colloidal gold nanoparticles All the solvents were prepared with ultrapure water and passed through a 0.22-µm filter membrane. Chloroauric acid (0.1 g/L, 25 mL) was heated to boiling under constant stirring. Following the addition of 1.0 mL of 1% w/v sodium citrate tribasic dehydrate, the mixture was stirred for 30 min. When the colour of the solution turned wine-red, it was allowed to cool to room temperature, and stored at 4°C. Observations by transmission electron microscopy revealed that the gold nanoparticles (GNPs) had a nearly uniform particle size of 15 nm.

Test procedure and principle
Prior to the test, of gold labelled mAbs (50 µL) was mixed with sample solution (150 µL), allowed to react for about 5 min, and added onto the sample pad. The solution migrated along the absorbent pad, and the test results are visualized within 5 min. In FB 1 -positive samples, FB 1 competes with FB 1 -BSA conjugates embedded in the test (T) line for the finite amount of anti-FB 1 mAb. When a sufficient amount of FB 1 is present, free FB 1 binds to gold-labelled mAb, preventing mAb from binding to FB 1 -BSA in the T line. Therefore, the higher the FB 1 content in the sample, the weaker the colour of the T line. In FB 1 -negative samples, the limited amount of colloidal gold-labelled mAb is trapped by the immobilized FB 1 -BSA conjugate, and a clearly visible red T line appears.
The sample must reach the control (C) line, which contains goat anti-mouse IgG antibody. Therefore, the C line must always appear in a successful test, whereas the T line will only appear when the sample is negative (Figure 3(A)). The appearance of the C line alone is indicative that FB 1 is present (Figure 3(B)), whereas if neither the C line nor the T line appears (Figure 3(C)), the test procedure is incorrect or the strip is invalid, indicating that the test should be repeated with a new strip.

Sensitivity of the test strip
The sensitivity of the test strip was determined by testing FB 1 -containing samples. FB 1 standard was diluted to 0, 1, 2.5, 5, 10, 25, and 50 ng/mL in 0.01 M PBS (pH 7.4), and the detection limit was determined. The sample solution (150 µL) was mixed with gollabelled mAb (50 µL), allowed to react for 5 min, and applied onto the sample pad. After 5 min, the color intensities of the different strips were recorded using a test strip reader. The lowest detection limit (LDL) that could be detected by the naked eye was defined as the amount of FB 1 that produced a colour reaction on the strip that was visibly different in intensity from that obtained from 0 ng/mL FB 1 . Six replicates for each concentration were analysed on the same day.

Detection of FB 1 in corn
We obtained corn from a local market. To 5 g of finely ground corn samples 25 mL of 70% methanol was added. The supernatant was obtained and diluted 10× with 0.01 M PBS (pH 7.4), and the samples were spiked with FB 1 solution (10 µg/mL, prepared with 0.01 M PBS, pH 7.4). The FB 1 concentrations in the corn sample were 0 (control), 0.5, 1, 2.5, 5, 10, and 25 ng/mL. Six replicates for each concentration were analysed using the test strips.

Optimization of the strip test
The materials for the strip test must be hydrophilic and have consistent flow characteristics in the immunochromatographic assay. NC must be hydrophobic; however, it can be rendered hydrophilic with the addition of surfactants. It is important to choose the appropriate surfactant when developing these assays because not every protein is compatible with every surfactant. In this study, suspension buffer (0.02 M PBS, 5% sucrose, 2% sorbitol, 1% mannitol, 0.1% PEG, 0.1% tween, and 0.04% NaN 3 ) was added to 12 kinds of reagent (polyvinylpyrrolidone [PVP], polyethylene glycol [PEG], BSA, casein, sucrose, trehalose, sorbitol, mannitol, tween-20, brij-35, triton X-100, and Rhodasurf® On-870 [an ethoxylated oleyl alcohol]). As shown in Figure 4, reagent BSA produced the best and most stable colour reactions and was used for the subsequent experiment.
The concentration of mAbs in GNPs and of the coating antigen affects the sensitivity of the assay. Two different concentrations of mAbs (8 and 10 µg/L) were allowed to react with FB 1 -negative samples (0 ng/mL) and FB 1 -positive samples (5 ng/mL). Figure 5 shows that there were significant differences in colour intensity between 8 and 10 µg/L. We observed that the optimum concentration of mAb in GNP was 10 µg/L based on the colour intensities on both lines (FB 1 -positive and FB 1 -negative samples).
The sensitivity of the assay was investigated with a series FB 1 standards diluted in using 0.01 M PBS (pH 7.4). The LDL with naked eyes was obtained at the amount of FB 1 producing a significant difference in colour intensity of the test strip in comparison with negative control (no FB 1 added). Figure 6 shows that the signal colour on the test lines changes from strong (0 ng/mL) to weak and finally disappeared completely at 50 ng/mL FB 1 .

Detection of FB 1 in corn samples
Optimize the strip test The concentration of mAbs in GNPs of the coating antigen affects the sensitivity of the assay. In this study, two different concentrations of mAbs (8 or 10 µg/mL) were allowed to react with FB 1 -negative (0 ng/mL) and FB 1 -positive spiked in the corn sample. Figure 7 shows that there were slight differences in colour intensity between 8 and 10 µg/mL. We observed that the optimum concentration of mAb in GNP was 8 µg/ mL. Additionally, there is a significant difference in colour intensity at 0.1 and 0.25 mg/  mL. It was found that the coating antigen with a concentration of 0.25 mg/mL had a deeper colour intensity than at 0.1 mg/mL, suggesting that more sensitive detection could be achieved at 0.25 mg/mL. Therefore, the optimum conditions for determination of FB 1 in corn sample were 0.1 mg/mL coating antigen and the concentration of mAb in GNP was 8 µg/mL. Figure 6. Colloidal gold immunochromatography assay for FB 1 in 0.01 M PBS (pH 7.4). FB 1 concentration: 1= 0 ng/mL; 2 = 1 ng/mL; 3 = 2.5 ng/mL; 4 = 5 ng/mL; 5 = 10 ng/mL; 6 = 25 ng/mL; and 7 = 50 ng/mL. In this study, we determined the effect of matrix on our immunochromatographic assay for FB 1 using corn samples. The advantages of immunochromatographic strip assay are its rapidity and ease of use. The corn samples were spiked with FB 1 standard solution (10 µg/ mL, prepared with 0.01 M PBS pH 7.4) at final FB 1 concentrations of 0.5, 1, 2.5, 5, 10, and 25 ng/mL. Six replicates were determined at each concentration. The spiked samples were analysed using our developed method. Figure 8 shows that colour intensity decreased with increasing FB 1 concentrations. The signal colour on the T line changed from strong to weak with increasing FB 1 concentrations and completely disappeared at 25 ng/mL.

Conclusions
A sensitive anti-FB 1 mAb 1H1 with a half-maximal inhibitory concentration (IC 50 ) of 5 ng/ mL and sensitive immunogen (FB 1 -BSA) was obtained following mouse immunization and cell fusion. A simple, rapid, and sensitive analytical method for determination of FB 1 mycotoxin was developed by colloidal gold-based lateral-flow immunochromatography assay. Under optimize conditions by using BSA as reagent and 10 µL as concentration of mAb in GNP, the cut-off values in 0.01 M PBS (7.4) was 50 ng/mL. Meanwhile, the optimum conditions for determination of FB 1 in corn sample were 0.1 mg/mL of coating antigen and the concentration of mAb in GNP was 8 µL. The assay has a cut-off values of 25 ng/mL spiked in corn samples and the results can be evaluated by the naked eye in 5 min. The lateral-flow immunochromatography assay represents a sensitive, simple, and rapid method to detect FB 1 in 0.01 M PBS and corn samples.

Funding
This work was financially supported by the National Key R&D Program [2016YFF0202300] and grants from Natural Science Foundation of Jiangsu Province and MOF [BE2016307, BK20150145, BX20151038, BK20140003, BE2014672, CMB21S1614, 201513006], and Taishan Industry Leading Talent Special Funds.