Detection Of Two Kinds Of Mycotoxins In Agricultural Products By Protein Microarray

Mycotoxins are secondary metabolites produced by different fungi which contaminate a large variety of agricultural products and foodstuffs. They may not only decrease the quality of products, cause serious foodborne poisoning, but also lead to acute or chronic toxicity to human and animals, such as immune suppression, tissue necrosis, liver and kidney damage, reproductive disorder, carcinogenesis and cacogenics and so on. They have caused highly worldwide food security concerns owing to their threats to human and animal health. In recent years, the problems and risks associated with mycotoxins contamination of animal feed have promoted the development of a variety of analytical methods for their determinations including gas chromatography (GC), high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), etc. Though high sensitivity, chromatography analytical method has shortcomings:pretreatment cumbersome, complicated operation, and time consuming which make it difficult to be applied extensively. ELISA has become more and more popular screening tool of detecting mycotoxins because of its sensitivity, specificity, stabilization and simply, but the defect is that it can be tested only one mycotoxin for a time. However, as the agricultural products are always contaminated by many mycotoxins, not only by one kind of mycotoxin, the protein microarray technology with sensibility and high-throughput screening advantages has become the rapid detection tool for detecting many kinds of mycotoxins simultaneously. In this research, based on two kinds of mycotoxins (Ochration A and Fumonisin B1) complete antigens and their complementary monoclonal antibodies, indirect competitive operating mode were adopted with fluorescence as testing signal to explore the feasibility of detecting these mycotoxins in protein microarray.TestⅠ. Preparation and detection of OTA complete antigenIn this test, active ester method was carried out for the synthesis of protein conjugate of OTA with OVA for perparing the probe of protein microarray.2 mg OTA was dissolved in 0.5 mL of anhydrous THF (THF), and then 2mg N-hydroxysuccinimide (NHS),3 mg N, N-Dicyclohexyl carbodiimide (DCC) were added respectively. Oscillating reaction at room temperature for 24 h activation,10000 r·min-1 centrifuged 15 min, the supernatant was obtained and the sediment was abandoned. Vacuum drying the supernatant, the activation product residue was dissolved in 0.5 mL dimethylformamide (DMF). The solution was slowly dropped in the carrier protein OVA solution (15 mg OVA was dissolved in 2 mL 0.13 mol·L-1 NaHCO3). Oscillating reaction at room temperature for 4 h, after reaction, the product was dialysis in 0.01 mol·L-1 pH 7.4 PBS solution at 4℃for 72 h. Dialysate was concentrated by PEG 20000, using Coomassie brilliant blue protein assay kit to detect protein content.The complete antigen was detected by UV scanning, mass spectrometry and protein microarray analysis. The UV scanning result showed that with different wave type, OTA and OVA conjugates were observed significant displacement, so it indicated the successful coupling. According to the UV scanning, the conjugation ratio of OTA and OVA was analyzed. Under the wavelength of 280 nm and 379 nm, the A values was measured 0.389 and 0.875 respectively. So the conjugation ratio of OVA and OTA was 10:1. According to mass spectrometry, the conjugate molecular weight was 50350.141. The conjugation ratio was calculated 13:1. Protein microarray analysis also showed that specific reaction occurred between OTA McAb and conjugate, suggesting the existence of complete antigen. Through the detection, the concentration of conjugate protein was 1.28 mg·mL-1.TestⅡ. Preparation and detection of FB1 complete antigenIn this test, One-step glutaraldehyde method was carried out for the synthesis of protein conjugate of FB1 with OVA for perparing the probe of protein microarray.1 mg FB1 was dissolved in protein suspension by the magnetic stirring. And the same volume of glutarldehyde (2%, V/V) was dropwise added at room temperature for 1 h. The reaction was stopped with sodium borohydride with final concentration of 10 mg·mL-1 at room temperature for 1 h. Then, the product was dialysis in PBS at 4℃for 72 h by Magnetic stirring. Dialysate was concentrated by PEG 20000, using Coomassie brilliant blue protein assay kit to detect protein content.The complete antigen was detected by UV scanning, mass spectrometry and protein microarray analysis. The UV scanning result showed that OVA and conjugate had different wave type. So it indicated the existence of complete antigen. According to mass spectrometry, the conjugate molecular weight was 51928.612. The conjugate ratio of FB1 and the carrier protein OVA was calculated 9.6:1. Meanwhile, protein microarray analysis also showed that specific reaction occurred between FB1 McAb and conjugate, suggesting the coupling was successful. Through the detection, the concentration of conjugate protein was 1.08 mg·mL-1.TestⅢ. Peraperation and condition optimization of mycotoxin protein microarrayIn this test, ascites preparation technology and saturated ammonium sulfate precipitation method were used to prepare and purify a large number of OTA and FB1 McAb. First of all, two kinds of hybridomas were recovered with sterile culture.20 BALB/c female mice were selected and injected with sterilized liquid paraffin, each 0.5 mL.1～2 weeks later, the mice were injected with hybridoma cell suspension, each 0.5 mL, inoculation of 5×105 cells. During the test, the growth condition was observed everyday. About 10 d later, the ascites was sterile collected when the mice were swollen abdomen. Collected ascites was purified by saturated ammonium sulfate precipitation method. Through the detection of protein content, the concentrations of these two kinds of monoclonal antibody were 1.6 mg·mL-1 and 1.2 mg·mL-1 respectively.Orthogonal experiment was adopted to ensure the lattice concentrations of OTA and FB1 complete antigen and dilution ratios of monoclonal antibody, and optimize five single factors, including the fixing condition of complete antigen, blocking solution, blocking time, antigen-antibody reaction time and the concentration of goat anti-mouse IgG-CY3 secondary antibody. The results showed that the best lattice concentrations of OTA and FB1 complete antigen were 30μg·mL-1 and 40μg·mL-1 respectively; the dilution ratios of monoclonal antibody were 1:1600 and 1:1200 respectively.4℃overnight incubation,0.1% BSA blocking solution,37℃0.5 h blocking time,1 h antigen-antibody reaction time and 2μg·mL-1 goat-anti-mouse IgG-CY3 secondary antibody were the best conditions of these five single tests. Indirect competitive inhibition test was processed with OTA and FB1 standard samples to establish OTA and FB1 standard curves in protein microarray detection. The sensitivity (IC50) were 2.1 ng·mL-1 and 41.5 ng·mL-1, the linear range were 0.5～10 ng·mL-1 and 10～200 ng·mL-1 with good logistic correlation and linear correlation coefficient (R2>0.99) TestⅣ. Comparison of the detecting methods between protein microarray and HPLCIn this test, based on the detection of protein microarray of OTA and FB1, two kinds of mycotoxins were detected by protein array simultaneously. Indirect competitive inhibition test was processed with OTA and FB1 standard samples to plot OTA and FB1 standard curves in protein microarray detection. The sensitivity (IC50) were 0.795 ng·mL-1 and 20.1 ng·mL-1, the linear range were 0.1～5 ng·mL-1 and 6.25～100 ng·mL-1 with good logistic correlation and linear correlation coefficient (R2>0.99).By the spiked detection of samples, the lowest limitations were 0.5μg·kg-1 and 12.5μg·kg-1 respectively. When the concentrations of OTA standard sample were 1,5 and 10μg·kg-1, the average detection contents were 0.82,4.34 and 8.24μg·kg-1 respectively, the highest recovery ratio achieved 86.7% and the coefficient of variation was less than 10%. When the concentrations of FB1 standard sample were 20,50 and 100μg·kg-1, the average detection contents were 16.46、44.75 and 85.72μg·kg-1 respectively, the highest recovery ratio achieved 89.5%, the coefficient of variation was less than 10% with good logistic correlation.The HPLC methed for the detection of OTA and FB1 was developed, and the corresponding standard curves were plotted. The detection range of two kinds of mycotoxins were 1～100 ng·mL-1 and 0.1～20μg·mL-1 and the linear correlation coefficient (R2) were 0.9999. By the spiked detection of samples, the lowest limitations were 1μg·kg-1 and 50μg·kg-1 respectively. When the concentrations of OTA standard sample were 5,10 and 50μg·kg-1, the average detection contents were 3.76,9.08 and 43.2μg·kg-1 respectively, the highest recovery ratio achieved 90.8% and the coefficient of variation was less than 10%. When the concentrations of FB1 standard sample were 50, 100 and 500μg·kg-1, the average detection contents were 37.5,84.5 and 411.5μg·kg-1 respectively, the highest recovery ratio achieved 84.5%, the coefficient of variation was less than 10% with good logistic correlation.46 samples of corn, wheat and rice collected from Nanjing, were extracted and purified. Then protein microarray and HPLC were carried out for detecting the concentrations of OTA and FB1 in these samples. The protein microarray result showed high levels of toxins in corn and the highest levels of OTA and FB1 were 9.58μg·kg-1 and 469μg·kg-1 respectively. In the three kinds of samples (corn, wheat and rice), the average detection rates of these two kinds of toxins were 23.4% and 29.8% respectively. The HPLC result showed the levels of toxins in corn were 9.75μg·kg-1 and 438μg·kg-1. In the three kinds of samples, the average detection rates of these two kinds of toxins were 19.3% and 21.2% respectively. By comparison, the detection limit of protein microarray was obviously lower than the HPLC method. While the highest detection value were similar of these two methods, but the detection ratio of OTA and FB1 using the HPLC method was lower than the protein microarray method. It demonstrated within the linear range, the protein microarray can be used for detection of both mycotoxins simultaneously in agricultural products.