Development Of Monoclonal Antibody Against Fumonisin B 1 And Its Establishment

Fumonisins is a kind of double ester metabolites produced by Fusarium moniliforme, So far, 28 kinds of Fumonisins have been discovered. The main component of Fumonisins is Fumonisin B1, which usually appears in grain crops and feed. Fumonisin not only has the neurotoxicity, liver and kidney toxicity, but also has the lung toxicity and carcinogenicity. Moreover, Fumonisin also has serious threat on the development of animal husbandry and human health. Several methods are known to detect FB1 such as the thin layer chromatography(TLC), gas chromatography(GC) and High Performance Liquid Chromatography(HPLC) etc. A rapid, simple and sensitive detection method for FB1 is of practical significance while all the methods mentioned before require special instruments, much time and professional operations. This study used monoclonal antibody preparations and enzyme labeled immunosorbent assay(ELISA) to detect the FB1, which have characteristics such as easy operations, low cost, strong specificity, high efficient and are suitable for rapid detection of large quantities of samples.1. Synthesis and identification of FB1 artificial antigens. According to the characteristics of the molecular structure of fumonisin B1, FB1 was conjugated with BSA and OVA proteins by GA method to compose the immune antigen FB1-BSA and detect the antigen FB1-OVA. We could preliminary believe that the FB1 was coupled with carrier protein successfully by UV scanning, SDS-PAGE and immunological method. The protein concentrations of FB1-BSA and FB1-OVA were 0.7 mg/mL and 1.83 mg/mL with the help of BCA method.2. Preparations of the anti-FB1 monoclonal antibodies.6-8-week-old female BALB/c mice were immunized with FB1-BSA as a routine immunization. After the fifth immunization, the titer of blood serum of these mice were over 1:10,000. The spleen cells of immunized mice were taken to mix up with the SP2/0 on the third day after the strengthened immune. The positive hybridoma were screened using the HAT medium and enzyme-linked immune technology. With the help of the limited dilution method, we got 2 strains hybridoma cells of secreting anti-FBI monoclonal antibodies which named 5F5 and 2C6 both with the titer of 1:256000. The concentration of Ascites protein were 10.79 mg/mL and 12.21mg/mL respectively.3. The establishment of ELISA detection method.5F5 hybridoma cells were selected to product monoclonal antibodies and establish indirect competitive ELISA detection method for FB1. After using the matrix method, the optimal working concentration of antigen was found as 1:2,000 and the optimal concentration of antibody was 1:8,000. The linear equation:y=0.1648x +0.3706 (R2=0.9845) was determined after drawing the FB1 inhibition standard curve. The IC50 was 6.1ng/mL, LOD was lower than 0.05ng/mL. The cross experiment showed that, the monoclonal antibody had an inhibition rate of 100% for FB1, and it had no cross reaction with Voitoxin, Zearalenone, Aflatoxin B1 and α-Zearalenol.4. Recovery test. The FB1 recovery test was performed to the feed corn through applying the indirect competitive ELISA method. The results of the simulated extraction of corn samples of FB1 showed that, when the extracting solution was diluted for 8 times, the intervention disappeared. In the rang of 0.05～500ng/mL, a linear equation of y=0.1381x+0.4225(R2= 0.9822) was obtained. The recovery rate was 84.2-102.6% and the coefficient of variation was 2.48%～8.79% when adding standard FB1 of 5,50,200,500ng/mL to the corn.