Development Of An Immunoassay For Ochratoxin A And Identification Of NJA-1, A Biotransformational Microorganism For Vomitoxin

Mycotoxin was secondary metabolite by spicies of some fungi, and contaminated a wide range of food and feed. Many kinds of mycotoxin existed in nature with different and complex toxicities which would cause acute or chronic toxicity to human and animals. Ochratoxin A (OTA) and vomitoxin (VT), two major membors of mycotoxin, could pose hazard on human health and be harmful to agricultural economics development. Immunoassay detection and biotransformation degradation were regarded as two major important measures for prevention and detoxification, therefor their reseach work was significance.Enzyme linked immunoassay, a classic representation of immunoassay, was not only easier to use than physics and chemical tests, but also more economical than microassay tests. ELISA detection for mycotoxins would have a very good application perspect.The single chain fragment antibody was a member of genetic engineering antibody, has been regarded as one direction of immunoassay development, because of its stable and easy to save nature. The study of single chain fragment antibody would be the basis work for its immunoassay application.Biotransformation was a measure of detoxification. The indentification and classification were major parts of biotransformation research. It was necessary to taxonomy and study on characteristic of biotransformation microorganism named NJA-1.This research was aiming to develop an enzyme-linked immunosorbent assay based on monoclone antibody for OTA determination, to produce the single chain fragment variable region antibody of OTA as a component for immunoassay, and to identify NJA-1 strain which had an ability biotransformation for vomitoxin.Test I Preparation of a monoclone antibody for OTAUsing active ester method and carbodiimide method, prepared six complete immunogens synthesized of OTA conjugated with 3 proteins, respectively: keyhole limpets hemocyanin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA). The absorption was tested to caculate the ratio of OTA to protein in conjugation using ultraviolet spectrum scan method. Compared the ratios of conjugation with different carrier proteins prepared by the same method, and compared the ratios of conjugation with the same protein prepared by the different methods. The antibody titers in mice serum were determined which were immunized with six complete antigens, respectively. According to the ratios of conjugations and the titers of mice, the superior carrier protein and the better prepared method for OTA were concluded. Immunized mice with OTA-BSA, prepared the spleen cells and complished a fusion with Sp2/0. Screened the optimize hybridoma cells using indirect ELISA. Test the group of antibody and the isotype of light chain and verified the weight of Mab by SDS-PAGE.The results showed that the active ester method was better than the carbodiimide method by comparing the conjugations ratio and the titer of immunized mice for the same protein as carrier to OTA, and KLH was the best carrier protein for OTA than OVA and BSA by the same preparation method. Four positive hybridoma cells were obtained after the fusion of spleen and Sp2/0 and ciELISA screening, one cell named D6D was been studied. The group of anti-OTA monoclonal antibody was IgG2b and the light chain wasκ. Two peptide chains were presented of anti-Mab in SDS-PAGE, about 25kD and 50kD, respectively, which consistant with the size character of IgG after denaturation.TestⅡDevelopment of an enzyme-linked immunosorbent assay for ochratoxin AOptimized the key reaction conditions of indirect complete enzyme linked immunosorbent assay (ciELISA) for OTA, including the coating time and temperature, the incubation time and temperature of antigen and antibody, and the reaction time of substrate. Established the stand curve of ciELISA for OTA. Determinated the 50% inhibition concentration of OTB, AFB1? FB1 and VT and calculated the cross-reaction ratio, both indicating the specificity of ciELISA. By spicked 2.5,5,10 ng·g-1 OTA to corn samples, respectively, calculated the recovery and coefficient of variability of intra and inter-batch, indicated the accuracy of ciELISA.65 samples of agricultural products:corn, rice and wheat, collected from Nanjing, were detected by ciELISA.The results showed that the optimization condition of ciELISA were 4℃overnight for coating,37℃incubation 1h for antigen and antibody reaction, and 10 min for substrate reaction. The 50% inhibition concentration (IC50) for OTA was 1.70 ng·mL-1, ranging from 0.55～6.75 ng·mL-1, limitation of detection was 0.15 ng·mL-1. The IC50 for OTB was 10 ng·mL-1, cross-reaction(CR) ratio was 17%. However, the cross-reaction for VT, AFB1 and FB1 all were below 10%. All of these datas indicated the ciELISA for OTA was specific. The recovery was 89～105%, with the coefficient of variability of intra and inter-batch were below 4% and 8%, which indicated the high accuracy and stability of the ciELISA for OTA. The survey of corn, wheat and rice in Nanjing showed the frequency of contamination was 26.08%,36.36% and 15.00%, with the mean concentration was 1.920, 1.545 and 0.407 ng·g-1, respectively,. Among the samples contaminated by OTA, one sample was detected over 10 ng·g-1 and two samples above 5 ng·g-1. Other 62 samples were detected to be lower than 5 ng·g-1, the limitation of contamination by OTA in European Union.Test III Production of a single chain fragment variable region of anti-ochratoxin A antibody.This test was processed to design the primers for colone the variable region of heavy chain and light chain of mouse monoclonal antibody. The overlap extension PCR (soe-PCR) was used to conjunct the heavy chain fragment and the light chain fragment with a 15 peptide linker to form the single chain fragment variable region (ScFV) antibody. The cloning vector pMD18-T-ScFV was constructed, following with ScFV sequenced. The expression vector pET-22b(+)-ScFV was constructed as well and ScFV was expressed in BL21 (DE). The Expazy-prot software was applied to predict the physico-chemical property of ScFV.The results showed that the gene of heavy chain variable region and the gene of the light chain variable region were about 300～400 bp and 200～300 bp, respectively. The ScFV was about 500～700 bp. Sequencing results indicated that the gene coded 203 amino acids, including the FR１, CDR１, FR2, CDR2, FR3 regions of both the variable region of heavy chain and the light chain. The variable region of heavy chain and the variable region of light chain were connected with 15 peptides. The ScFV protrein was expressed as inclusion body form in BL21 (DE) prokaryotic expression system. The physico-chemical property of ScFV predicted by Epazy-prot software as follows: molecular weight was 21250.2, pI was 7.85, the half-time in Escherichia coli. was 10 h. ScFV was recognized as unstable protein.TestⅣIdentification of NJA-1, a biotransformational microoganisim for VomitoxinThe NJA-1 strain had the ability of biotransformated Vomitoxin. Using the traditional morphological method, described and classified the NJA-1 strain, including colony morphology, colony growth characteristics, strain morphology, etc. Using the uniform pairs of primer, ITS domain sequence of NJA-1 was amplified and blasted in NCBI GeneBank to compare and assessment the taxonomy of NJA-1. Cultivation conditions were optimized in the carbon source, the nitrogen source, and the pH of medium.The results showed that NJA-1 presented the way of asexual spores, with carbon black conidial heads in shade of globose, smooth brown conidiophore hyaline, conidia in shade of globose, echinulate or verruculose, in chain. According the characters, NJA-1 was classified as a genus of Aspergillus niger group. The phylogenetic tree constructed by rDNA ITS sequence with comparing the sequence in NCBI GeneBank, demonstrated the genetic relationship of NJA-1 with Aspergillus tubingensis greater than 99.7%. It was concluded that the genus of NJA-1 was Aspergillus niger forma tuebingensis. The optimization of cultivation conditions was glucose as carbon source, ammonium chloride as nitrogen source, in pH 4 medium, cultivate for 10 d, the NJA-1 appearanced the best growth performance.