Preliminary Study On Testing Method Of Three Mycotoxins By Protein Microarray And Preparation Of Anti-TC Polyclonal Antibodies

1 Mycotoxin is the secondary metabolic product of fungus,often existing in moldy grains.Once entering human's body,it may cause serious danger,like deformity,mutation or cancer and so on.Therefore,it is fully concerned in global food security.At present,HPLC and ELISA are adopted in the test of Mycotoxin,of which the former is of high sensitivity but the pretreatment of the sample is trivial and the manipulation is so complicated that it needs more time,while the latter is easy to operate but it can test only one for a time.So protein microarray technology of high throughput will be the tendency of the rapid test of the protein microarray.Based on three prepared mycotoxins(ZEN,AFB₁ and DON)artificial antigen and related monoclonal antibody,this research adopts similar indirect competitive ELISA operating mode,to do research into ZEN,AFB₁ and DON protein microarray method with fluorescence as testing signal.The results are as follows:(1)The research of single standard testing method of ZEN,AFB₁ and DON protein microarrayOrthogonal experiment is adopted to ensure the lattice concentration of ZEN,AFB₁ and DON:40μg/mL,40μg/mL and 20μg/mL.The single factor optimization of the fixed method of artificial antigen,blocking mode and the second antibody concentration determines that 4℃overnight incubation,37℃20 min blocking and 2μg/mL goat-anti-mouse second antibody IgG-CY3 are the best conditions of the three single factor,which primarily establishes the manipulative mode of protein microarray detcetion.Respective indirect competitive restrain test,with the samples of ZEN,AFB₁ and DON enables us to draw the standard curves of ZEN,AFB₁ and DON protein microarray detection,and the sensitivity(IC₅₀)are 2.1 ng/mL,0.29 ng/mL and 86.8 ng/mL respectively and the linear range are 0.5-10 ng/mL,0.125-1.0 ng/mL and 50-400 ng/mL respectively. (2)The research of simutanous testing method of ZEN,AFB₁ and DON protein microarrayBased on the first result,the research of triple standard testing method of ZEN,AFB₁ and DON protein microarray is conducted,thus determining the best concentration of goat anti-mouse IgG-CY3 is 4μg/mL.Respective indirect competitive inhibition test,with the samples of ZEN,AFB₁ and DON,make it possible for us to draw the standard curves of ZEN,AFB₁ and DON by protein microarray detection and the sensitivity(IC₅₀)are 1.3 ng/mL,0.154 ng/mL and 70.4 ng/mL respectively and the linear range are 0.75-6 ng/mL,0.125-0.6 ng/mL and 25-200 ng/mL respectively.2 Tetracycline is an alkaline broad-spectrum antibiotic produced by streptomyces.Because of its low price and good result of antibacterial,it is widely used in poultry breeding industry,but it unavoidably has great potential possibility of tetracycline residue in animals' bodies.A little the residue though it exists,if eaten for a long term,it may cause great potential danger to human's health.So the timely test of the residue of teracycline is an effective way to prevent and control it.In order to establish the method of immunological detection of Tetracycline,the research conducts the preparation for anti-tetracycline polyclonal antibodies.The results are as follows:Mannich reaction is used to prepare TC-cBSA and TC-OVA.After the identification of UV-spectrogram,back subcutaneous immunization is done on three BALB/C female mice,with TC-cBSA as immunogen.By strengthening immunization three times,with TC-OVA as testing detectable antigen,indirect ELISA is used to test the titer of antiserum and indirect competitive ELISA to test the specificity of antiserum.Competitive inhibition curve indicates the three mice all have anti-TC polyclonal antibodies,of which the No.2 mouse gets the highest antiserum titer 1:32000,half inhibitory concentrations(IC₅₀)is 173.78 ng/mL.The cross reaction rate between this antiserum and oxytetracycline is 44.7%,and that between streptomycin,chloramphenicol and amoxicillin are all less than 0.01%.