Study Of Substitution Technology For Ochratoxin A In Immunoassay And Development Of Innocuous ELISA-Kit Quantitative Analysis

Background:The ochratoxin is produced by Aspergillus species andPenicillium species ,inchulding ochratoxin A,B,C,D. The ochratoxin A is themost poisonous and the worst contamined to crops.Being the closest tohuman,it is also distributed the most widly.As a mytoxin ,it has strong effect ofhepatotoxicity and nepHrotoxicity and also has effect of carcinogenicity,teratogenecity and mutagenicity.Ochratoxin A was first discovered in maize and was considerd naturalcontaminant of maize.then it also was foud in many other crops andfoodstuff ,for example corn ,dry fruit,grape,wine,coffee,cocoa, legume,beer,tea etc.Human being exposure to OA through taking crops and animalorganism polluted by OA.OA could affect kidney and liver function ofhuman and animal,and induces disease .Ochratoxin A could pollute many kinds of corns and foods.To the point of pollution rate,corn and its productions ,chocolate and meatare higher than others. But to the point of pollution degree,The contaminationof corn ,chocolate and meat is higher than others. Our country is powerful inagriculture. The residents, traditional food relies mainly on the corn and it'sproductions.So it is very important to establish a safe and reliable detectionmethod.The enzyme-linked immunosorbent method(ELISA) has high sensitivityand high specificity,and it can simply and quicly detect a great dealsamples at the same time . General evaluating,it has extensively potential.But this method has a common problem in detection process,thatpersons must contact OA of high concentration. Because OA has stronglyhepatotoxicity ,nepHrotoxicity and also has carcinogenicity,teratogenecityand mutagenicity,detection persons has potential fatalness if touching OA for along time.In addition to,the higher price of OA results in higher cost .A newimmunity reagent-anti-idiotype anti-body has mirror image with OA inspatial structure ,so we can substitute OA in immunoassay and realizeinnocuous detection and reduce cost.Purpose:To substitute ocharatoxin A in enzyme-linked immunosorbentmethod (ELISA)through preparing anti-idiotype anti-body and establishinnocuous ELISA –kit to detect OA in food supplies and corns.Method: 1. The hybridoma cell line A9B5 excreting monoclonalanti-body againt ochratoxin A was resuscitated;The ascites monoclonalanti-bodies were obtained by hybridoma cloning technology and inoculatinghybridoma cell into abdominal cavity of BALB/c mice.Chracater ofmonoclonal anti-bodies were identified by ELISA.2. The F(ab')2 fragments were prepared by digestion of ficin andseparated by FPLC;The character was identified by ELISA.3. The polyclonal an-idiotype anti-bodies against monoclonal anti-bodyof OA were prepared by immunising rabbits and identified by ELISA.4. The innocuous ELISA –kit of OA was developed with the polyclonalan-idiotype anti-body and the parameters of the kit were studied.Results:1.The ascites monoclonal anti-bodies were obtained byhybridoma cloning technology and inoculating hybridoma cell into abdominalcavity of BALB/c mice.2.characterization of monoclonal antibody :antibody subclass IgG1,ascites value 1:1.28×106;molecular weight 150kD;reference workconcentration 1:2.56× 105;protein concentration of IgG 6mg·ml-1;nocross-reaction with other analogue;affinty constant 7.94×10-10L· mol-1。3.using ficin digestion to gain F(ab')2 fragment, characterization:molecular weight 100 KD;reference work concentration 1:5.12× 104;proteinconcentration of fragments 7mg·ml-1;affinty constant 3.14×10-10L· mol-1。4.characterization of anti-idiotype antibody : antibody subclass Ab2β,nocross-reaction with other analogue .affinity constant is 4.48 × 108L· mol-1.5.prepare the corresponding curve between anti-idiotype antibody(AID )and OA, logarithmic transformation equations: Y=1.2651914X-3.2597689,R2=1.0000.6.innocuous ELISA -kit ,parameters :anti-idiotype antibody(AID )substitute OA ,competition standard curve : Y=-0.5488204X-1.26981,R2=0.9938. AID detection limit 993ng·ml-1,linearity range 546.875～17500ng·ml-1,50% inhibition ratio 5260ng·ml-1。Logarithmic transformationequations with standard toxin,toxin detection limit 3.40ng·ml-1,linearity range1.6～128.38 ng·ml-1。50% inhibition ratio 16.3ng·ml-1。Recovery test for wheatincluded three level: 10ng·ml-1,20ng·ml-1 and 50ng·ml-1.recovery range :80.97%～108.58%,average recovery is 95.60%,101.58%,94.91%,CV is15.06%,5.11%,6.69%,4℃ to preserve stably, 37℃ to preserve for half ayear.