Preparation Of The Monoclonal Antibody Against Citrinin And Application In Hongqu

Hongqu is a traditional fermented food in China for thousands of years,which is produced by Monascus and has been widely used to produce food fermentation starter, food colorant,food antiseptic,food flavor enhancer and Chinese traditional medicine. However,Professor Blanc discovered and demonstrated that some strains of Monascus spp.could produce citrinin.The mycotoxin citrinin has nephrotoxic effects and genotoxicity,hence this triggered a controversy about the safety of Hongqu.Up to now,the major ways to detect citrinin need large-scaled apparatus and exorbitant expenses.So it is urgent to establish a cheap,convenient,quick method for the qualitative and quantitative analysis of citrinin and can be applied to practice.In this paper,citrinin-protein conjugates were prepared by coupling citrinin to BSA or OVA byuh activated ester method,then through the hybridoma technology to prepare a specific McAb to citrinin using.With the indirect competed ELISA,the method for detecting citrinin in Hongqu product was established.The main results were followed.1.Preparation for CIT-BSA(OVA)Due to its low molecular weight(250.3),citrinin(CIT) is a hapten with only reactiongenicity and no immunogenicity.It can stimulate animal to produce antibody only after it is changed into complete antigen by conjugating with carrier-protein.In this study,activated ester method was applied to construct the CIT complete antigens by conjugating with bovine serum blbumin(BSA) and ovalbumin(OVA),respectively, because of the active carboxyl belong to citrinin.In this foundation,UV-Vis and FT-IR were applied to analyze,which the calculation of conjugate molar rations were immunogen(CIT-BSA,8.4:1) and coating antigen(CIT-OVA,6.3:1),respectively. 2.Preparation the monoclonal antibody and establishment of ELISAThe splenic cells from BALB/c mice immunized by CIT-BSA were fused with the murine Sp2/0 myeloma cells.As a result,the fusion ratio of the cells was up to 61.3%.Two hybridoma cell lines(named as 5H1 and 7B7,respectively),which were capable of continually secreting high specificity and sensitivity McAb against CIT, were screened out by the limited dilution method.Working parameters in indirect ELISA technique for detection of CIT was optimized,including selection of coating method,optimum dilution ratio of ascites. An indirect ELISA method for the detection of CIT was developed.Calibration graphs was formed by the logarithm of the CIT concentration(x) against the inhibitory rate(y),the equation was as follow:y=-7.14x+72.97,(R²= 0.962),y=-7.83x+77.56,(R²=0.982),the half inhibitory concentration(IC₅₀) reached 24.95 ng/mL and 33.78 ng/mL,respectively.The linearity range for detection of CIT were both 2 ng/mL～200 ng/mL.Different cross-reactions with the toxins in our laboratory(AFB₁,AFB₂,AFG₁, AFG₂,AFM₁ and patulin) were observed,which results described the antibodies from 5H1 and 7B7 with no cross-reactions with CIT.So indicated the antibodies were nice specific.3.Recovery test of CIT in Hongqu with indirect competed ELISAThe spiked recoveries(0.05μg/g～0.5μg/g) were used to represent the accuracy of this CI-ELISA.Therefore,citrinin in samples of Hongqu were detected using the optimized CI-ELISA.In these results,the average recoveries of spiked Hongqu were from 83.2%to 94.4%,with coefficients of variation(CVs) between 6.3%～9.6%.The other,selected 28 Monascus species from different sources,with the ELISA and HPLC together,found the results between them with little differences.