Development And Priliminary Application Of The Monoclonal Antibody Against Patulin

Patulin(Patulin,PAT)was a mycotoxin,which were produced by penicillium,sickle bacteria and aspergillus.It had been found in the fruits,cereals,cheese,bacon,and other food.PAT was widely disseminated in nature,especially in corruption,deciduous hawthorn,apple and other fruits and their products.And it had the toxicity of genetic material damage and destruction of the intestinal mucosa cell,etc.To ensure the food safety,and establish a rapid detection method which was high sensitivity,simple operation,low price,high efficiency and can deal with large quantities of samples in detecting patulin.After the analysis of molecular structure and physicochemical properties of PAT,we used the technology of artificial antigen synthesis and monoclonal antibody,and assembled the rapid detection kit by indirect competitive enzyme-linked immune sorbent assay(ELISA).The main research was as follows: 1 Preparation and identification of artificial antigenWe used the method of carbonyl imidazole to couple hapten molecules with egg white protein(OVA)and the method of mixed anhydride to couple PAT with bovine serum albumin(BSA),then we got the complete antigen PAT-OVA and PAT-BSA.We used the method of ultraviolet scanning(UV),sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and ELISA to identify artificial antigens and get the sensitivity and titer of mices’ polyclonal serum.The UV scanning spectrum showed that comparing with the scantling and carrier protein two ultraviolet absorption peaks of the artificial antigens obviously displaced.Gel electrophoresis showed that two kinds of artificial antigen were weighter than carrier protein molecule.What’s more,the antiserum was susceptibility to PAT obviously.Above all,the preparation of artificial antigen was successful.2 Preparation and identification of monoclonal antibodyAfter immunization of 3 Balb/c mice by artificial antigen,we selected 1 mice which had the high titer and curbing good to improve the cell,its spleen cells fused with tumor cell NS0.Then,HAT medium was used to screen and culture hybridoma,PAT-OVA was used to detection the supernatant of hybridoma.Finally,we screened the hybridoma cell lines which can secrete monoclonal antibody with strong specificity and good sensitivity.The titer of our hybridoma’s supernate was 1:8.0×102.Hybridoma was administered by intraperitoneal injection to mice,the titer of ascite was 1:5.12×105.After appraisal by indirect competing ELISA,we got the half inhibition(IC50)of monoclonal antibody to patulin(anti-PAT)was of 29.69 ng/m L.3 Establishment of the rapid detection methodAccording to the principle of enzyme-linked immunosorbent assay,we assembled an indirect competing ELISA detection kit,which was used to detect the content of patulin in apple juice.The optimal concentration of PAT—OVA was 0.4μg/m L,the the optimal concentration of anti-PAT was 0.5μg/m L,and the detection range was 4.14 ~ 4.14 ng/m L,half inhibitory concentration was 13.14 ng/m L,the detection limit was 2.82 ng/m L,the testing experiments can be completed within 1 h.The recovery rate of our reagent kit was 93.34%,and the kit could be saved for six months under 4 ℃ condition.We made a kind of immune chromatography test strip which was a prompt,half quantitative detection of PAT in apple juice.It had the character of accuracy and repetition rate higher.The strip provided a quick,convenient and able to bulk samples detection technology of PAT test out of the laboratory conditions.