Studies On Enzyme-Linked Immunosorbent Assay For The Pyrimethanil

Pyridamil is a kind of aniline fungicide with both internal absorption and fumigating effect.Because of its good control effect on the grey mould of cucumber and tomato and other vegetables,the irrational application of pyridamil residues in fruits and vegetables often exceeds the standard,thus endangering the environment and human health.At present,LC-MS,GC,BS and TLC are used to detect the residues of pyrimimamine,but these methods have the disadvantages of complex pre processing,high requirements for professionals and expensive equipment.Therefore,it is necessary to establish a fast,sensitive and efficient detection method for pyrimidine.Enzyme linked immunosorbent assay?ELISA?has advantages as well as low cost and suitable for rapid on-site detection of large quantities of samples.Therefore,enzyme-linked immunosorbent assay?ELISA?is of practical significance for the determination of pyrimidine residues.This study explored the synthesis route of four azimipyridine hapten?PMTH?,and finally determined that p-nitroaniline and 2-chloro-4,6-two methyl pyrimidine were used as starting materials to synthesize azimipyridine hapten by amino substitution,nitro reduction and ester ammoniation,and confirmed by ¹H-NMR,TLC and MS.PMTH and bovine serum protein?BSA?were coupled with mixed anhydride method;PMTH and chicken ovalbumin?OVA?were coupled by active ester method.The coupling was confirmed by UV.The coupling ratio of PMTH-BSA?immunogen?and PMTH-OVA?envelope antigen?was successful,in which the coupling ratio of PMTH to BSA was 14:1,and the coupling ratio of PMTH and OVA was 6:1.The synthetic pyrimidine immunogen and Freund's adjuvant were fully emulsified to the W/O state.The adult male new blue rabbit was immunized with the adjuvant antigen of the W/O emulsification state.The first immunization was used by the Freund complete adjuvant and the effective dose of the immunogen was 0.5mg.The rabbits were immunized at intervals of two weeks.The other immunization times were treated with Freund's incomplete adjuvant.The effective dose of the immunogen was 175?g.When the rabbit serum reached a reasonable titer,the whole blood could be collected.The titer of the last immunized rabbit serum was 64000 by indirect ELISA.The cross reaction rates of antiserum with fluoridazine,chlorphenazine,pyrimidimoxime,dioxethers,cyczoxoxethers,azoximethers and atrazine were less than 1%,and the specificity of antiserum was better.In this experiment,the antibody was purified by the method of octanoic acid ammonium sulfate precipitation,and the optimum working concentration was determined with the concentration of coated antigen of 0.8 mu g/m L and the dilution ratio of antibody to 4000.The antibody was diluted to a certain concentration and packed in a number of 1m L centrifuge tubes,so as to facilitate the follow up and use.To determine the optimal working conditions of the kit,two in distilled water and normal saline,sodium carbonate,phosphate,borate and phosphate citric acid hydrogen six buffer,to determine the concentration of carbonate buffer 0.05mol/L,pH=9.6 kit as the best buffer;in 0.1,0.2,0.4,0.6,0.8 and 1?g/m L six concentration gradient coating antigen,identified 0.6?g/mL as the best concentration of coating antigen concentration was 1;temperature and 2,4,8,and 12h coated temperature in 4^oC and 37^oC two package,37^oC package is a combination of 3h to determine the optimal coating concentration and temperature,if not considering the time factor,can choose 4 ^oC package by 12h;at the concentration of PBS solution is 1%gelatin,skim milk powder,BSA and OVA,identified 1%gelatin PBS as the best material in 1000,closed;2000,4000,8000,16000 and 32000 six two anti dilution,1:2000 As the best dilution ratio of two resistance,2mol/L is selected as the best concentration of termination liquid in 0.5,1,1.5,2 and 2.5mol/L sulfuric acid,and the indirect competition ELISA is the best competition type in screening the best competition type.HPLC confirmed that in ELISA test and recovery test,the average recovery rate of ELISA was 96.58%,and the average recovery rate of HPLC was 104.28%,and the coincidence rate was 92.62%.According to the standard curve of pyrimidine,the value of IC₁₅ was 0.01g/m L,which was determined as the detection limit and sensitivity of the kit.Intra plate variation and interboard test results showed that10-10⁶ng/mL had good linearity,indicating that the kit had good accuracy.Three samples of cucumber,grape and tomato were tested,and the test results showed that the recovery rate of the kit was 98.35%±11.84%.It meets the requirements of accuracy.