| Sulfonamides(SAs)is a class of chemical drugs for the prevention of bacterial infection of human and animal diseases,because of its stability,cheapness,broad-spectrum antibacterial,is used widely for a long time in veterinary clinic and feed additives.However,due to the abuse of sulfa drugs is more serious,resulting in excessive residues in the animal body,and then through meat,eggs,milk and other animal foods intake of the human body,the solubility of the product is low,and it is excreted by the kidney.If the urine is acidic,it can be crystallized in the renal pelvis and ureter,which can cause the obstruction of urinary system.It also can cause allergic reactions,bacterial resistance,hematopoietic system disorders,teratogenic,carcinogenic and other hazards.SAs residues in animal food is one of the most serious veterinary drugs residues in the past decades,and it is also a major obstacle in the import and export trade of animal foods.Therefore,each country pay attention to the study of SAs residue detection,and the ministry of agriculture of China has also made the SAs residue in animal food as a monitor focus following the clenbuterol.Detection method of SAs are commonly used in microbiological assay,high performance liquid chromatography,high performance liquid chromatography-mass spectrometry,diode-high performance liquid chromatography,high performance liquid chromatography fluorescence spectrometry,UV spectrometry and high performance liquid chromatography,thin layer chromatography etc..However,the physical and chemical detection methods are expensive,the process is complex,and the requirements of operators are higher,some of the methods have the disadvantages of poor anti-interference ability,low accuracy and so on.The residues of SAs also can be detected by radioimmunoassay,enzyme-linked immunosorbent assay,immunochromatography assay and immunosensor method.These methods have their own advantages,but still need to be further improved,improve the stability and specificity of the antibody.To simplify the sample processing and operation steps,high throughput and multi residue screening will be the main direction of development the rapid detection of SAs residues in the future.This study used the most serious three SAs residual sulfadiazine(SD),sulfamethazine(SM2)and sulfamethoxazole(SMZ)as the research object,three kinds of SAs artificial antigen were prepared and hypodermic injected BLAB/c mice.Based on the SD,SM2 and SMZ monoclonal antibody,we prepared single and triple test strip and used to detect the three SAs.The strip able to complete the SAs screening of samples in a short period of time,provided a new method for immunology rapid detection technology of SAs,also provided an effective monitoring technical support for SAs residues in animal products.The main results of this study are as follows: 1.In this study,the artificial antigens of SD,SM2 and SMZ of three kinds of SAs were prepared by diazotization method.Adopt appraisal by full wavelength UV scanning and SDS-PAGE gel electrophoresis,the characteristic absorption peaks of SD,SM2,SMZ and BSA were significantly different from those of SD-BSA,SM2-BSA,and SMZ-BSA in UV scanning,the mobility of SD-BSA,SM2-BSA,SMZ-BSA was less than SD,SM2,SMZ and BSA were identified by SDS-PAGE gel electrophoresis.The results of the two methods showed that the coupling preparation of the artificial antigen had a good effect,and provided the basis for the preparation of the antibody.2.In this study,three kinds of SAs antigens were prepared,SD-BSA,SM2-BSA,SMZ-BSA were injected subcutaneously to immunize BLAB/c mice,through the rational design of immunization procedures and methods.After the four immunization,tail tip blood of mice were collected and the mice antiserum identified by ELISA.Determination of the titer of antiserum by indirect ELISA,results showed that the titer of BLAB/c antiserum which injected SD-BSA were greater than 1:1.28 × 104,the titer of BLAB/c antiserum which injected SM2-BSA were greater than 1:2.56×104,the titer of BLAB/c antiserum which injected SMZ-BSA were greater than 1:1.28×104.The antiserum sensitivity was determined by competition ELISA,the result showed that the SD-BSA,SM2-BSA and SMZ-BSA mice antiserum IC50 were 31.9 ng/m L,22.8 ng/m L and 47.1 ng/m L.The test showed three SAs of SD,SM2,SMZ antiserum were high titer,high sensitivity,and high specificity.3.According to the identification results of immunological characteristics of mice antiserum,the best characteristics mice(No.6 of SD mice,No.1 of SM2 mice and No.5 of SMZ mice)were selected as target mice for fusion.Then the spleen cells were fused with SP2/0 myeloma cells by hybridoma technology,with several repeated and repeated ELISA screening,1B3,1G9,2F11,3D2 and 4D8 five strains of SD monoclonal antibodies were obtained,in which the 2F11 property was the best,the titer was 1:5.12×105,the affinity constant was 2.13×1010 L/mol,the half inhibitory concentration was 4.36 ng/m L;2E7,2F10 and 4B11 three strains of SM2 monoclonal antibodies were obtained,in which the 2E7 property was the best,the titer was 1:1.024×106,the affinity constant was 2.96×1010 L/mol,the half inhibitory concentration was 1.71 ng/m L;1C6,1F7,3G6 and 5D10 four strains of SMZ monoclonal antibodies were obtained,in which the 3G6 property was the best,the titer was 1:5.12×105,the affinity constant was 9.26×109 L/mol,the half inhibitory concentration was 9.19 ng/m L.4.In this study,the three monoclonal antibody of 2F11,2E7 and 3G6 not only have high efficiency,high affinity and high susceptibility,but also three subtypes of monoclonal antibodies are Ig G1 type,and with similar SAs drugs,carrier protein BSA and OVA,as well as beta agonist drugs had no obvious cross reaction showed that three strains of monoclonal antibodies have high specificity.2F11,2E7 and 3G6 three monoclonal antibodies with high specificity can be used to establish a rapid screening method for SAs drug residues.5.The study based on the colloidal gold nanoparticles which preparation of citric acid three sodium method,using the pre prepared antigens and high specific monoclonal antibodies,developed single test strip and triple test strip for SD,SM2 and SMZ residues detection.Performance test of test paper the positive detection rate was 100%,when the concentrations of SD,SM2 and SMZ were greater than 50 ng/m L,20 ng/m L and 50 ng/m L,respectively.Results show that detection sensitivity of the test strip for SD,SM2 and SMZ were 50 ng/m L,20 ng/m L and 50 ng/m L,respectively.The test results show that the test paper has the characteristics of high sensitivity,strong specificity and strong stability,and can be used for the rapid and accurate detection of three kinds of SAs residues in milk,muscle and feed samples. |
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