Preparation Of Monoclonal Antibodies Against Indomethacin And Initially Establishment Of An ELISA Test Method

Indomethacin(IDM)is a non-steroidal anti-inflammatory drug,which is widely used in human medicine and veterinary clinics.The adverse reactions of indomethacin can cause damage to multiple systems such as digestion,nerves,circulation,and blood.The EU has stipulated the maximum residual amount of some non-steroidal anti-inflammatory drugs,but there is no maximum residual amount of indomethacin in animal foods and the drug holiday period after food animal medicines,and indomethacin is easy to purchase.The clinical application effect is good,so it is easy to be abused by farmers,and then cause a large amount of residue in animal products.Therefore,the detection of indomethacin residues is essential to ensure food safety.At present,domestic and foreign methods for detecting indomethacin residues are diversified.Among them,methods that rely on instruments such as high-performance liquid chromatography can quickly analyze chemical substances with high boiling points,large molecular weights,and unstable chemical properties.The disadvantage is that the processing of samples is complicated,results cannot be obtained quickly,and high-throughput samples cannot be tested on site.In contrast,immunological detection methods have the advantages of high throughput and low cost,and have broad application prospects in the detection of drug residues.Therefore,this test aims to prepare indomethacin monoclonal antibodies with high specificity and high affinity,and then establish a rapid and efficient immunological detection method to provide theoretical and technical reference for the residues of indomethacin in animal-derived foods.1.Synthesis and identification of artificial antigen of IDMIn In this experiment,the carbodiimide method was used to synthesize the artificial antigen immunogen IDM-BSA and the detection original IDM-OVA.The UV scanning and SDS-PAGE gel electrophoresis were used to identify the artificial antigen.IDM-BSA is routinely immunized to BALB/c mice.The serum antibody titer measured by ELISA is 1:64000,which can judge the success of artificial antigen synthesis.The protein concentration of IDM-BSA measured by the BCA kit was 5.20 mg/mL,and the protein concentration of IDM-OVA was 3.26 mg/mL.The coupling ratio of IDM-BSA measured by ultraviolet spectrophotometry was 5.7:1,and the coupling ratio of IDM-OVA was 6.2:1.2.Preparation and identification of monoclonal antibodies against IDMIDM-BSA used as the immunogen,the mice was immunized according to the conventional animal immunization protocol,and the serum of the mice was collected 7d after five immunizations.Then its serum titer was determined and drug inhibition experiments were conducted.Taking mice with better immune effect,its spleen taken and SP2/0 cells were used for cell fusion experiment.ELISA and CiELISA were used for cell screening,and finally a hybridoma cell line that was continuously secreted and stabilized against IDM monoclonal antibody was obtained,which was named 5D8.About 10 mL of ascites was collected using the in vivo induction method of ICR mice.Purified ascites by affinity column,antibody titer measured up to 1:128 000,and significantly reduced contaminants by SDS-PAGE.Cross-experiment results showed that the monoclonal antibody did not cross-react with meloxicam,flunixin meglumine,carprofen,and naproxen,and the inhibition rate of indomethacin was 100%.3.The establishment of the ELISA detection methodUsing purified ascites,a CiELISA method for detecting IDM residues was established.The best blocking solution is 0.5%gelatin,the best working concentration of the original coating is 1:1500,and the best working concentration of the antibody is 1:16000.When the concentration of IDM is 5?10 000 ng/mL,the linear relationship is good.The linear equation is y=0.216 5 x+0.010 9,the correlation coefficient R2=0.9931.The IC50 of monoclonal antibody for IDM is 180.61 ng/mL.The LOD is 1.01 ng/mL,The LOQ is 8.77 ng/mL.4.The addition and reclamation test of IDM in fresh milkAdding the recovery experiment in the fresh milk sample,when the fresh milk is diluted 16 times,the influence of matrix interference can be ruled out.The concentration of indomethacin is in the range of 5?10000 ng/mL,and the linear relationship is good.The linear regression equation is y=0.224 8 x-0.048 7(R2?0.9869),IC50 is 275.05 ng/mL,LOD is 2.19 ng/mL,The LOQ is 40.88 ng/mL.Three kinds of IDM solutions with different concentrations of 50 ng/mL,500 ng/mL,and 2000 ng/mL were used for drug addition recovery experiments.After detection,the recovery rate of indomethacin in fresh milk was between 85%and 100%.