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

Dipyrone,also known as noramidopyrine,is a pyrazolone antipyretic analgesic and anti-inflammatory drug that has been used for nearly a hundred years.In recent years,the toxic side effects of various drugs have gradually attracted attention of people.Since the 1970s,the clinical research of dipyrone has gradually deepened,this research found there are a large number of patients with serious side effects and even life-threatening in the vast majority of users.Therefore,many developed countries have begun to gradually reduce the use of dipyrone.Despite this,the use of dipyrone is still very large in some areas.As early as 1982,tablets of dipyrone were classified as banned drugs in china,but other preparations such as dipyrone tablets and injections are still in use today.In view of the growing problem of veterinary drug residues of animal-derived food,how to screen this foods containing residues and avoid they entering the markets is very important,and detection and analysis means are an essential part.At present,the analytical methods for the residues of dipyrone and its metabolites are mainly physical and chemical analysis methods.These methods have highly accurate and specific,but the instruments to be used are expensive,the sample preparation process is complicated,the operation is complicated,and the time is too long.There are certain requirements for the professional level of the operators,and they cannot be popularized at the grassroots level.The immunological detection method is a rapid detection and analysis method based on the principle of antigen-antibody specific reaction.Compared with the physical and chemical analysis method,this method is convenient to use,the detection process is fast and efficient,the detection result is accurate,and is suitable for the detection of a large number of samples.This experiment aims to establish a rapid immunoassay for the detection of dipyrone residues,and provide a scientific basis for the monitoring and detection of animal-derived food residues.1.Synthesis and identification of complete antigen of DipyroneDipyrone quickly decomposes after entering the body,and the prototype drug of dipyrone can't be detected in the body's plasma.Therefore,this experiment was carried out by the residue of 4-aminoantipyrine,a metabolite of dipyrone.4-aminoantipyrine is a small molecule compound.This experiment was prepared by coupling 4-aminoantipyrine with carrier bovine serum albumin(BSA)and ovalbumin(OVA)by two-step synthesis of glutaraldehyde.Two artificial antigens were produced,namely immune antigen(AA-BSA)and coated antigen(AA-OVA).The results were determined by UV spectrophotometry and immunological methods,and the results showed that the artificial antigen was successfully coupled.2.Preparation of Dipyrone monoclonal antibody against DipyroneThe artificial antigen AA-BSA was used as an immune antigen,and the mouse plasma was collected from the vaccinated mice to prepare serum and subjected to indirect ELISA.The serum titer was up to 1:256000.The spleen of mice with high serum titer after the end of the immunization program was fused with mouse myeloma cells cultured in the logarithmic growth phase by the monoclonal antibody technique,after three sub cloning and all the cells of the well.When the supernatants were all positive,two hybridoma cell lines were screened and named as 3B6 and 7G6.After further screening,7G6 monoclonal cell lines were injected into the peritoneal cavity of mice,and 16 mL of ascites was prepared by in vivo induction method.The protein concentration of ascites was determined to be 24.32 mg/mL,and the ascites protein concentration after purification was 0.72 mg/mL.The aspergic water purification effect was detected by SDS-PAGE.The results showed that the ascites purification effect was good.The drug cross-test results showed that the AA monoclonal antibody enrofloxacin,cephalexin,tetracycline,ciprofloxacin,BSA and OVA had no obvious cross-reaction,and the cross-reaction rate with dipyrone was 86.3%,and the cross-reaction rate of 4-methylaminoantipyrine was 79.1%.3.The establishment of ELISA detection methodThe conditions in the ELISA assay were optimized to determine that 2%gelatin solution was the best blocking solution,and the optimal working concentrations of antigen and antibody were 1:3000 and 1:16000,respectively.The linear equation of ELISA standard inhibition curve is y=0.2894x-0.1085,the correlation coefficient R2=0.995,and the linear relationship of AA drug concentration is 10?5000ng/mL.The calculated data shows that the IC50 is 120.79ng/L.The LOD is 7.1 ng/mL and the LOQ is 51.76 ng/mL.4.The addition and reclamation test of AA in the porkIn this experiment,pork samples were set and the pork samples were pretreated by the relevant steps.When the sample solution was diluted 16 times,the interference of the sample matrix was excluded.Through the mapping of the competition inhibition curve,it can be seen that the linear relationship between the drug concentration is 20?2000ng/mL,the linear regression equation is y=0.3489x-0.2311,the correlation coefficient R2=0.9907,and the IC50 of the drug half inhibition concentration is 124.58ng/mL.The LOD is 9.52 ng/mL and the LOQ is 22.68 ng/mL.Three drug concentration gradients were set at 50 ng/mL,400 ng/mL and 800 ng/mL for the recovery test.Finally,the recovery of 4-aminoantipyrine in pork samples was calculated from 87.70%to 101.57%.