Preparation Of Oleic Acid Monoclonal Antibody And Establishment Of CiELISA Detection Method

Fatty liver is one of the major metabolic diseases susceptible to dairy cows in the perinatal period,which will affect the health and reproductive performance of the dairy cows,and cause huge economic losses.Due to the production and lactation needs of dairy cows during the perinatal period,the hormone levels in the body change drastically,resulting in a decrease in feed intake.Ascending,when the rate of free fatty acid output by the liver is less than the input rate,it will accumulate in the liver in the form of triglycerides,triggering fatty liver.At present,liver biopsy is the gold standard for diagnosing fatty liver,but this method has irreversible damage to cows,and is expensive.The detection process is cumbersome and complicated,and it is not suitable for large-scale diagnosis in actual production.Oleic acid(OA)is the main component of free fatty acids,which has a certain relationship with the formation of triglycerides and the reproductive performance of dairy cows.In actual production,the content of oleic acid in blood has a certain reference role for the diagnosis of the degree of infiltration of fatty liver in dairy cowsAt present,the methods of detecting OA are mainly gas,liquid chromatography and liquid chromatography-mass spectrometry.However,the equipment is expensive and the sample preparation process is cumbersome,which will increase the detection time of the sample and affect the accuracy of the detection results.ELISA is an immunological detection method widely used today.It has the characteristics of simple operation,strong specificity and high degree of automation.It is suitable for the rapid detection of large batches of samples.In this experiment,the active ester method was used to synthesize OA complete antigen,and then monoclonal antibody technology was used to prepare highly sensitive and specific OA antibody,and on this basis,an OA competitive ELISA detection method was successfully established.The establishment of the detection method has important practical value and application prospect for production practice,and is expected to become a new method for clinical diagnosis of dairy cow fatty liver.1.Synthesis and identification of oleic acid complete antigenOA is a hapten.Only the reactive activity does not have immunological activity,and it needs to be coupled with a large molecule to stimulate the body to produce antibodies.According to its molecular structure,OA and dicyclohexylcarbodiimide(DCC)are synthesized into a hydrophilic intermediate product by active ester method,and then coupled with carrier protein BSA to prepare OA-BSA immune antigen.At the same time,in order to avoid the cross reaction of carrier protein,OA-OVA was synthesized as the coating antigen by the same method.Synthetic antigens were tested for protein concentration using BCA kits,and identified by thin layer chromatography(TLC),SDS-PAGE,infrared spectroscopy,and ELISA.The results showed that the concentration of OA-BSA detected by BCA was 2.24 mg/mL,and the concentration of OA-OVA was 1.83 mg/mL;TLC monitored the formation of new substances during antigen synthesis;SDS-PAGE analysis revealed changes in protein molecules;infrared spectrum showed OA-BSA Pop shape was similar to BSA and has the characteristic peak of OA;the multi-antibody titer of mice reaches 1:6400 and has a competition rate.These results indicate that OA was successfully coupled with carrier proteins BSA and OVA,which laid the foundation for the preparation of OA monoclonal antibodies in the next step.2.Preparation of oleic acid monoclonal antibodyUsing OA-BSA as the immunogen,routinely immunize BALB/c mice,use hybridoma technology to perform cell fusion,and screen out a monoclonal cell line that can stably secrete antibodies against OA by ELISA and limiting dilution The ascites was prepared by inducing method,the ascites was purified with HiTrap Protein G HP purification column,and the purified ascites was used for antibody subtype identification by antibody subtype identification kit,and the monoclonal antibody was specifically identified by competitive ELISA.The results showed that the serum titer of the mouse was 1:12800 after five immunizations;and a monoclonal cell line G-1 was selected;the mouse ascites was successfully prepared.The purified ascites protein concentration was 2.27 mg/mL,and the antibody titer was 1:25600;the antibody was identified as IgGl;the results of competitive ELISA showed no cross-reactivity with niacin and palmitic acid.The preparation of the OA monoclonal antibody provides necessary materials for the establishment of CiELISA method for detecting OA.3.Establishment of CiELISA detection method for oleic acidThe G-1 strain was used to prepare ascites and purified antibody.The optimal dilution concentration,optimal blocking solution and blocking time of coating and antibody were determined by square array ELISA,and the standard curve and parameters of OA were prepared by CiELISA.The results showed that the optimal dilution concentration of the coating was 1:100;the optimal working concentration of the antibody was 1:200;the optimal blocking solution was 1%gelatin with a reaction time of 30 min;when the OA concentration was 10?800 ng/mL,the linear relationship was good.The linear equation was y=0.3504x-0.197(R2=0.9943),IC50 was 95.16 ng/mL,LOD was 5.16 ng/mL,and LOQ was 20.52 ng/mL.These results show that the CiELISA method established in this paper has the advantages of good stability,high sensitivity and strong specificity,and has important application value for the detection of oleic acid content in dairy cow blood and the clinical diagnosis of dairy cow fatty liver.