Preparation Of Anti-ADMA Rabbit Monoclonal Antibody Based On Single Cell Technology

Objective:At present,cardiovascular disease(CVD)is still the main cause of death caused by global diseases,while cardiovascular disease in China accounts for the top cause of total deaths of urban and rural residents.Clinical trials have shown that inhibitors of endogenous nitric oxide synthase(NOS),asymmetric dimethylarginine(ADMA)and monomethylarginine(L-NMMA),can compete for inhibition of NOS binding sites and reduce the production of endogenous NO,leading to the development of endothelial dysfunction and cardiovascular related diseases,such as hyperlipidemia disease,diabetes,hypertension,heart failure,etc.ADMA concentration in the body within a narrow range,with an increased prevalence of elevated risk of cardiovascular disease associated ADMA concentration.At present,the method of detecting ADMA at this stage is complicated and time-consuming,which brings difficulties for ADMA detection.Compared with mouse monoclonal antibodies,rabbit monoclonal antibodies have the advantages of higher affinity and specificity,recognition of more novel epitopes,and recognition of mouse-derived protein antibodies.Therefore,the preparation of anti-ADMA rabbit monoclonal antibodies can be used to improve the current ADMA detection technology,and can also provide new ideas and help for the preparation of other clinical small molecular markers monoclonal antibodies.At the same time,it also provides clinicians with accurate,rapid,stable and reliable test results,and provides new evidence for the early diagnosis and treatment of cardiovascular-related diseases.Methods:1.Detection of animal immunity and antibody titerImmunize New Zealand white rabbits with KLH-SMCC-Cys-ADMA.While detecting antibody production,peripheral blood was collected from rabbits for flow cytometric analysis to detect the presence of antigen-specific cells.Immunospecific and antibody titers were analyzed by ELISA and Western blot.2.Antibody variable region and constant region sequence acquisitionRabbit lymph nodes and spleen were processed into single cells,and specific plasma cells and B cells were sorted by flow.Total RNA was extracted and cDNA was synthesized using a 5'RACE rapid amplification technique(5'RACE),and a variable region sequence and a constant region sequence were obtained by PCR.3.Obtain the antibody light chain and heavy chain expression frameThe correct sequence of the constant region was subjected to PCR,and the variable and constant regions of the antibody were ligated using the Target-Selective Coupling PCR(TS-jPCR)technique to construct expression cassettes for the light and heavy chains for further protein expression.Results:1.Flow cytometric analysis showed that before immunization,the percentage of antigen-specific B cells in t.wo rabbits was 0.946%and 0.403%,and that of antigen-specific plasma cells was 0.403%and 0.881%,respectively,and there was a small amount of antigen-specific cells.The percentage of specific plasma cells and B cells produced after immunization was increased compared with that before immunization,indicating that rabbits produced a certain amount of antigen-specific cells after immunization.2.Western blot immunospecific analysis showed that specific bands were generated near 64 kD,antibody titers were detected,and non-specific binding was reduced after dilution of 1:500,indicating that rabbits were immune to anti-ADMA antibodies produced specifically after immunization with high potency.3.The results of ELISA detection of antibody titer showed that the OD value after immunization was more than 2 times the OD value before immunization,that is,there was a difference,indicating that the rabbit produced specific antibodies after immunization and the titer was as high as 1:50000.4?Rabbit lymph node and spleen cells were collected for flow cytometric sorting,and antigen-specific cells were sorted to obtain their variable and constant region sequences using 5'RACE technology.As a result of agarose gel electrophoresis,specific bands were found in the variable regions.5?he result of sequencing in the constant region showed that the light chain constant region is completely identical to the reference sequence,and the heavy chain constant region differs from the reference sequence by two amino acids.Obtaining the constant region expression vector box experimental results showed that the light chain band was approximately 4000 bp by agarose gel electrophoresis,and the heavy chain band was approximately 5000 bp,which is close to the theoretical value.Conclusions:1?The antigens used in this study can produce anti-ADMA-specific antibodies in rabbits.The designed primers can successfully obtain the constant region sequences of light chain and heavy chain of anti-ADMA antibody,and use TS-jPCR to link the variable region and constant region of the antibody to construct the expression frame of the light chain and the heavy chain,which lays the foundation for the protein expression in the next step.2?The 5'RACE technology can be used to obtain the sequences of the variable regions of the antibody light and heavy chains,laying the foundation for the subsequent acquisition of single-cell variable region sequences and the further establishment of clinical testing ADMA methods.