Preparation Of Monoclonal Antibody Against Human Cystain C And Preliminary Establishment Of Fluorescence Immunochromatography Detection

Abstract:To better evaluate early renal injury,we chose the cystatin C with good clinical sensitivity as a detection index.The human cystatin C protein was constructed and expressed by gene recombination technology,and the cystatin C monoclonal antibody was prepared by hybridoma technology.The cystatin C fluorescence immunochromatographic assay was established to provide a new method for rapid detection of early renal injury in clinical practice.Methods:1.Construction of recombinant plasmid:Synthesize the target gene according to the GenBank human cystatin C(NM 000099.3)sequence and design the subcloning primers.Connect double digestion PCR amplification products with PET-28a,transform into BL21(DE3)Competent cells,and construct recombinant BL21(DE3)-pET28 a-CysC strains,dentified by double digestion and gene sequencing.2.Protein expression and purification:Induce recombinant bacteria to express cystatin C protein,optimize the concentration and induction time of inducer IPTG,use Ni~+affinity chromatography to purify the expressed product.To dialysis against purified recombinant Cystatin C protein reactivate it,through ELISA identify the antigenic activity of the recombinant protein.3.Monoclonal antibody preparation:The mice were immunized with the above cystatin C protein,and the hybridoma cells were prepared by fusion of myeloma cells and mouse spleen cells with the best immune effect,and select monoclonal cell lines of stably secreting specific antibodies with cloning.Injected monoclonal cell into the abdominal cavity of the mice to prepare monoclonal antibodies.purified ascites by n-caprylic-ammonium sulfate precipitation and determined the titer,purity,and subtypes of the purified monoclonal.4.Establishment of detection methods:By preparing indoor reference products and screening for cystatin C pairing antibodies to establish fluorescence immunochromatographic assays.Study and determ the best test strip preparation process and the corresponding components,and test the fluorescence tomography strip with linearity,low detection limit,precision,accuracy,stability,etc.A cystatin C fluorescence chromatographic method was initially established.Results:1.The recombinant BL21(DE3)-pET28a-Cys C was successfully constructed.The optimal expression conditions were 37°C,0.2 mmol/L IPTG induced for 6 h,the target protein was inclusion body,dissolved by 8 mol/L urea,affinity Chromatographic purification,dialysis and dialysis,ELISA determination of cystatin C antigen response titers above 10~4.2.Five monoclonal antibodies against IgG1 subtypes of cystatin C were selected and five monoclonal antibodies were prepared.The titer was detected at1.28×10~5-1.024×10~6.The purity of the antibodies detected by SDS-PAGE was greater than 95%.3.The cystatin C fluorescence immunochromatographic assay was established using 3H4 as the coating antibody and 1A7 as the labeled antibody.A rapid diagnostic test strip with a detection time of 5 min,a linear range of 0.05 mg/L to 20mg/L and an R~2 of greater than 0.99 was prepared with good precision,high stability,and high accuracy.Conclusion:The cystatin C protein with biological activity was prepared by genetic engineering method.Five monoclonal antibodies against cystatin C were obtained by hybridoma technique.A fluorochrome immunochromatographic assay for cystatin C was established.