Preparation Of Human Procalcitonin Monoclonal Antibody And Preliminary Study Of Its Fluorescent Immunochromatography Assay

Procalcitonin?PCT?is currently recognized as an inflammatory marker and has important value in the differential diagnosis,therapeutic monitoring and prognosis of infectious diseases.The combined application of immunofluorescence and electrochemical technology enables quantitative and rapid detection of PCT.At present,the detection kits of PCT are based on the specific binding reaction of antigen and antibody,antigens and corresponding antibodies are derived from the importer.Preparation of stable antigens and highly specific monoclonal antibodies is the basis for the realization of domestically produced products.Objective:To express human procalcitonin?PCT?recombinant protein in prokaryocytes and optimize protein expression conditions.To prepare and identify high-purity PCT monoclonal antibodies.To establish PCT fluorescence immunochromatography assay and detect clinical specimens to evaluate performance.Methods:The constructed recombinant expression strain E.coli BL21?containing PCT-pET22b?+??was activated to induce expression of PCT protein,and concentration of IPTG that induced protein expression was optimized.The target protein was purified by Ni-NTA affinity chromatography column and identified by Western blot and colloidal gold method.Obtained PCT recombinant protein and adjuvant were fully ground and emulsified,and the Balb/c mice were injected subcutaneously by long-term immunization.The serum titer of the mice was detected after 4 immunizations,after reached the fusion condition,the splenocytes and myeloma cells were taken?SP2/0?fusion,then positive hybridoma cell lines were screened by indirect ELISA and subcloned 3⁴ times.The monoclonal cell lines was injected into the peritoneal cavity of mice to produce ascites to prepare monoclonal antibody,which were purified by ammonium octoate ammonium sulfate and Protein G affinity chromatography column,finally these monoclonal antibodies were identified about purity,subtype,titer,affinity,etc.The prepared monoclonal antibody was subjected to fluorescent latex labeling,and activity of the labeled antibody was judged by fluorescence value detection and indirect ELISA.The antibody pairing results were analyzed,and fluorescent latex immunochromatographic method was established.Working concentration of capture antibody and labeled antibody was optimized,and then PCT content was detected.Results:Recombinant expression strain was subjected to ultrasonic lysis,the supernatant and precipitate were subjected to SDS-PAGE electrophoresis.The results showed a significant band at a relative molecular weight of approximately 14 kDa,which was consistent with the size of the target protein,and mainly existed in the supernatant,indicating that the target protein was a soluble protein.Different concentrations of IPTG induction results showed that the protein expression was highest when the final concentration of IPTG was 0.8 mmol/L.Target protein in the supernatant was purified by Ni-NTA column and results of SDS-PAGE showed that the purity of the target protein could meet the requirements of animal immunity.Western blotting and colloidal gold showed that the soluble protein was PCT and contained his-tag.The purified PCT was used as an immunogen to immunize Balb/c mice,and serum antibody titers of the three mice after immunization were 10⁶,10⁶,10⁵,respectively,meet the cell fusion conditions.After immunization,the mouse spleen cells were fused with myeloma cells?SP2/0?,and 7 positive hybridoma cells?B3,C4,C7,E9,F8,F10,G2?were successfully screened by indirect ELISA.Two strains?B3and C4?were selected for further cultured and injected into the peritoneal cavity of mice to generate monoclonal antibody by in vivo induction method.The ascites was purified by ammonium octoate ammonium sulfate and Protein G strain,SDS-PAGE showed that there were obvious bands at 25kDa and 55kDa,which were the light chain and heavy chain of immunoglobulin,the purity can reach more than 95%.The subclass identification of two antibodies were IgG1 type,indirect ELISA method showed that B3 and C4 ascites antibody titers was 10⁸ and 10⁷,respectively,the antibody titers after purification reached 10⁷,and the affinity constants were 5.06 x10⁸ and 7.3 x 10⁸,respectively.The fluorescence value of the antibody after fluorescent latex labeling can reach about 250,000.After labeled antibody was diluted 10⁵ times,the results of indirect ELISA showed that OD₄₅₀ was above 1.5,indicating that the labeled antibody still has good activity.B3-C4 antibody was successfully paired and the antibody working concentration was optimized.Coated antibody concentration was 1.5 mg/ml,the latex-labeled antibody concentration was 400?g/ml,and optimal dilution ratio of the sprayed film was 1:10.Compared with chemiluminescence immunoassay,the correlation coefficients of the two batches of clinical samples were 0.9871 and 0.9984,respectively.Conclusions:High purity PCT recombinant protein was obtained by optimizing the induction conditions.PCT monoclonal antibody was successfully obtained by cell fusion technology.Fluorescence immunochromatography assay was initially established,which has good detection performance.