| Objective: In the case of infection,a variety of tissues can be induced to release Procalcitonin(PCT)to the blood. The content of PCT in blood of healthy people is less than 0.1ng/m L, the change of PCT concentration in the blood of the specific response of the body is subject to severe bacterial, fungal, parasitic infection, invasion and sepsis. It has been widely used in Clinical detection and lots of detection methods and products has been emerged, as it’s concentration changing can reflect the infection of severe bacteria and fungi parasites. However, the PCT antibodies quality largely determines by the performance and feasibility of the detection method of the product. Preparation of monoclonal antibodies for clinical and establishment of ELISA and other immune detection method is one of the hotspots. Therefore we use monoclonal antibody preparation technology to product the PCT which meet the requirement of Clinical application. Morever, these PCT antibody establish enzyme immunoassay quantitative determination method can be applied for the Clinical diagnosis of bacterial infection. Methods: This experiment used His-hrPCT antigen as an immunogen to immunize BABL/c mice, use the indirect ELISA method to detect mice serum titers. Using immune mouse spleen cells with myeloma cells prepared by fusion of hybridoma cells and by semi solid culture method fusion cell culture and as coating antibody using protein trx-hrPCT and anti mouse IgG-HRP as enzyme standard antibody and by indirect ELISA screening positive cell lines and screened monoclonal antibodies by limited dilution method, separation to obtain stable monoclonal cell line, and preparation of monoclonal antibody. Searching the paired PCT antiobody screening using double antibody sandwich method,and in order to establish double antiobody sandwich method based detection method.This process include board selection, coating liquid, coating condition selection, selection of concentration of antibody and enzyme labeled antibody concentration, reaction time and reaction model, linear survey, sensitivity and specificity, the intra and inter assay values, kit detecting clinical serum contrast detection experiments by paired PCT monoclonal antibody, the eventual establishment of detection method. Results: The titer of the conditions of cell fusion in mice after immunized which titer was up to 1/105.The preparation of monoclonal antibody obtained 4 strains available cell line from 10 strains of positive cell lines by the 3 cloned with limited dilution method, named F1D5, F2H3, F3B7, F3D2. 4 strains of positive cells injected into mouse peritoneal culture and aspirate ascites, measuring the titer over 3.2×105. By PCT monoclonal antibody screening more than 4 strains with immunological activity, obtain a set of paired antibodies F1D5 and F3B7 by means of paired experiment, the establishment of double antibody sandwich ELISA method for detection of the antibody.The PCT method we establish through the research: coated and the liquid are conditions of 0.05 mol / L, pH 9.6 CB buffer at 4℃ package by 12 h; sealing liquid and closed condition for 0.6% BSA at 37℃ closed 2 hours; package best antibody F1D5 concentration was 400 ng / mL, enzyme labeled concentrated antibody F3B7 degree 1:8000; reaction mode selection 37℃ samples incubated 30 min, enzyme labeled secondary antibody was incubated for 30 min, color for 20 min two-step response patterns. Established detection method, the linear range is 0.5 ng/mL-10 ng / ml( R2=0.9912); within batch precision less than 10%; high, medium and low value recovery rate was 99.00% and 98.68%, 108.69%; stability results are shown detection reagent at 37℃ can be placed for 9 d; sensitivity of 95.6%, specificity was 97.8%. Detection of PCT negative and positive interfering substances(calcitonin, calcium inhibitory peptide, bilirubin, heparin sodium, triglyceride, heme) results showed that the has no effect on the results; simultaneous determination of 200 patients sera with similar kit. The results showed good correlation( R2=0.9391). Conclusion: The preparation of monoclonal antibody obtained 1 pairs of antibodies, which initially established the PCT ELISA detection method. |
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