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Tissue factor pathway inhibitor -2 (tissue factor pathway in Inhibitor-2 of TFPI-2), also known as placental protein The -5 (placental protein 5, PP-5), or matrix-associated serine protease inhibitor (matrix-associated serine protease inhibitor , MSPI), is a protease inhibitor with a Kunitz-type domain, belong to the serine proteinase inhibitor super family and may be involved in the tissue by affecting the extracellular matrix remodeling, embryonic development and differentiation, wound healing, tumor invasion transfer and atherosclerotic plaque rupture and other physiological and pathological processes. TFPI-2 in human blood in very low concentrations, currently used detection method of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (by day3 post, RIA). ELISA was influenced by many factors, likely to cause distortion of the results of the sensitivity is not ideal. RIA method has high sensitivity and accuracy, wide linear range, but the reagent to the presence of radioactive contamination, and the short half-life of the marker reagents do not keep well. The time-resolved fluorescence immunoassay (time-resolved fluorescence immunoassay, TRFIA) enzyme markers and isotope labeling technique advantages, with high sensitivity, specificity, stability, markers preparation is simple, long storage time, non-radioactive pollution detection repeatability, operational processes, the wide range of the standard curve, in particular, to eliminate the high background and the advantages of conventional fluorescence measurement, non-RIA analysis of the great potential for development of analytical methods. To help TFPI-2 basic and clinical research, and to promote the application and development of TFPI-2 in the prevention and treatment of related diseases, we have established a TFPI-2 time-resolved fluorescence immunoassay. The system with anti-TFPI-2 polyclonal antibody as a solid-phase antibody, TFPI-2 monoclonal antibody (McAb) 3C8 as primary antibody of Eu-conjugated goat anti-mouse monoclonal antibody as the detection antibody, prokaryotic expression recombinant TFPI-2 as analysis of the standard product. TFPI-2 the antigen BalB / C mice were separated spleen cells with myeloma cells, cell fusion, by the three clones screened, obtained three stable hybridoma cells secrete anti-TFPI-2 McAb were named 3C8, 2F11 and 1C4; three hybridoma cell immunized mice were prepared by ascites, r-protein-A affinity purified, to give a concentration of about 1.5mg/ml TFPI-2 McAb mother liquor was concentrated, by SDS-PAGE purity of 98%; affinity identification McAb 3C8 affinity, was chosen as our time-resolved fluorescence immunoassay anti; immunoglobulin subtype identification display the McAb to 3C8 heavy chain isoforms IgG1 type, light chain subtypes k type. Western-blotting confirmed that three monoclonal antibodies can specifically bind to TFPI-2 immunohistochemistry confirmed that they are able to detect the normal breast and breast cancer cells expressing TFPI-2. People TFPI-2 antigen to immunize New Zealand white rabbits, antiserum, monoclonal antibody purification methods purified rabbit polyclonal anti-serum antibody titers of 1:32. This multi-resistant as coating antibody, the best package concentration 4ug/ml. McAb 3C8 as primary antibody, the optimal concentration of 400ng/ml. The production of recombinant TFPI-2 standard curve linear range between in 0.1-50ng/ml, the correlation coefficient was 0.999 sensitivity 0.048ng/ml, intra-and inter-coefficients of variation were ≤ 8.39%. TFPI-2 TRFIA will have been prepared successfully stored for 6 months at 4 ℃ determination of the standard curve had no significant drift, basically meet the stability requirements. Application homemade TFPI-2 TRFIA ELISA kit simultaneous determination of 56 patients with coronary heart disease patients and 24 normal controls the TFPI-2 TRFIA detection results were from 10.58 ± 7.12ng/ml and 0.53 ± 2.32ng/ml,. ELISA detection of the same sample, the results of 27 were negative. Application of two methods for detecting the remaining 56 specimens, on the results of the correlation analysis, ELISA assay value of X, Determination of TRFIA is Y, Y = 1.1182x 2.4225, r = .9363, both have a significant correlation (P lt; 0.01). Conclusion: We established TFPI-2 time-resolved fluorescence immunoassay method better than the enzyme-linked immunosorbent assay sensitivity, specificity and stability to meet the requirements, can be used to determine the blood vessels and tumor-related diseases TFPI-2 detection.
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