Development And Evaluation Of Time-resolved Fluorescence Immunoassay For Tissue Factor Pathway Inhibitor-2 (tfpi-2)

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein-5 (PP-5) or matrix associated serine protease inhibitor (MSPI), is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor super-family. TFPI-2 may be involved in, by affecting the remodeling of extracellular matrix, tissue occurrence, differentiation of embryonic development, wound healing, tumor invasion transfer and atherosclerotic plaque rupture, such as physiological and pathological processes.Concentration of TFPI-2 is very low in the human circulation. Currently, enzyme-linked immune-absorbent assay (ELISA) and radioimmunoassay (RIA) are commonly used in detecting protein concentration. However, the sensitivity of ELISA is not satisfactory for trace protein and RIA has radioactive contamination. Compared with ELISA and RIA, Time-resolved fluorescence assay (TRFIA) has not only the advantages of enzyme marker technology, but also the isotope labeling. TRFIA has high sensitivity and specificity, good stability and easy preparation of markers, long storage time, no radioactive contamination, detection repeatability, short time operational processes, the wide standard curve range, especially to the elimination of conventional high fluorescence background and so on. So TRFIA is a great potential analytical method in non-RIA immunoassay.In order to contribute to the basic and clinical research of TFPI-2, we have established TRFIA method for quantitative TFPI-2 detection. In this detection system, anti-TFPI-2 polyclonal antibody was used as a solid phase antibodies, TFPI-2 monoclonal antibody (McAB) 3C8 as the first antibody, and Eu3+labeled goat anti-mouse monoclonal antibody as a detection antibody, recombinant TFPI-2 expressed by Prokaryotic as the standard.First of all, BalB/C mice were immunized with human TFPI-2 as foreign antigen, and cell fusion was proceeded. After cloning screening on the integration of positive hybridoma cells for three times, we obtained three hybridoma cell lines that could secret the anti-TFPI-2 McAb stably, named 3C8,2F11 and 1C4. Mice were immunized with these hybridoma cells to prepared ascites. By r-protein-A affinity purification, we obtained about 1.5mg/ml of each McAb and the purities were all above 98%.3C8 was chosen as our time-resolved fluorescence detection for it was the best at affinity activities. Identification of immunoglobulin heavy chain subtype showed 3C8 was IgGl and the light chain was kapa. Western-blotting confirmed 3 of McAbs could bind TFPI-2 specifically. Immunohistochemical method confirmed the McAb could identify the expression of TFPI-2 in normal breast tissue and breast cancer.New Zealand White rabbits were immunized with human TFPI-2 for the preparation of anti-serum. The titer of rabbit TFPI-2 polyclonal antibody was 1:32 and we purified it in accordance with the purified protocol of TFPI-2 monoclonal antibodies. The best coating concentration of TFPI-2 polyclonal antibody was 4ug/ml, and the best concentration of second monoclonal antibody was 400ng/ml. The best linear range of the standard was 0.1-50ng/ml and the correlation coefficient was 0.999. The sensitivity was 0.048ng/ml. The intra and inter coefficient of variation were less than 8.39%.After storage at 4 degree for six months, the standard curve of TFPI-2 TRFIA kit had no significant drift and met the basic requirements of stability. By cmparing self-produced TFPI-2 TRFIA with the ELISA kit through simultaneous determination of 56 cases of coronary heart disease patients and 24 normal controls, TFPI-2 TRFIA test results were 10.58±7.12 ng/ml and 0.53±2.32ng/ml. But by ELISA detection of the same samples, the results of 27 were negative. Then the other 56 samples were detected with the two methods and the results were analyzed, with the value of ELISA as X axis and the value of TRFIA as y axis making relevance analisis. The relevance of both group data show that Y=1.1182x+2.4225 r= 0.9363. The relevance of two group data is remarkable.In conclution, the result proved that TFPI-2 TRFIA kit we developed is better than ELISA kit in sensitivity. The specificity and stability can meet the requirements and can be used to the TFPI-2 detection of the blood vessels and tumor-related diseases.