病院検査室で実践できる分子病態検査法開発 転写因子活性化検査の臨床応用に向けて

摘要

The nuclear factor-kappa B (NF-B) family of transcription factors is known to play an important role in the regulation of the immune system. NF-B is activated by bacterial and viral antigens, which lead to the production of proinflammatory cytokines and chemokines. The rapid detection of activated NF-kB by systemic inflammatory response syndrome (SIRS) is considered to be crucial for the treatment of patients with septicemia. The aim of the present study was to evaluate the sensitivity of two methods, electrophoretic mobility shift assay (EMSA) and transcription factor enzyme-linked immunoassay (TF-ELISA). TF-ELISA detected 25ng of recombinant human NF-B p50 (rhp50) and 5g of TNFa-stimulated HeLa nuclear protein, while EMSA detected approximately lOOng of rhp50 and 10g of HeLa nuclear protein. We found that TF-ELISA was more sensitive than EMSA in detecting NF-k-B; however, it was judged that the 3~6 hour measuring time required in TF-ELISA was excessively long for patients with SIRS. Therefore, the development of new analytical methods with improved sensitivity and measurement time was necessary for the detection and quantification of activated NF-B protein in the hospital laboratory. Consequently, we have developed a new NF-B analyzer based on surface plasmon resonance (SPR), which is recognized as one of the most sensitive direct optical detection methods. This method can detect nanomolar concentrations of NF-B within 15 minutes. In addition, we have developed new experimental apparatus for the detection of NF-B based on fluorescence correlation spectroscopy (FCS), which is able to analyze binding between DNA and protein in the liquid phase. At present, we are carrying out clinical trials using this new transcription factor analysis apparatus for SIRS patients.