2008 年 18 巻 1 号 p. 25-29
Toll-like receptors (TLRs) play essential roles in the innate immune system, which recognize pathogen-asso ciated molecular patterns and initiate the acquired immune responses. Previous studies revealed that expres sion levels of TLR2 mRNA and protein on monocytes from sepsis patients are significantly up-regulated com pared to those from healthy subjects. In those studies, flow cytometric analysis was used to examine the mod ulation of TLR2 protein levels. Generally, flow-cytometer is a high precision tool to quantify the expression level of an antigen on cell sur face. However, it is hard to compare the measurements assayed at different time-points, because the inter assay variations are inevitable by the fluctuating factors, such as cytometerʼs performance and other assay conditions. To perform a longitudinal follow-up study for TLR2 protein levels on monocytes from the same patients, we developed a new quantitative analysis system for TLR2. For the first step, we created a set of standard beads which bore 4 differing amounts of recombinant TLR2 protein on their surface. On every assay, mean fluores cence intensity (MFI) of these standard beads was assessed by flow-cytometer to draw an analytical curve. Simultaneously, TLR2 levels on monocytes from the subjects were assayed, and the MFI values were convert ed to the numbers of anti-TLR2 monoclonal antibody binding sites with the analytical curve. By using this method, we followed-up TLR2 modulation of patients with bacterial infections. Through this follow-up study, some characteristics of TLR2 modulation on monocyte were exposed, which provide critical information about clinical conditions and medical treatment. Consequently, in clinical our developed TLR2 quantitative assay system is likely to provide more reliable measurement than conventional methods. Our methodology may be applicable to quantify not only TLR2 level but also other useful cell-surface markers, which are worth carrying out time-interval assay during clinical courses.