Establishment of an enzyme-linked immunosorbent assay system for determining soluble CD96 and its application in the measurement of sCD96 in patients with viral hepatitis B and hepatic cirrhosis.

CD96, previously named T cell activation increased late expression (Tactile), is a transmembrane molecule that functions as an activated receptor on natural killer cells. It is well known that many transmembrane molecules have soluble forms, which were either shed from the cell surface or spliced at mRNA level. In many cases, the levels of soluble forms in the circulation could be used as biomarkers of lymphocyte activation in bacterial or virus infection, tumour, transplantation and autoimmune disease. To investigate whether CD96 could be released into the sera and the possible biological function of soluble hCD96 (sCD96), we generated and characterized five clones of anti-hCD96 mouse monoclonal antibodies (mAb) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) system based on two anti-hCD96 mAbs with different epitope specificities. Using this ELISA system, sCD96 in serum samples from 99 healthy individuals could be detected. Furthermore, we found that the level of sCD96 in serum samples from patients with chronic viral hepatitis B or classes B and C of hepatic cirrhosis classified using the Child-Pugh score was much higher (P < 0.001 versus healthy individuals; P = 0.006 versus healthy individuals respectively) than that from healthy individuals (0.98 ng/ml). Our study demonstrates for the first time that sCD96 existed in sera, and suggests that sCD96 may be used as a serous marker for some diseases such as chronic viral hepatitis B infection or hepatic cirrhosis in classes B and C. The level of sCD96 in patients' serum may have some relationship with a chronic inflammatory reaction.