A modification of the ELISA-double sandwich method for estimating the concentration of habutobin

A monoclonal antibody to the thrombin-like enzyme, habutobin, was produced. An ELISA-double sandwich method employing this monoclonal antibody was devised as a method for determining the habutobin concentration in vitro. However, the absorbance of the control sample in such an ELISA-double sandwich procedure was too high to estimate low levels of the enzyme. The present study therefore attempted to establish a reliable ELISA-double sandwich method in which the absorbance of the control sample was lower than previously, and which had a high sensitivity, in order to determine the habutobin concentration in vitro and in vivo. The modification of the ELISA-double sandwich technique employing the monoclonal antibody against habutobin, the purified rat IgG against habutobin and POD-mouse anti-rat IgG2a, provided a reliable means of determining the habutobin levels in the circulating blood of rabbits.