The Rat-MID? Osteocalcin EIA is based upon the competitive binding of a monoclonal antibody to soluble osteocalcin or to immobilized osteocalcin. Briefly, the monoclonal antibody is raised against human osteocalcin and recognizes the mid-molecular part (amino acids 21-29) of the molecule (11). Synthetic human osteocalcin is used for standardization. Parallelism is observed with purified rat osteocalcin and rat serum. During the Pre-incubation step, biotinylated osteocalcin is immobilized by binding to the streptavidin-coated microtitre wells. The wells are emptied and washed, and standards, control, or unknown serum samples are pipetted into appropriate wells, followed by a solution of monoclonal antibody. Following the Primary-incubation step the wells are emptied and washed. In the Secondary-incubation step a solution of peroxidase conjugated anti-mouse immunoglobulins is added and binds to the monoclonal antibody. After the third washing step a chromogenic substrate (TMB) is added and the colour reaction is stopped with sulfuric acid. Finally, the absorbence at 450 nm is measured. The absorbence level is inversely related to concentration of osteocalcin in the sample.