The N-MID? Osteocalcin ELISA is based upon the application of two highly specific monoclonal antibodies (Mabs) against human osteocalcin. An antibody recognizing the midregion (amino acids 20-29) is used as the capture antibody and for detection a peroxidase conjugated antibody recognizing the N-terminal region (amino acids 10-16) is used. In addition to intact osteocalcin (amino acid 1-49) the N-terminal-Mid fragment (amino acids 1-43) is also detected. Standards, control and unknown samples are pipetted into the appropriate microtitre wells coated with streptavidin. Then a mixture of a biotinylated antibody and a peroxidase conjugated antibody is added. Following incubation for 2 hours at room temperature the wells are washed and a chromogenic substrate is added and the colour reaction is stopped with sulfuric acid. Finally, the absorbance is measured.