An enzyme immunoassay for the quantitative determination of free prostate specific antigen (f-PSA) in serum or plasma. This f-PSA ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA). The microtiter wells are coated with an anti-f-PSA monoclonal antibody, directed towards an epitope of an antigen molecule. An aliquot of patient serum is incubated in the coated well with enzyme conjugated second antibody (E-Ab), directed towards a different region of the antigen molecule. After incubation the unbound E-Ab is washed off: The amount of bound E-Ab is proportional to the concentration of antigen in the sample. After adding the substrate solution, the intensity of colour developed is proportional to the antigen concentration in the sample. The measured ODs of the standards are used to construct a calibration curve against which the unknown samples are calculated.