Prolactin (PRL) is a 199 aa, 23kD peptide hormone that is secreted primarily by the pituitary gland in both males and females, though its major roles are in pregnancy and lactation. Prolactin may have a role in breast cancer development, with higher prolactin levels correlating with postmenopausal breast cancer risk. The sensitive quantitative measurement of total human prolactin antigen in plasma, serum, or breast milk samples is easily performed with this 96 well strip format ELISA kit. The concentration of prolactin in normal human plasma ranges from 2-18 ng/ml in males, 2-29 ng/ml in nonpregnant females, and 10-209 ng/ml in pregnant females. The assay measures total human prolactin in the 0.1-100 ng/ml range. Samples with human prolactin levels above 100ng/ml should be diluted in blocking buffer before use. Normal plasma should not require dilution before use in this assay. A 1:2 to 1:4 dilution for breast milk is suggested for best results. Human prolactin will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, biotin labeled anti-human prolactin primary antibody binds to the captured protein. Excess primary antibody is washed away and bound antibody is reacted with peroxidase conjugated streptavidin. Following an additional washing step, TMB substrate is used for color development at 450nm. Color development is proportional to the concentration of prolactin in the samples. A standard calibration curve is prepared using dilutions of purified prolactin and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.