Cancer Antigen ELISA - CA 15-3

Enzyme Immunoassay for the Quantitative Determination of CA 15-3 in Serum

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

SUMMARY
CA15-3 (Cancer Antigen 15-3) is a tumor marker used to monitor certain cancers, especially breast cancer. It is found on the surface of many types of cancer cells and shed into the blood stream. It is used to monitor advanced, i.e. metastatic, cancer. Elevated CA15-3, in conjunction with alkaline phosphatase (ALP), was found to be associated with an increased chance of early recurrence in breast cancer. CA15-3 and associated CA27.29 (aka BR27.29) are different epitopes on the same protein antigen product of the breast cancer-associated MUC1 gene. CA27.29 has enhanced sensitivity and specificity and has therefore surpassed CA15-3 as a serum tumor marker. CA27.29 is elevated in 30% of patients with low-stage disease and 60 to 70% of patients with advanced-stage breast cancer. Both CA27.29 and CA15-3 may be elevated in patients with benign ovarian cysts, benign breast disease, and benign liver disease. Elevations may also be seen in cirrhosis, sarcoidosis and lupus. CA27.29 levels over 100 U/mL or CA15-3 levels over 25 U/mL are rare in benign conditions. CA27.29 may be elevated in non-breast malignancies including colon, stomach, pancreas, prostate and lung.

PRINCIPLE OF THE TEST
The CA15-3 ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a monoclonal antibody directed against a distinct antigenic determinant on the intact CA15-3 molecule is used for solid phase immobilization (on the microtiter wells). A rabbit anti-CA15-3 antibody conjugated to horseradish peroxidase (HRP) is in the antibody-enzyme conjugate solution. The test sample is allowed to react sequentially with the two antibodies, resulting in the CA15-3 molecules being sandwiched between the solid phase and enzyme-linked antibodies. After two separate 1-hour incubation steps at 37°C, the wells are washed with Wash Buffer to remove unbound labeled antibodies. A solution of TMB Reagent is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of CA15-3 is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.

EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450 nm shown in the Y axis against Cancer Antigen concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.