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J Immunol Methods ; 295(1-2): 169-82, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15627622

ABSTRACT

Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells, and is being studied in clinical trials for therapy of other types of cancer. In the present work, we developed a Herceptin enzyme-linked immunosorbent assay (ELISA) from commercially available reagents to meet the growing needs of clinical studies. In this immunoassay, a mixture of monoclonal antibodies specific for the cytoplasmic domain of human HER2 (676-1255 amino acids) is adsorbed onto a microtiter plate, followed by addition of full-length HER2 protein, which is captured by the antibodies. Herceptin in the serum that is added to the microwells binds to the extracellular domain (ECD) of the captured HER2. The Herceptin bound to HER2 is detected by an antihuman IgG-horse radish peroxidase (HRP) conjugate and a (3, 5, 3', 5')-tetramethylenbenzidine (TMB) substrate. The calibration range of the assay is 5-100 ng/mL after 1:2000 sample dilution corresponding to 10-200 microg/mL Herceptin in undiluted serum. The intra- and interassay CVs ranged from 4.56% to 13.3% and from 9.9% to 18.9%, respectively. The assay shows dilutional linearity and specificity. Soluble p105 HER2, which can be shed into serum does not interfere with the assay. The analytical performance of the Herceptin ELISA indicates that this assay can be used for monitoring concentration levels of Herceptin in human serum.


Subject(s)
Antibodies, Monoclonal/blood , Antineoplastic Agents/blood , Enzyme-Linked Immunosorbent Assay/methods , Antibodies, Monoclonal, Humanized , Humans , Reproducibility of Results , Sensitivity and Specificity , Trastuzumab
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