title: Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma.
abstract: Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to support these pharmacokinetic studies. For this immunoassay, we raised anti-idiotype antibodies in rabbits. After purification of the rabbit material, the anti-idiotype antibodies are used as capturing antibodies on the ELISA plate. After trastuzumab has bound to the catcher antibody, a sandwich ELISA procedure is followed whereby biotinylated anti-idiotype antibodies can bind to trastuzumab. Detection is performed by streptavidin-polyHRP (poly-horseradish peroxidase) conjugate and (3,5,3',5')-tetramethylbenzidine (TMB) substrate. The reaction is stopped using sulfuric acid, and the absorbance is measured at 450 nm. The calibration range of the assay is 0.039 to 5 ng/ml in well. Because samples are analyzed in multiple dilutions, the validated range corresponds to 1.6 to 1600 ng/ml in undiluted serum. Samples above the upper limit of quantification (ULOQ) can be diluted before transfer to the assay plates. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum and plasma. The assay is now used to support pharmacokinetic studies with trastuzumab in human serum and plasma.
authors.affiliation: Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, 1066 EC Amsterdam, The Netherlands.
authors.forename: Carola W N
authors.initials: CW
authors.lastname: Damen
authors.forename: Els R
authors.initials: ER
authors.lastname: de Groot
authors.forename: Marianne
authors.initials: M
authors.lastname: Heij
authors.forename: David S
authors.initials: DS
authors.lastname: Boss
authors.forename: Jan H M
authors.initials: JH
authors.lastname: Schellens
authors.forename: Hilde
authors.initials: H
authors.lastname: Rosing
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: Lucien A
authors.initials: LA
authors.lastname: Aarden
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000097:blood* / Q000493:pharmacokinetics; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000097:blood* / Q000493:pharmacokinetics; D001943:Breast Neoplasms / Q000188:drug therapy; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D011817:Rabbits; D000068878:Trastuzumab
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D000068878:Trastuzumab