Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma.
Anal Biochem
; 391(2): 114-20, 2009 Aug 15.
Article
en En
| MEDLINE
| ID: mdl-19464994
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ensayo de Inmunoadsorción Enzimática
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Anticuerpos Monoclonales
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Antineoplásicos
Límite:
Animals
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Female
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Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2009
Tipo del documento:
Article
País de afiliación:
Países Bajos
Pais de publicación:
Estados Unidos