The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry.
For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays--especially enzyme-linked immunosorbent assays (ELISAs)--are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3',5'-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r=0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.
Damen
Carola W N
CW
Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. carola.damen@slz.nl
Speijer
Han
H
Hermens
Wim T
WT
Schellens
Jan H M
JH
Rosing
Hilde
H
Beijnen
Jos H
JH
D000911:Antibodies, Monoclonal / Q000097:blood*; D061067:Antibodies, Monoclonal, Humanized; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000068878:Trastuzumab