RESUMEN
Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales , Immunoblotting/métodos , Preparaciones Farmacéuticas/análisis , Acridinas/química , Antibacterianos/química , Antibacterianos/inmunología , Antiinflamatorios/química , Antiinflamatorios/inmunología , Anticuerpos Monoclonales/química , Anticonvulsivantes/química , Anticonvulsivantes/inmunología , Carbamazepina/química , Carbamazepina/inmunología , Colodión/química , Ciclosporina/química , Ciclosporina/inmunología , Gentamicinas/química , Gentamicinas/inmunología , Hidrocortisona/química , Hidrocortisona/inmunología , Inmunosupresores/química , Inmunosupresores/inmunología , Metotrexato/química , Metotrexato/inmunología , Reproducibilidad de los Resultados , Sirolimus/química , Sirolimus/inmunología , Tacrolimus/química , Tacrolimus/inmunologíaRESUMEN
BACKGROUND: Pro-gastrin releasing peptide (ProGRP) concentrations in blood play an important role in the diagnosis and treatment of patients with small cell lung cancer (SCLC). The automated quantitative ARCHITECT ProGRP assay was developed to aid in the differential diagnosis and in the management of SCLC. The purpose of this study was to evaluate the analytical performance of this chemiluminescent microparticle immunoassay at multiple sites. METHODS: ARCHITECT ProGRP measures ProGRP using a two-step sandwich using monoclonal anti-ProGRP antibodies coated on paramagnetic microparticles and labeled with acridinium. Analytical performance of the assay was evaluated at four sites: Abbott Japan, Denka Seiken, the Johns Hopkins University, and the University of Munich. RESULTS: Total precision (%CV) for nine analyte concentrations was between 2.2 and 5.7. The analytical sensitivity of the assay was between 0.20 pg/mL and 0.88 pg/mL. The functional sensitivity at 20% CV was between 0.66 pg/mL and 1.73 pg/mL. The assay was linear up to 50,000 pg/mL using a 1:10 autodilution protocol. The calibration curve was stable for 30 days. Comparison with the Fujirebio microtiter plate enzyme-linked immunosorbent assay (EIA) ProGRP assay gave a slope of 0.93 and a correlation coefficient (r) of 0.99. CONCLUSIONS: These results demonstrate that the ARCHITECT ProGRP assay has excellent sensitivity, precision, and correlation to a reference method. This assay provides a convenient automated method for ProGRP measurement in serum and plasma in hospitals and clinical laboratories.