RESUMO
OBJECTIVE: To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. METHODS: rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot. RESULTS: The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. CONCLUSIONS: The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Eritropoetina/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas RecombinantesRESUMO
Anti-rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti-rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme-linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS-PAGE, and Western-blotting. Experimental results showed that the subclass and the light chain of anti-rhEPO McAb was IgG1 and kappa light chain. The molecular weight of anti-rhEPO McAb was 166,000 Daltons. The affinity constant (K(aff)) of anti-rhEPO McAb with coated antigen was 5.0 x 10(5)L/mol.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Eritropoetina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Western Blotting , Fusão Celular , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Eritropoetina/análise , Feminino , Humanos , Hibridomas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo , Proteínas RecombinantesRESUMO
AIM: The pharmacokinetics and biodistribution of cisplatin encapsulated in polyphase liposome (KM-1) were compared with those of free drug in rats. METHODS: The platinum levels in serum and normal organs, after a single dose of iv injection of free or encapsulated cisplatin to rats, were determined by induced coupled plasma atomic emission spectrometry. RESULTS: Serum platinum concentration-time curve after a single iv dose of KM-1 4.5 mg/kg in rats was fitted with an open three-compartment model. The pharmacokinetic parameters were as follows: Vc=0.10 L/kg, T1/2pai=0.3 h, T1/2alpha=3.5 h, T1/2beta=2.7 h, AUC=265 mg.h.L(-1), and CL(s) =0.02 g.L.h(-1). KM-1 was cleared from the circulation much more slowly than free cisplatin. Liver and spleen had the highest concentration of platinum after KM-1 treatment. CONCLUSION: KM-1 remained in the bloodstream longer than its free drug, and was taken mainly by the reticuloendothelial system.