RESUMO
Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.
Assuntos
Anticorpos Monoclonais/biossíntese , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB CRESUMO
cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.
Assuntos
Bioensaio , Fator A de Crescimento do Endotélio Vascular/análise , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/análise , Biotinilação , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Humanos , Proteínas Imobilizadas/análise , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Cinética , Ligantes , Medições Luminescentes , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.
Assuntos
Anticorpos Monoclonais/biossíntese , Encéfalo/metabolismo , Expressão Gênica , Transportadores de Ânions Orgânicos/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Escherichia coli/genética , Vetores Genéticos , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Imunização , Imunoensaio , Camundongos , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
Polyethylene glycol (PEG)ylated (stealth) immunoliposomes directed against human gliofibrillary acidic protein (GFAP) were prepared by coupling the thiolated monoclonal anti-GFAP antibodies with a maleimide derivative of phosphatidyl ethanolamine of the liposomal membrane. Experiments with cell cultures demonstrated specific and competitive binding of these immunoliposomes to embryonic rat brain astrocytes. Administered intravenously into rats, the immunoliposomes displayed typical kinetics with elimination half-lives of 8-15 hr. Being incapable of penetrating the unimpaired blood-brain barrier (BBB), these immunoliposomes, nevertheless, may be useful in delivering drugs to glial brain tumors (which continue to express GFAP) or to other pathological loci in the brain with a partially disintegrated BBB.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Astrócitos/metabolismo , Encéfalo/metabolismo , Proteína Glial Fibrilar Ácida/imunologia , Lipossomos , Animais , Anticorpos Monoclonais/metabolismo , Barreira Hematoencefálica , Portadores de Fármacos , Polietilenoglicóis , Ratos , Ratos WistarRESUMO
Preparations of I(125)-labeled monoclonal antibodies against neurospecific enolase and mouse plasma IgG1 were injected intravenously to rats immediately after unilateral occlusion of the middle cerebral artery. Radioactivity of I(125)-labeled monoclonal antibodies against neurospecific enolase in the brain tissue progressively increased, reached a maximum by the 48th hour, and remained practically unchanged after 72 h. At the same time radioactivity of labeled IgG1 in the brain tissue and radioactivity of both preparations in the blood, liver, spleen, kidneys, heart, and lungs decreased over 72 h. Selective accumulation of I(125)-labeled monoclonal antibodies against neurospecific enolase was less significant in the brain tissue of the contralateral hemisphere and cerebellum not exposed to ischemia.
Assuntos
Anticorpos Monoclonais/farmacocinética , Encéfalo/enzimologia , Encéfalo/imunologia , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/imunologia , Fosfopiruvato Hidratase/imunologia , Animais , Anticorpos Monoclonais/sangue , Barreira Hematoencefálica , Isquemia Encefálica/sangue , Isquemia Encefálica/enzimologia , Isquemia Encefálica/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Infarto da Artéria Cerebral Média/sangue , Radioisótopos do Iodo , Masculino , Camundongos , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos WistarAssuntos
Astrócitos/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Imunoconjugados/química , Imunoconjugados/farmacologia , Lipossomos/química , Lipossomos/farmacologia , Polietilenoglicóis/química , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/administração & dosagem , Astrócitos/imunologia , Encéfalo/citologia , Células Cultivadas , Imunoconjugados/imunologia , Isótopos de Iodo , Cinética , Lipossomos/imunologia , Ratos , Especificidade por SubstratoRESUMO
Methods of GFAP purification and obtaining of hybridoma cells producing monoclonal anti-GFAP antibodies and properties of GFAP preparation were described. The immunobloting data on specificity of obtained monoclonal antibodies are presented. A new method of GFAP immunoenzyme assay based on GFAP preparation and anti-GFAP antibodies was elaborated. Standardization of the immunoenzyme system was shown in tests for specificity, accuracy, and reproducibility.
Assuntos
Anticorpos Monoclonais/imunologia , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/imunologia , Técnicas Imunoenzimáticas/métodos , Animais , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Humanos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Enzyme immunoassay showed penetration of two glia-specific antigens, glial fibrillar acid protein GFAP and specific brain glycoprotein alpha(2)GP, through the blood-brain barrier in rats treated with toxic doses of sodium barbital. The permeability of the blood-brain barrier was completely normalized 3 days after treatment. This method can be used in clinical practice for evaluation of the severity of impairment and dynamics of normalization of blood-brain barrier properties during acute intoxication with barbiturates.
Assuntos
Barreira Hematoencefálica/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Neuroglia/imunologia , Animais , Barbitúricos/toxicidade , Proteínas Sanguíneas/biossíntese , Calibragem , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/sangue , Hipnóticos e Sedativos/toxicidade , Masculino , Pentobarbital/toxicidade , Ratos , Fatores de Tempo , alfa-2-Glicoproteína-HSRESUMO
Hydrophobized and non-hydrophobized Fab fragments of human antibodies against gliofibrillar acid protein (GFAP) and brain specific alpha 2-glycoprotein (alpha 2GP) were used to study their penetration through the blood-brain barrier (BBB). These Fab fragments were modified by stearoyl chloride in reversed micelles of aerosol OT in octane (one or two fatty acid residues attached to protein molecule). Modified and non-modified 125I-labelled Fab fragments were intracardially administered to rats. The amount of label accumulated in brain was 55% higher than the total amount in all other organs. In contrast, non-hydrophobized Fab fragments did not penetrate through the BBB. We assume that the artificial hydrophobization of Fab fragments can increase their capability to penetrate through the cell membranes and, in particular, the BBB.
