RESUMO
The present report describes a simple and rapid dot-immunobinding assay combined with a chemiluminescence detection system for screening hybridoma supernatants for specific monoclonal antibodies (MAbs). Small rectangular nitrocellulose filters dotted with either crude mixtures of antigens, or with control samples, were placed in six well plates, incubated with hybridoma supernatants, then stained with peroxidase-conjugated anti-mouse IgG. The reaction was performed with a chemiluminescence detection system. We used this method to screen hybridoma supernatants for MAbs against a 354 amino acid polypeptide of hog cholera virus (HCV) gp33-gp55 protein expressed as a fusion protein. We also extended it for the screening of MAbs against foot-and-mouth disease virus (FMDV). The chemiluminescence dot-immunobinding assay (CDIA) was compared with neutralization (N) and immunofluorescence (IF) screening tests and some FMDV seroneutralizing MAbs were found to be either poorly reactive or undetected by the IF test. The advantage of the present method is that it detects in only one step all MAbs detected in the IF and/or N tests together with some MAbs not detected by either of these methods. The present method is at least 356 times more sensitive than the IF test.
Assuntos
Anticorpos Monoclonais/análise , Hibridomas/imunologia , Immunoblotting/métodos , Medições Luminescentes , Animais , Antígenos Virais/imunologia , Aphthovirus/imunologia , Western Blotting , Vírus da Febre Suína Clássica/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Sensibilidade e Especificidade , Proteínas Virais de Fusão/imunologiaRESUMO
Probes were prepared from genomic RNA of Hog Cholera Virus (HCV) after synthesis of cDNA and cloning. Six probes were selected according to their place on the viral genome determined by sequencing and comparison with BVDV sequence. These probes were hybridized with two strains of HCV (Alfort and Nord), two strains of Bovine Viral Diarrhea (BVDV) (NADL, New York) and four strains of Border Disease (BD) (Lyon 1, Lyon 2, Aveyron, IEMVT). This panel of six probes seem to be able to differentiate pestiviruses but some differences rely only on slight intensity of the hybridization.
Assuntos
Vírus da Febre Suína Clássica/genética , Sondas de DNA , DNA Complementar/genética , Pestivirus/isolamento & purificação , Animais , Bovinos , Linhagem Celular , Hibridização de Ácido Nucleico , SuínosRESUMO
Cis-acting elements involved in the control of rat alpha-fetoprotein gene expression in the liver and its modulation by glucocorticoid hormones were detected after transfection of chloramphenicol acetyltransferase constructs and their transient expression into two hepatoma cell lines. The proximal promoter region (-324 to -15) was found to contain all the information necessary for tissue-specific expression. It is also involved in the negative gene modulation by glucocorticoids and includes an activating regulatory domain allowing efficient expression in the HepG2 cells. Three regions within 7 kilobase pairs of the 5' extragenic sequences are capable of stimulating the chloramphenicol acetyltransferase activity driven by the alpha-fetoprotein promoter sequence. One of these regions, at about -2.5 kilobase pairs, contains a short indivisible 170-base pair DNA element that fulfills all the criteria of a tissue-specific enhancer, i.e. orientation and position independence, as well as cell-specific stimulation of gene expression driven by a homologous or heterologous promoter. The enhancing properties of this element are totally abolished by glucocorticoids. DNase I footprinting experiments indicate that several rat liver nuclear proteins interact with this enhancer element.
