RESUMO
The purification of Japanese quail IgG from serum was performed using caprilic acid and ammonium sulfate, and from egg yolk using PEG-6000 and ethanol. After confirming the purification and the concentration of IgG, the yolk samples had twice the amount of protein compared to serum samples. The IgG extracts were analyzed by SDS-PAGE and western blot showing similar results as the ones of chicken IgG used as the standard. So, these methodologies can be used for purifying quail serum or egg yolk IgG, which would enable the development of diagnostic assays.(AU)
Assuntos
Animais , Imunoglobulina G/isolamento & purificação , Coturnix , Gema de Ovo/imunologia , Meios de Cultura Livres de SoroRESUMO
An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV) was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI) test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100 percent). These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation(AU)
Assuntos
Animais , Vírus da Doença de Newcastle/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina G/isolamento & purificação , CoturnixRESUMO
An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV) was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI) test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100 percent). These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation
Assuntos
Animais , Coturnix , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/isolamento & purificação , Vírus da Doença de Newcastle/isolamento & purificaçãoAssuntos
Baculoviridae/genética , DNA Recombinante/análise , Transfecção , Animais , Células Cultivadas , DNA Recombinante/isolamento & purificação , Desoxirribonuclease EcoRI , Desoxirribonucleases de Sítio Específico do Tipo II , Vetores Genéticos , Reação em Cadeia da Polimerase , Spodoptera/metabolismoRESUMO
A large number of DNA sequences corresponding to human and animal transcripts have been filed in data banks, as cDNAs or ESTs (expression sequence tags). However, the actual function of their corresponding gene products is still largely unknown. Several of these genes may play a role in regulation of important biological processes such as cell division, differentiation, malignant transformation and oncogenesis. Elucidation of gene function is based on 2 main approaches, namely, overexpression and expression interference, which respectively mimick or suppress a given phenotype. The currently available tools and experimental approaches to gene functional analysis and the most recent advances in mass cDNA screening by functional analysis are discussed.
Assuntos
Elementos Antissenso (Genética) , Expressão Gênica , RNA Catalítico , Transgenes , Animais , DNA Recombinante , HumanosRESUMO
A large number of DNA sequences corresponding to human and animal transcripts have been filed in data banks, as cDNAs or ESTs (expression sequence tags). However, the actual function of their corresponding gene products is still largely unknown. Several of these genes may play a role in regulation of important biological processes such as cell division, differentiation, malignant transformation and oncogenesis. Elucidation of gene function is based on 2 main approaches, namely, overexpression and expression interference, which respectively mimick or suppress a given phenotype. The currently available tools and experimental approaches to gene functional analysis and the most recent advances in mass cDNA screening by functional analysis are discussed