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1.
J Vet Diagn Invest ; 19(2): 180-4, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17402613

RESUMO

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.


Assuntos
Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/isolamento & purificação , Animais , Cobaias/virologia , Imunoensaio/métodos , Coelhos/virologia , Sorotipagem/métodos
2.
Am J Med Genet A ; 127A(2): 158-66, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15108204

RESUMO

We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child.


Assuntos
Gangliosidoses GM2/genética , Mutação/genética , RNA Mensageiro/genética , beta-N-Acetil-Hexosaminidases/deficiência , beta-N-Acetil-Hexosaminidases/genética , Processamento Alternativo/genética , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Células COS , Chlorocebus aethiops , Primers do DNA , Evolução Fatal , Fibroblastos/metabolismo , Hexosaminidase A , Humanos , Lactente , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , beta-N-Acetil-Hexosaminidases/metabolismo
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