RESUMO
A colorimetric method, reverse transcriptase PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was evaluated for ease of use, reliability, and sensitivity when detecting known human pathogenic virus present in shellfish, using a traditional polyethylene precipitation or immunocapture virus concentration method. The newly developed ELISA method could successfully detect enteroviruses and noroviruses in artificially and naturally contaminated shellfish. Overall, ELISA was shown to be a robust and sensitive method, which had a detection limit of 10 to 100 50% tissue culture infective dose enterovirus per gram of Crassostrea gigas (Pacific oyster) digestive gland and whole Mytilus edulis (common blue mussel). The technique was easily established in a new laboratory and required no specialized equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Moluscos/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frutos do Mar/virologia , Animais , Qualidade de Produtos para o Consumidor , Enterovirus/isolamento & purificação , Humanos , Norovirus/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
A new method, termed RT-PCR-ELISA, was evaluated for ease of use, reliability and sensitivity when detecting infectious pancreatic necrosis virus (IPNV) present in trout kidney tissue. The method had comparable sensitivity to existing PCR assays and could successfully detect 1.5 x 10(4) pfu IPNV in artificially contaminated trout kidney samples. The technique was easily established in a new laboratory and required no specialised equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. The RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.
Assuntos
Infecções por Birnaviridae/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Peixes/diagnóstico , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Oncorhynchus mykiss/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Doenças dos Peixes/virologia , Rim/virologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
AIMS: The purpose of this study was to comprehensively compare the response of nine biosensors capable of being induced by Hg. Induction by Hg was based upon the insertion of merR, merB, zntA and zntR promoter genes. LuxCDABE or lucFF reporter genes expressed luminescence, and host organisms were Escherichia coli, Vibrio anguillarum and Pseudomonas fluorescens. The role of transcriptional switches, reporter mechanism and host organism was to be investigated. METHODS AND RESULTS: All biosensors were subjected to the same assay conditions. Sensors had their own individual growth characteristics and response to the doses of Hg tested. Maximum bioluminescence response was induced by concentrations of Hg between 2.5 nm and 5 microM. E. coli pRB28 was found to detect levels of Hg as low as 1.6 nm and yet was capable of operating in a concentration range of up to 12.5 microM. CONCLUSIONS: The response of the sensors demonstrated their suitability for analysis under environmentally relevant concentrations. The sensitivity of the sensors, the optimum range and the expediency of the assay could not be related to a single sensor trait. It may be concluded that biosensor performance is dependent on more than one of the single factors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that comparative testing of sensors is an important step in evaluating the relevance and performance of biosensors prior to routine environmental application.
Assuntos
Genes Bacterianos/genética , Genes Reporter/genética , Bactérias Gram-Negativas/genética , Mercúrio , Transcrição Gênica/genética , Disponibilidade Biológica , Técnicas Biossensoriais/métodos , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Medições Luminescentes , Mercúrio/análise , Mercúrio/farmacocinética , Regiões Promotoras Genéticas/genética , Pseudomonas fluorescens/genética , Vibrio/genéticaRESUMO
The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.
Assuntos
Técnicas Biossensoriais/métodos , Cianobactérias/fisiologia , Herbicidas/análise , Microbiologia da Água , Poluentes Químicos da Água/análise , Cianobactérias/efeitos dos fármacos , Cianobactérias/genética , Genes Reporter , Herbicidas/química , Herbicidas/metabolismo , Luciferases/genética , Luminescência , Plasmídeos/genética , Reação em Cadeia da Polimerase , Poluentes Químicos da Água/metabolismoRESUMO
Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Escherichia coli/genética , Haptenos/imunologia , Biblioteca de Peptídeos , Peptídeos Cíclicos/imunologia , Anticorpos Monoclonais/genética , Ligação Competitiva , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Haptenos/química , Hemocianinas , Humanos , Toxinas Marinhas , Microcistinas , Peptídeos Cíclicos/química , Soroalbumina BovinaRESUMO
Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.