RESUMEN
A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed ( 2 h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography-mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.
Asunto(s)
Toxinas Marinas/química , Neurotoxinas/química , Ostreidae/química , Oxocinas/química , Extractos de Tejidos/análisis , Animales , Electroquímica/métodos , Humanos , Inmunoensayo/métodos , Luminiscencia , Toxinas Marinas/sangre , Toxinas Marinas/aislamiento & purificación , Toxinas Marinas/orina , Modelos Moleculares , Estructura Molecular , Neurotoxinas/aislamiento & purificación , Oxocinas/sangre , Oxocinas/aislamiento & purificación , Oxocinas/orina , Reproducibilidad de los Resultados , RutenioRESUMEN
Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.
Asunto(s)
Toxinas Bacterianas/análisis , Proteínas de Unión al Calcio/análisis , Clostridium perfringens , Electroquímica/métodos , Mediciones Luminiscentes/métodos , Fosfolipasas de Tipo C/análisis , Anticuerpos , Toxinas Bacterianas/inmunología , Proteínas de Unión al Calcio/inmunología , Modelos Moleculares , Sensibilidad y Especificidad , Fosfolipasas de Tipo C/inmunologíaRESUMEN
We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5-1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE's in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.
Asunto(s)
Enterotoxinas/orina , Ensayo de Inmunoadsorción Enzimática/métodos , Staphylococcus aureus/fisiología , Enterotoxinas/inmunología , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.