RESUMEN
AIM: Development of micro technologies based approach for express diagnostics of toxigenic C. diphtheriae strains. MATERIALS AND METHODS: Corynebacterium diphtheriae 10648 (tox+) and C. diphtheriae NCTC 10356 (tox-) from Central Health Laboratory (London) reference strains were used as positive and negative controls respectively. Diagnostic kit was created by using fractions of antibodies with high avidity that were obtained by consecutive fractioning of positive antitoxic blood sera and then loaded onto polyacrylamide latex particles with the diameter of 0.81 microm. 20 Elek test positive C. diphtheriae strains and 20 tox gene PCR negative C. diphtheriae strains (i.e. non toxigenic) (Pasteur Research Institute of Epidemiology and Microbiology) were used as control. Indirect hemagglutination with anti-diphtheria antibody diagnostic kit was used as a quantitative control. PCR, Elek test and ICS test were used as quality control. RESULTS: The diagnostic kit obtained had specificity of 97%, sensitivity of 98%. Specimen preparation time is 15 - 20 minutes, reaction time - 2 - 3 minutes, and up to 93 specimens can be analyzed on a single microchip. CONCLUSION: The developed approach has high sensitivity and specificity, is easy to use, and fast in regard to preparation and reaction time. Portability of the apparatus allows the use of reagents in micro volumes.
Asunto(s)
Proteínas Bacterianas/análisis , Toxina Diftérica/análisis , Difteria/diagnóstico , Ensayos Analíticos de Alto Rendimiento/métodos , Procedimientos Analíticos en Microchip/métodos , Complejo Antígeno-Anticuerpo/inmunología , Antitoxinas/sangre , Antitoxinas/inmunología , Corynebacterium diphtheriae/patogenicidad , Difteria/sangre , Difteria/inmunología , Difteria/microbiología , Pruebas de Hemaglutinación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Dispositivos Laboratorio en un Chip , Microesferas , Reacción en Cadena de la Polimerasa , Estándares de Referencia , Sensibilidad y EspecificidadAsunto(s)
Proteínas Sanguíneas/efectos de los fármacos , Cloruros , Cobalto/farmacología , Compuestos de Manganeso , Animales , Proteínas Sanguíneas/análisis , Cromatografía Líquida de Alta Presión/métodos , Cobalto/fisiología , Ácido Edético/farmacología , Femenino , Técnicas In Vitro , Masculino , Manganeso/farmacología , Concentración Osmolar , Presión Osmótica/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
A method of determination of molecular masses of globular proteins based on microcolumn size-exclusion HPLC on modified silica sorbent (TSK-GEL 3000 SW) with refractometric detection has been developed. Molecular masses in the range 10 to 200 kD were determined with the accuracy of +/- 3%.
Asunto(s)
Cromatografía Líquida de Alta Presión , Proteínas/análisis , Glicina/análisis , Peso Molecular , RefractometríaRESUMEN
A highly sensitive method for the determination of myoglobin in serum is described, based on high-performance size-exclusion chromatography with chemiluminescence detection. Serum proteins are separated according to their molecular masses on columns packed with TSK-SW gel and those containing haem are detected selectively by post-column chemiluminescence reaction with luminol using a conventional fluorimetric detector. The method is rapid (30 min) and sufficiently sensitive for the diagnosis of myocardial infarction. The minimum detectable myoglobin concentration is 10 ng/ml.