Validación de un ensayo inmunoenzimático tipo ELISA para cuantificar niveles de antitoxina diftérica en suero humano / Validation of an ELISA-type immune-enzymatic test to quantify levels of diphtheria antitoxin in human serum

Introducción: La difteria aún persiste en numerosos países. En Cuba, estudios realizados en diferentes grupos etarios han demostrado que existen niveles no protectores de antitoxina diftérica en la población, por lo que es necesario contar con métodos que permitan la estimación serológica de la inmunidad poblacional. La cuantificación de anticuerpos contra antígenos vacunales como la toxina diftérica es además un método útil, rápido y económico para evaluar la respuesta inmune. Objetivo: Validar un ensayo inmunoenzimático tipo ELISA para cuantificar los niveles de antitoxina diftérica en suero humano. Material y Método: Se realizó un estudio experimental de desarrollo tecnológico, en el cual se determinaron los valores óptimos de las variables que influyen en el resultado de un ensayo inmunoenzimático heterogéneo indirecto para la cuantificación de antitoxina diftérica, desarrollado en el laboratorio de Inmunología del Centro Nacional de Genética Médica de Cuba. La curva de calibración se evaluó contra el estándar de la OMS (Diphtheria Antitoxin Human Serum 00/496). Se realizó la validación analítica del método estandarizado. Resultados: Los coeficientes de variación intraensayo e interensayo fueron inferiores a 10 por ciento y 20 por ciento, respectivamente. En la exactitud y selectividad se encontraron valores de recobrado entre 90 y 110 por ciento. El paralelismo entre la curva estándar y las muestras estudiadas presentó un coeficiente de variación menor o igual a 10 por ciento. El límite de cuantificación fue 0,015 UI/mL y el de detección 0,0039 UI/mL. Conclusiones: El resultado obtenido en la precisión, exactitud y selectividad del ensayo inmunoenzimático tipo ELISA desarrollado demostró que puede ser utilizado en la práctica clínica para cuantificar los valores de antitoxina diftérica en suero humano(AU)

Introduction: Diphtheria still persists in many countries. In Cuba, studies conducted in different age groups have demonstrated that there are non-protective levels of diphtheria antitoxin in the population, so it is necessary to have methods that allow the serologic survey of population immunity. The quantification of antibodies against vaccine antigens such as diphtheria toxin is also a useful, rapid and economic method to evaluate the immune response. Objective: To validate an ELISA-type immune-enzymatic test to quantify the levels of diphtheria antitoxin in human serum. Material and Method: An experimental study of technological development was carried out in the Immunology Laboratory of the National Medical Genetics Center, Havana, Cuba. The optimal values ​​of the variables that influence on the result of the indirect heterogeneous immune-enzymatic test for the quantification of diphtheria antitoxin were determined. The calibration curve obtained was evaluated against the WHO standard (Diphtheria Antitoxin Human Serum 00/496). The analytical validation of the standardized method was performed. Results: The intra-assay and inter-assay coefficients of variation were less than 10 percent and 20 percent, respectively. Recovery values ​​between 90 and 110% were found in accuracy and selectivity. The parallelism between the standard curve and the samples studied showed a coefficient of variation lower or equal to 10 percent. The limit of quantification was 0,015 IU/mL and the one of detection was 0,0039 IU/mL. Conclusions: The result obtained in the precision, accuracy and selectivity of the ELISA-type immune-enzymatic test developed and validated in the National Medical Genetics Center demonstrated that it can be used in the clinical practice to quantify the values ​​of diphtheria antitoxin in human serum(AU)

In vivo assay to identify bacteria with ß-glucosidase activity

Background: ß-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with ß-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo ß-glucosidase assay as a fast method to find a ß-glucosidase producer strain. Results: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-ß-glucopyranoside (pNPG). The presence of ß-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks ß-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows ß-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The ß-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher ß-glucosidase activity among several lactobacillus species. Conclusion: This in vivo ß-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with ß-glucosidase activity but also with high ß-glucosidase activity.