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)

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Potency assay of diphtheria antitoxin in vero cell micro-cultures

Se establecieron condiciones para llevar a cabo la titulación de antitoxina diftérica en células Vero (TCC), y comparar los resultados con la prueba intradérmica (TID) tradicionalmente empleada en todo el mundo para determinación de potencia de toxoide diftérico y de antitoxina de uso terapéutico. Los resultados de titulaciones en pruebas intradérmicas y en cultivos celulares mostraron gran repetibilidad y detectabilidad a concentraciones muy bajas de toxina en la prueba de cultivos celulares, así como una correlación satisfactoria en la titulación de antitoxinas preparadas por inmunización con Patrón Internacional de OMS y con antitoxina de uso terapéutico, pero en los sueros de cobayo se observó un título aproximadamente 10 veces menor al de la prueba intradérmica. Se discuten las posibles causas de estas diferencias de títulos. Esta prueba se podrá adoptar en la forma propuesta para determinar potencia de antitoxina diftérica. En la prueba de potencia del toxoide diftérico, se podría usar si el límite de título en los cobayos se ajusta a 0.2 UIAD en lugar de 2.0 UIAD/ml que se emplea actualmente, considerando la equivalencia obtenida entre las dos pruebas y tomando en cuenta una relación 1:10 entre las pruebas TCC y TID

Immunity against diphtheria in the age group 15 to 25 years.

Levels of diphtheria antitoxin in 254 serum samples were estimated in subjects in the age group 15 to 25 years with no previous history of immunisation against diphtheria using indirect haemagglutination test. All the individuals showed more than 0.03 IU/ml of diphtheria antitoxin level. This indicates that the age group studied was not susceptible to diphtheria. No significant relationship could be, however, ascertained between diphtheria antitoxin level and epidemiological factors like age, area (rural/urban), socioeconomic status, family size, education status and religion.

Incidence of cutaneous diphtheria in Bangladesh.

Corynebacterium diphtheriae (C. diphtheriae) was isolated from 23 (7%) skin ulcers out of 327 cases studied. Eight (33%) of the 23 positive cases belonged to the age group of 6 to 10 years. Of the 19 strains of C. diphtheriae typed, 18 (95%) were gravis and of which 14 (77%) were toxigenic. More than 90% of the C. diphtheriae positive ulcer cases had serum anti-toxin level at or above the protective level as against 49% of control. No diphtheria bacilli were isolated from throat of any of the skin positive cases. All the ulcers from which C. diphtheriae were isolated also harboured other definite wound pathogens. It is believed that the presence of toxigenic C. diphtheriae in skin wound might play an important role in the spread of faucial diphtheriae. It is evident from serum anti-toxin level that infected persons develop natural immunity.

Diphtheria and tetanus antitoxin levels in Thai children.

Determination of diphtheria and tetanus antitoxin levels by an indirect haemagglutination method were conducted in 101 nonimmunized schoolchildren, 155 pediatric patients and 102 blood donors. Diphtheria and tetanus antitoxin levels were found mostly adequate among immunized children. Diphtheria antitoxin levels were found adequate in 68.3% of the non-immunized schoolchildren. Tetanus antitoxin levels were found inadequate for protection in the non-immunized children and adults. Immunization of children and adults with diphtheria and tetanus toxoid are highly recommended.