Introducción de un ELISA como ensayo alternativo en la determinación de la potencia de vacunas antitetánicas.

Introduction of an ELISA as an alternative assay for potency testing of tetanus toxoid vaccines.

Juan Carlos Ramírez, Esther María Fajardo, Beatriz Romeu, Ubel Jesús Ramírez, Eduardo Álvarez, Rolando Ochoa, Alicia Perojo, Regla Sosa, Deolinda Silva, Irma Labrador, Adina García, Pablo González, Luis Izquierdo.

Instituto Finlay. Centro de Investigación-Producción de Vacunas y Sueros. Ciudad de La Habana, Cuba. E-mail: jcramirez@finlay.edu.cu

RESUMEN

Por más de medio siglo de uso, la vacunación con el toxoide tetánico ha mostrado un elevado porcentaje de eficacia en la prevención del tétanos. Este trabajo pretende introducir un ensayo inmunoenzimático en fase sólida (ELISA) como método alternativo a la prueba de seroneutralización in vivo utilizada en la evaluación de la potencia de las vacunas antitetánicas.Se desarrolló un ELISA de tipo indirecto para la cuantificación de antitoxina tetánica en suero de curiel a partir de un estándar con 29 UI/mL, previamente calibrado. Se determinó la precisión, exactitud y linealidad del ensayo. Se analizó la correlación entre el ELISA y la prueba biológica mediante la evaluación de un total de 75 muestras de sueros por ambos métodos. Por último, se estudió la respuesta individual de un grupo de animales contra 15 lotes de toxoide tetánico. El ensayo demostró ser preciso y exacto, con imprecisiones inferiores al 20% y valores de recuperación entre el 90–110%. Las desviaciones del paralelismo mostraron coeficientes de variación alrededor del 10%. El análisis por regresión lineal mostró una buena correlación entre el ELISA y el ensayo biológico (R2= 0,989). El método alternativo desarrollado probó ser una herramienta útil para la determinación de la potencia de vacunas antitetánicas a partir de la evaluación independiente de la respuesta de cada animal contra el toxoide tetánico.Los niveles de seroprotección alcanzados se encontraron entre el 83–100%.

Palabras claves: ELISA, métodos alternativos, ensayos in vitro, toxoide tetánico.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to quantify tetanus antitoxin activity in guinea pig sera based on a previously calibrated standard. This test is proposed as an alternative to the traditional in vivo neutralization assay used for potency evaluation of tetanus vaccines.The accuracy, precision and linearity of our ELISA were determined. Both methods were used to evaluate 75 guinea pig sera and the results were compared by linear regression analysis. The individual immunological responses against different lots of tetanus vaccine were also studied. The ELISA showed an excellent accuracy, with recovery values between 90-110%; intra, inter and total-assay imprecision ranging around 10% and parallelism deviations below 10%. A high correlation was found between our ELISA and the in vivo neutralization assay (R2= 0,989). The seroprotection levels of the tetanus vaccines tested were over 83%. All those results indicate our ELISA is an excellent alternative procedure for potency testing of tetanus toxoid vaccines.

Keywords: ELISA, alternative methods, in vitro tests, tetanus toxoid

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