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¿Qué es el ELISA?

El ensayo inmunoabsorbente ligado a enzimas (ELISA) es un ensayo basado en placa que detecta y cuantifica péptidos, proteínas, anticuerpos y hormonas.
Este ensayo depende de anticuerpos específicos para unir el antígeno objetivo y un sistema de detección para indicar la presencia y la cantidad de unión al antígeno.

Aplicabilidad

ELISA puede usarse para:

detectar la presencia de alérgenos en los alimentos
determinación de concentraciones de drogas ilícitas
monitorear los niveles de concentraciones farmacéuticas de drogas (medicamentos)
prueba de embarazo
diagnóstico de diversas enfermedades que inducen la producción de inmunoglobulinas, como enfermedades infecciosas, autoinmunes o alergias.

Como es el procedimiento?

En este ensayo, un antígeno se inmoviliza en una superficie sólida y luego forma un complejo con un anticuerpo que está unido a una enzima. La detección se logra evaluando la actividad enzimática conjugada mediante incubación con un sustrato para producir un producto medible.
El elemento más importante de la estrategia de detección es una interacción anticuerpo-antígeno altamente específica.
La siguiente figura resume los pasos del método ELISA.





Este método puede ser complejo e incluir múltiples pasos intermedios, especialmente cuando se mide la concentración de proteínas en muestras heterogéneas (como la sangre). El paso más complejo y variado en el proceso general es la detección, donde se pueden usar múltiples capas de anticuerpos para amplificar la señal.


Modificaciones en procedimiento básico

ELISA se puede realizar con algunas modificaciones al procedimiento básico:

Directo - utiliza un anticuerpo primario marcado que reacciona directamente con el antígeno. La inmovilización del antígeno de interés se realiza mediante adsorción directa a la placa de ensayo.

Indirecta - utiliza un anticuerpo secundario marcado para la detección. La inmovilización del antígeno de interés se realiza indirectamente a través de un anticuerpo de captura que se ha unido a la placa.

Sandwich - el analito a medir se une entre dos anticuerpos primarios (el anticuerpo de captura y el de detección).

Competitivo - se usa comúnmente cuando el antígeno es pequeño y tiene un solo epítopo o sitio de unión de anticuerpos.


Referencias
British Society of Immunology. Enzyme-linked immunosorbent assay (ELISA). Available at: https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay. Accessed on: 02/04/2020.
John R. Crowther. Methods in Molecular Biology, The ELISA Guidebook. Second Edition. Humana Press, a part of Springer Science + Business Media, LLC 2009.
Jianwen He. Practical Guide to ELISA Development. The Immunoassay Handbook (Fourth Edition), 2013.
Boster. ELISA Fundamental Principle, How ELISA Works. Available at: https://www.bosterbio.com/protocol-and-troubleshooting/elisa-principle. Accessed on: 02/04/2020.
R., Crowther, J. (1995). ELISA : theory and practice. Totowa, N.J.: Humana Press. ISBN 978-0896032798.
Lima, Raquel. LABORATORY ANALYSIS OF ISOPROSTANES. 1º Edition. Amazon, 2020.
Lima, Raquel. ANALYSIS OF ANTIOXIDANTS IN FOOD. 1º Edition. Amazon, 2020.




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