quantification of autoantibodies in the serum of experimental autoimmune encephalomyelitis immunized mice using elisa

Investigating Humoral Response in Autoimmune Demyelination

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal infiltration of immune cells into the brain parenchyma, breakdown of myelin sheaths wrapping axons, glia activation, and neuronal loss1. In addition to the well-established role of pathogenic T cells, multiple lines of evidence have highlighted the involvement of B cells in mediating the autoimmune response against the CNS. B cells undergo clonal expansion in the MS brain and antibodies against myelin components have been detected within demyelinated lesions2,3. The selective activation of peripheral B cells at disease onset has been recently documented, suggesting a putative role for this immune cell compartment in disease initiation as well4. The success of B cell-depleting therapies such as anti-CD20 monoclonal antibodies further corroborates the mechanistic connection between aberrant B cell functioning and autoimmune demyelination5,6. From a molecular standpoint, B cells can contribute to disease via autoantigen presentation, pro-inflammatory cytokine secretion, and autoreactive antibody production.
Multiple animal models have been developed to recapitulate specific features of the complex MS phenotype. Among them, experimental autoimmune encephalomyelitis (EAE) is the most widely used in vivo paradigm and relies on the immunization of experimental animals with short peptides derived from myelin proteins such as myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP)7. EAE immunized animals develop a demyelinating pathology that resembles MS in many aspects, including a robust humoral response against the encephalitogenic peptide8. For this reason, EAE studies have been instrumental in dissecting the function of B cells and autoantibodies in the context of disease. For instance, it was demonstrated that MOG-specific antibodies isolated from MS patients can aggravate the clinical course in EAE models9. Notably, the proline residue at position 42 in human MOG was shown to be critical for determining autoantibody pathogenicity10. More recently, MOG-specific autoantibodies have been found to promote disease not only by mediating myelin loss but also via boosting the reactivation of autoreactive T cells within the CNS11. 
Considering the importance of antibody responses in CNS autoimmunity, this article presents an ELISA-based protocol to efficiently measure the serum levels of autoreactive antibodies in C57BL/6J mice EAE immunized with MOG35-55 peptide. In the first part of the protocol, the method to collect serum via intracardiac puncture will be described. Subsequently, the procedures to set up the ELISA assay and acquire the data will be detailed. Lastly, data analysis and interpretation will be discussed.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

So the focus of our laboratory is to understand the molecular basis of multiple sclerosis, a immune disease of the central nervous system that is characterized by demyelination, neuroinflammation, and axonal injury. So this protocol represent an economical and flexible alternatives to commercial kits. Our can be easily adjusted to test B-cell antibody responses against different EEA peptides.My lab is devoted to further understanding the connection between neuroinflammation and neurodegeneration in chronic neurological diseases such as multiple sclerosis.

Quantification of Autoantibodies in the Serum of Experimental Autoimmune Encephalomyelitis Immunized Mice Using ELISA

Comience volviendo a suspender el péptido MOG 35 55 liofilizado en agua. Diluya el caldo de péptidos a 10 microgramos por mililitro en tampón de recubrimiento antes del experimento. Ahora pipetee 100 microlitros de la solución final en pocillos de una placa de 96 pocillos.Cubra simultáneamente un número igual de pocillos con 100 microlitros de BSA disueltos en tampón de recubrimiento. Selle la placa con una película adhesiva para evitar la evaporación e incube la placa durante la noche a cuatro grados centígrados. Al día siguiente, realice tres lavados de la placa con 200 microlitros de PBS suplementado con 0,5%tween 20.Posteriormente añadir 100 microlitros por pocillo de una solución de bloqueo compuesta por un 3%BSA en PBS. Incubar la placa sellada durante una hora a 37 grados centígrados en un horno de hibridación. Después de la incubación, lave la placa tres veces con 200 microlitros por pocillo de PBST.Diluir cada muestra de suero obtenida de la sangre de ratones inmunizados con EAE en solución bloqueante y añadir 100 microlitros por pocillo tanto a los pocillos MOG 35 55 como a los pocillos recubiertos de BSA. Agregue el mismo volumen de solución de bloqueo a los pozos en blanco designados. Incubar la placa sellada durante dos horas a temperatura ambiente con agitación constante.Después de la incubación, retire la placa de la incubadora y enjuague la placa tres veces con 200 microlitros por pocillo de PBST. Diluir el anticuerpo secundario conjugado HRP en una solución que contenga 0,2% de BSA en PBST y añadir 100 microlitros a cada pocillo. Incubar la placa sellada durante una hora a temperatura ambiente, manteniendo una agitación constante.Lave el plato tres veces con 200 microlitros por pocillo de PBST. Agregue 100 microlitros de sustrato de tetrametil bencidina de tres, tres primos, cinco, cinco primos a cada pocillo. Incubar en la oscuridad durante uno a cinco minutos, monitoreando el desarrollo de un color azul.Agregue 100 microlitros de solución de parada a cada pocillo. A continuación, utilice un lector de placas configurado a una longitud de onda de 450 nanómetros para medir la densidad óptica de cada pocillo. Los valores de densidad óptica de las muestras de EAE fueron significativamente más altos que los de las muestras de control, lo que indica una respuesta robusta de inmunoglobulina G contra el péptido MOG.

Research

JoVE Journal

Immunology and Infection

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Investigación

Inmunología e Infección

Serum Collection via Intracardiac Puncture in Experimental Autoimmune Encephalomyelitis (EAE) Immunized Mice