Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis
Summary
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.
Abstract
Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.
Introduction
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.
Protocol
Representative Results
Discussion
Here, a simple and efficient ELISA-based protocol was reported to accurately quantify the humoral response in a relevant animal model of MS pathology. This method has been recently employed to describe the novel role of the ataxin-1 protein in controlling the serum levels of autoantibodies in the MOG35-55/C57BL6J EAE paradigm12. In this regard, a number of factors should be taken into consideration, in order to obtain consistent and biologically meaningful results with this method.
<p class="j…Declarações
The authors have nothing to disclose.
Acknowledgements
This study was supported by the National Institutes of Health (R03NS131908) and the Department of Defense through the Multiple Sclerosis Research Program under Award No. W81XWH-22-1-0517. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This study was also supported by East Carolina University startup funds.
Materials
| 1 mL syringes | BD Biosciences | 309628 | |
| 1.5 mL microcentrifuge tubes | Fisher | 05-408-129 | |
| 25 G needles | BD Biosciences | 305122 | |
| 3,3',5,5'-tetramethylbenzidine (TMB) substrate | Thermo Fisher | N301 | Store at 4 °C |
| Adhesive seals | Thermo Fisher | AB0558 | |
| Bovine serum albumin (BSA) | Sigma | A7906 | Store at 4 °C |
| C57BL/6J female mice | The Jackson Laboratory | 000664 | Animals between 8-10 weeks of age should be used for EAE experiments |
| Cryogenic tubes | Fisher | 10-500-25 | |
| Dissection tray | Fisher | S111022 | |
| Dissector scissors | Fine Science Tools | 14082-09 | |
| ELISA coating buffer | BioLegend | 421701 | Store at 4°C |
| Excel software | Microsoft | Analysis spreadsheet | |
| Forceps | Fine Science Tools | 11152-10 | |
| Goat Anti-Mouse IgG, Human ads-HRP | SouthernBiotech | 1030-05 | Store at 4 °C |
| LED light source | Fisher | AMPSILED21 | |
| Microplate reader | Fisher | 14-377-575 | |
| Molecular biology grade water | Corning | 46-000-Cl | |
| Mouse MOG35-55 peptide | EZBiolab | cp7203 | Store at -80 °C |
| Multichannel pipette | Axygen | AP-12-200-P | |
| Noyes spring scissors | Fine Science Tools | 15011-12 | |
| Nunc MaxiSorp 96-well plates | BioLegend | 423501 | |
| Orbital shaker | Fisher | 88-861-023 | |
| Oven | VWR | 445-0024 | |
| Phosphate buffer saline (PBS) | Thermo Fisher | 14190144 | |
| Refrigerated tabletop centrifuge | Thermo Fisher | 75002441 | |
| Stop solution | Thermo Fisher | N600 | |
| Tween 20 | Bio-Rad | 1706531 |
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