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

Undersöker Humoral respons vid autoimmun demyelinisering

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.

Författare i fokus: Ny analys för att studera B-cellssvar vid forskning om multipel skleros

Kvantifiering av autoreaktiva antikroppar hos möss vid experimentell autoimmun encefalomyelit

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.

Kvantifiering av autoantikroppar i serum hos experimentella autoimmuna encefalomyelitimmuniserade möss med hjälp av ELISA

Börja med att åter suspendera den frystorkade MOG 35 55-peptiden i vatten. Späd peptidmaterialet till 10 mikrogram per milliliter i beläggningsbuffert före experimentet. Pipettera nu 100 mikroliter av den slutliga lösningen i brunnar på en platta med 96 brunnar.Betäck samtidigt ett lika stort antal brunnar med 100 mikroliter BSA upplöst i beläggningsbuffert. Försegla plattan med en självhäftande film för att förhindra avdunstning och inkubera plattan över natten vid fyra grader Celsius. Nästa dag tvättar du plattan tre gånger med 200 mikroliter PBS kompletterat med 0,5 % mellan 20 och 20 mikroliter.Tillsätt därefter 100 mikroliter per brunn av en blockerande lösning bestående av 3% BSA i PBS. Inkubera den förseglade plattan i en timme vid 37 grader Celsius i en hybridiseringsugn. Efter inkubation, tvätta plattan tre gånger med 200 mikroliter per brunn PBST.Späd varje serumprov som erhållits från blod från EAE-immuniserade möss i blockerande lösning och tillsätt 100 mikroliter per brunn till både MOG 35 55 och BSA-belagda brunnar. Tillsätt samma volym blockerande lösning till avsedda tomma brunnar. Inkubera den förseglade plattan i två timmar i rumstemperatur med konstant skakning.Efter inkubation, ta bort plattan från inkubatorn och skölj plattan tre gånger med 200 mikroliter per brunn PBST. Späd den HRP-konjugerade sekundära antikroppen i en lösning bestående av 0,2 % BSA i PBST och tillsätt 100 mikroliter till varje brunn. Inkubera den förseglade plattan i en timme vid rumstemperatur och skaka konstant.Tvätta plattan tre gånger med 200 mikroliter per brunn PBST. Tillsätt 100 mikroliter tre, tre prime, fem, fem primära tetrametylbensidinsubstrat till varje brunn. Inkubera i mörker i en till fem minuter och övervaka utvecklingen av en blå färg.Tillsätt 100 mikroliter stopplösning till varje brunn. Använd sedan en plattläsare inställd på en våglängd på 450 nanometer för att mäta den optiska densiteten i varje brunn. De optiska densitetsvärdena för EAE-proverna var signifikant högre än kontrollproverna, vilket indikerar ett robust immunglobulin G-svar mot MG-peptiden.

Research

JoVE Journal

Immunology and Infection

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

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

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

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

Seruminsamling via intrakardiell punktion hos experimentella autoimmuna encefalomyelit (EAE) immuniserade möss