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

Undersøgelse af humorær respons ved 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.

Forfatter Spotlight: Nyt assay til undersøgelse af B-celleresponser i multipel skleroseforskning

Kvantificering af autoreaktive antistoffer hos mus ved eksperimentel autoimmun 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.

Kvantificering af autoantistoffer i serum af eksperimentel autoimmun encephalomyelitis immuniserede mus ved hjælp af ELISA

Begynd med at re-suspendere det frysetørrede MOG 35 55 peptid i vand. Fortynd peptidstammen til 10 mikrogram pr. milliliter i belægningsbuffer før eksperimentet. Pipette nu 100 mikroliter af den endelige opløsning i brønde af en 96 brøndplade.Overtræk samtidig et lige antal brønde med 100 mikroliter BSA opløst i belægningsbuffer. Pladen forsegles med en klæbende film for at forhindre fordampning, og pladen inkuberes natten over ved fire grader Celsius. Den næste dag udføres tre vaske af pladen med 200 mikroliter PBS suppleret med 0,5% mellem 20.Derefter tilsættes 100 mikroliter pr. hul af en blokerende opløsning sammensat af 3% BSA i PBS. Inkuber den forseglede plade i en time ved 37 grader Celsius i en hybridiseringsovn. Efter inkubation vaskes pladen tre gange med 200 mikroliter pr. PBST-brønd.Fortynd hver serumprøve opnået fra blodet fra EAE-immuniserede mus i blokerende opløsning og tilsæt 100 mikroliter pr. Brønd til både MOG 35 55 og BSA-belagte brønde. Der tilsættes den samme mængde blokeringsopløsning til udpegede tomme huller. Inkuber den forseglede plade i to timer ved stuetemperatur med konstant omrystning.Efter inkubation fjernes pladen fra inkubatoren og skylles pladen tre gange med 200 mikroliter pr. PBST-brønd. Det HRP-konjugerede sekundære antistof fortyndes i en opløsning bestående af 0,2 % BSA i PBST, og der tilsættes 100 mikroliter til hvert hul. Den forseglede plade inkuberes i en time ved stuetemperatur under konstant omrystning.Vask pladen tre gange med 200 mikroliter pr. Brønd PBST. Tilsæt 100 mikroliter tre, tre prime, fem, fem prime tetramethylbenzidinsubstrat til hver brønd. Inkuber i mørke i et til fem minutter, der overvåger udviklingen af en blå farve.Tilsæt 100 mikroliter stopopløsning til hver brønd. Brug derefter en pladelæser indstillet til en bølgelængde på 450 nanometer til at måle den optiske tæthed i hver brønd. De optiske densitetsværdier af EAE-prøverne var signifikant højere end kontrolprøverne, hvilket indikerer et robust immunoglobulin G-respons mod MOG-peptidet.

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

Serumopsamling via intrakardial punktering i eksperimentel autoimmun encephalomyelitis (EAE) immuniserede mus