Name: 著者スポットライト:多発性硬化症研究におけるB細胞応答を研究するための新しいアッセイ
Uploaded: 2023-12-01T14:30:00
Description: Watch this Scientific Journal Video about Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis at JoVE.com

この研究は、米国国立衛生研究所(R03NS131908)および国防総省の多発性硬化症研究プログラムを通じて、賞番号W81XWH-22-1-0517の支援を受けました。意見、解釈、結論、および推奨事項は著者のものであり、必ずしも国防総省によって承認されているわけではありません。この研究は、イーストカロライナ大学のスタートアップファンドの支援も受けました。

マウスを含むすべての手順は、イーストカロライナ大学動物管理委員会(IACUC)によって承認された実験ガイドラインに準拠して実施されました。この研究では、8〜10週齢の野生型C57BL / 6J雌マウスを使用しました。動物は商業的な供給源から入手しました(資料の表を参照)。EAEは、以前に発表された報告12,13,14に続いて誘発されました。<su...

自己免疫性脱髄における体液性反応の調査

<ol>
	<li>Reich, D. S., Lucchinetti, C. F., Calabresi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis.">Multiple sclerosis.</a> N Engl J Med. 378 (2), 169-180 (2018).</li><li>Baranzini, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+repertoire+diversity+and+clonal+expansion+in+multiple+sclerosis+brain+lesions.">B cell repertoire diversity and clonal expansion in multiple sclerosis brain lesions.</a> J Immunol. 163 (9), 5133-5144 (1999).</li><li>Genain, C. P., Cannella, B., Hauser, S. L., Raine, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+autoantibodies+associated+with+myelin+damage+in+multiple+sclerosis.">Identification of autoantibodies associated with myelin damage in multiple sclerosis.</a> Nat Med. 5 (2), 170-175 (1999).</li><li>Ma, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+hypomethylation+programs+underpin+b+cell+activation+in+early+multiple+sclerosis.">Specific hypomethylation programs underpin b cell activation in early multiple sclerosis.</a> Proc Natl Acad Sci U S A. 118 (51), e2111920118 (2021).</li><li>Hauser, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocrelizumab+versus+interferon+beta-1a+in+relapsing+multiple+sclerosis.">Ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis.</a> N Engl J Med. 376 (3), 221-234 (2017).</li><li>Montalban, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocrelizumab+versus+placebo+in+primary+progressive+multiple+sclerosis.">Ocrelizumab versus placebo in primary progressive multiple sclerosis.</a> N Engl J Med. 376 (3), 209-220 (2017).</li><li>Didonna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+sclerosis:+Advantages+and+limitations+towards+better+therapies.">Preclinical models of multiple sclerosis: Advantages and limitations towards better therapies.</a> Curr Med Chem. 23 (14), 1442-1459 (2016).</li><li>Steinman, L., Zamvil, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+successfully+apply+animal+studies+in+experimental+allergic+encephalomyelitis+to+research+on+multiple+sclerosis.">How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis.</a> Ann Neurol. 60 (1), 12-21 (2006).</li><li>Khare, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein-specific+antibodies+from+multiple+sclerosis+patients+exacerbate+disease+in+a+humanized+mouse+model.">Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.</a> J Autoimmun. 86, 104-115 (2018).</li><li>Marta, C. B., Oliver, A. R., Sweet, R. A., Pfeiffer, S. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+myelin+oligodendrocyte+glycoprotein+antibodies+recognize+glycosylated+epitopes+and+perturb+oligodendrocyte+physiology.">Pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology.</a> Proc Natl Acad Sci U S A. 102 (39), 13992-13997 (2005).</li><li>Flach, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibody-boosted+t-cell+reactivation+in+the+target+organ+triggers+manifestation+of+autoimmune+cns+disease.">Autoantibody-boosted t-cell reactivation in the target organ triggers manifestation of autoimmune cns disease.</a> Proc Natl Acad Sci U S A. 113 (12), 3323-3328 (2016).</li><li>Didonna, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-1+regulates+b+cell+function+and+the+severity+of+autoimmune+experimental+encephalomyelitis.">Ataxin-1 regulates b cell function and the severity of autoimmune experimental encephalomyelitis.</a> Proc Natl Acad Sci U S A. 117 (38), 23742-23750 (2020).</li><li>Didonna, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+tau+methylation+patterns+and+synaptic+transcriptional+alterations+are+associated+with+neural+vulnerability+during+chronic+neuroinflammation.">Sex-specific tau methylation patterns and synaptic transcriptional alterations are associated with neural vulnerability during chronic neuroinflammation.</a> J Autoimmun. 101, 56-69 (2019).</li><li>Ma, Q., Matsunaga, A., Ho, B., Oksenberg, J. R., Didonna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte-specific+argonaute+profiling+identifies+micrornas+associated+with+experimental+autoimmune+encephalomyelitis.">Oligodendrocyte-specific argonaute profiling identifies micrornas associated with experimental autoimmune encephalomyelitis.</a> J Neuroinflammation. 17 (1), 297 (2020).</li><li>Lanz, T. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonally+expanded+b+cells+in+multiple+sclerosis+bind+ebv+ebna1+and+glialcam.">Clonally expanded b cells in multiple sclerosis bind ebv ebna1 and glialcam.</a> Nature. 603 (7900), 321-327 (2022).</li></ol>

ここでは、MS病理の関連する動物モデルにおける体液性反応を正確に定量化するために、シンプルで効率的なELISAベースのプロトコルが報告されました。この方法は、MOG35-55/C57BL6J EAEパラダイム12における自己抗体の血清レベルの制御におけるアタキシン-1タンパク質の新規の役割を説明するために最近採用されました。この点で、この方法で一貫した生物学的に意味のある結...

多発性硬化症(MS)は、中枢神経系(CNS)の慢性自己免疫疾患で、脳実質への免疫細胞の限局浸潤、軸索を包むミエリン鞘の破壊、グリアの活性化、およびニューロンの喪失を特徴とする1。病原性T細胞の確立された役割に加えて、複数の証拠が、CNSに対する自己免疫応答の媒介におけるB細胞の関与を強調しています。B細胞はMS脳でクローン増殖を起こし、ミエリン成分に対する抗体が脱髄病変内で検出されています2,3。疾患発症時の末梢B細胞の選択的活性化が最近文書化されており、疾患開始におけるこの免疫細胞コンパートメントの推定上の役割も示唆されています4。抗CD20モノクローナル抗体などのB細胞枯渇療法の成功は、異常なB細胞機能と自己免疫性脱髄との間の機構的関係をさらに裏付けています5,6。分子の観点から、B細胞は、自己抗原の提示、炎症誘発性サイトカイン分泌、および自己反応性抗体産生を介して疾患に寄与する可能性があります。
複雑なMS表現型の特定の特徴を再現するために、複数の動物モデルが開発されています。その中でも、実験的自己免疫性脳脊髄炎(EAE)は、 最も広く使用されているin vivo パラダイムであり、ミエリンオリゴデンドロサイト糖タンパク質(MOG)やミエリン塩基性タンパク質(MBP)7などのミエリンタンパク質に由来する短いペプチドによる実験動物の免疫に依存しています。EAE免疫動物は、脳原性ペプチド8に対する強力な体液性応答を含む、多くの面でMSに似た脱髄性病理を発症します。このため、EAE研究は、疾患の状況におけるB細胞と自己抗体の機能を解剖するのに役立ちました。たとえば、MS患者から単離されたMOG特異的抗体は、EAEモデル9の臨床経過を悪化させる可能性があることが実証されました。 特に、ヒトMOGにおける42位のプロリン残基は、自己抗体病原性を決定するために重要であることが示された10。最近では、MOG特異的自己抗体は、ミエリン喪失を媒介するだけでなく、CNS内の自己反応性T細胞の再活性化を促進することによっても疾患を促進することがわかっています11。 
CNS自己免疫における抗体応答の重要性を考慮して、この記事では、MOG35-55ペプチドで免疫したC57BL / 6JマウスEAEの自己反応性抗体の血清レベルを効率的に測定するためのELISAベースのプロトコルを紹介します。プロトコルの最初の部分では、心臓内穿刺 を介して 血清を採取する方法について説明します。その後、ELISAアッセイの設定とデータ取得の手順について詳しく説明します。最後に、データ分析と解釈について説明します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringes</td>
			<td>BD Biosciences</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needles</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB) substrate</td>
			<td>Thermo Fisher</td>
			<td>N301</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Adhesive seals</td>
			<td>Thermo Fisher</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>C57BL/6J female mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>Animals between 8-10 weeks of age should be used for EAE experiments</td>
		</tr>
		<tr>
			<td>Cryogenic tubes</td>
			<td>Fisher</td>
			<td>10-500-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tray</td>
			<td>Fisher</td>
			<td>S111022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissector scissors</td>
			<td>Fine Science Tools</td>
			<td>14082-09</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA coating buffer</td>
			<td>BioLegend</td>
			<td>421701</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td>Analysis spreadsheet</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Human ads-HRP</td>
			<td>SouthernBiotech</td>
			<td>1030-05</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>Fisher</td>
			<td>AMPSILED21</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Fisher</td>
			<td>14-377-575&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade water</td>
			<td>Corning</td>
			<td>46-000-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse MOG35-55 peptide</td>
			<td>EZBiolab</td>
			<td>cp7203</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Axygen</td>
			<td>AP-12-200-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15011-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp 96-well plates</td>
			<td>BioLegend</td>
			<td>423501</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Fisher</td>
			<td>88-861-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>VWR</td>
			<td>445-0024</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>Thermo Fisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated tabletop centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr>
			<td>Stop solution</td>
			<td>Thermo Fisher</td>
			<td>N600</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-Rad</td>
			<td>1706531</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of autoreactive antibodies in mice upon experimental autoimmune encephalomyelitis

実験的自己免疫性脳脊髄炎 (EAE) は、組織病理学的および分子レベルで自己免疫疾患である多発性硬化症 (MS) を再現する疾患モデルです。EAEは、短いミエリンペプチドと特異的なアジュバントを皮下注射 して 実験動物に免疫を与え、免疫応答を高めることによって誘導されます。ヒトマウスと同様に、EAEマウスは脱髄病変、中枢神経系(CNS)への免疫細胞浸潤、グリア活性化、および神経細胞損傷を発症します。一貫した一連の証拠は、MSとEAEの両方の病因におけるB細胞機能障害の機構的役割も支持しています。B細胞は、抗原提示細胞としてだけでなく、炎症誘発性サイトカインや自己抗体の主要な供給源としても機能します。EAEでは、病気を誘発するために使用されたミエリンペプチドに対する抗体が生成されます。このような自己抗体は、ミエリン喪失または病原性T細胞のCNSへの再活性化のいずれかを媒介することが示されています。この記事では、ミエリンオリゴデンドロサイト糖タンパク質35-55(MOG35-55)ペプチドで免疫したC57BL/6Jマウスの血清中の自己抗体を定量するための効率的なELISAベースのプロトコルについて説明します。提案された方法は、自己免疫脱髄の文脈における異常な体液性応答の特異性と大きさを調査するための強力なツールとして機能します。

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.

著者スポットライト:多発性硬化症研究におけるB細胞応答を研究するための新しいアッセイ

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

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.

本ELISAアッセイの頑健性を実証するために、この方法は、C57BL/6J雌マウスのコホートから分離された血清サンプルを、免疫後20日(dpi)に、100μgのMOG35-55ペプチドを完全フロイントアジュバント(CFA)に従ったもので、検証済みのEAE誘導プロトコル12,13,14に従って試験した。動物はまた、0日目と2日目に400ngの百日咳毒素を投与され...

Watch this Scientific Journal Video about Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis at JoVE.com

実験的自己免疫性脳脊髄炎(EAE)は、多発性硬化症(MS)の動物モデルであり、ヒトの疾患と強力な体液性自己免疫応答を共有しています。ここでは、EAE免疫マウスの血清中の自己抗体を定量するためのシンプルで柔軟なELISAプロトコルを報告します。

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

そこで、私たちの研究室では、脱髄、神経炎症、軸索損傷を特徴とする中枢神経系の免疫疾患である多発性硬化症の分子基盤を理解することに重点を置いています。したがって、このプロトコルは、市販のキットに代わる経済的で柔軟な代替品です。当社のものは、さまざまなEEAペプチドに対するB細胞抗体反応をテストするように簡単に調整できます。私の研究室では、多発性硬化症などの慢性神経疾患における神経炎症と神経変性の関係をさらに理解することに専念しています。

quantification-autoreactive-antibodies-mice-upon-experimental

Research

JoVE Journal

Immunology and Infection

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 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.

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.

免疫・感染学

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

実験的自己免疫性脳脊髄炎(EAE)免疫マウスにおける心臓内穿刺による血清採取

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

Begin by re-suspending the lyophilized MOG 35 55 peptide in water. Dilute the peptide stock to 10 micrograms per milliliter in coating buffer before the experiment. Now pipette 100 microliters of the final solution into wells of a 96 well plate.Simultaneously coat an equal number of wells with 100 microliters of BSA dissolved in coating buffer. Seal the plate with an adhesive film to prevent evaporation and incubate the plate overnight at four degrees Celsius. The next day, perform three washes of the plate with 200 microliters of PBS supplemented with 0.5%tween 20.Subsequently add 100 microliters per well of a blocking solution composed of 3%BSA in PBS. Incubate the sealed plate for one hour at 37 degrees Celsius in a hybridization oven. After incubation, wash the plate three times with 200 microliters per well of PBST.Dilute each serum sample obtained from the blood of EAE immunized mice in blocking solution and add 100 microliters per well to both MOG 35 55 and BSA coated wells. Add the same volume of blocking solution to designated blank wells. Incubate the sealed plate for two hours at room temperature with constant shaking.After incubation, remove the plate from the incubator and rinse the plate three times with 200 microliters per well of PBST. Dilute the HRP conjugated secondary antibody in a solution consisting of 0.2%BSA in PBST and add 100 microliters to each well. Incubate the sealed plate for one hour at room temperature maintaining constant shaking.Wash the plate three times with 200 microliters per well of PBST. Add 100 microliters of three, three prime, five, five prime tetramethyl benzidine substrate to each well. Incubate in the dark for one to five minutes monitoring the development of a blue color.Add 100 microliters of stop solution to each well. Then use a plate reader set at a wavelength of 450 nanometers to measure the optical density in each well. The optical density values of the EAE samples were significantly higher than the control samples indicating a robust immunoglobulin G response against the MOG peptide.