Name: 作者聚焦：在多发性硬化症研究中研究 B 细胞反应的新型检测方法
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

这项研究得到了美国国立卫生研究院 （R03NS131908） 和国防部通过多发性硬化症研究计划的支持，奖励编号为 W81XWH-22-1-0517。意见、解释、结论和建议是作者的观点、解释、结论和建议，并不一定得到国防部的认可。这项研究也得到了东卡罗来纳大学启动基金的支持。

所有涉及小鼠的程序均按照东卡罗来纳大学机构动物护理和使用委员会 （IACUC） 批准的实验指南进行。本研究使用8-10周龄的野生型C57BL/6J雌性小鼠。这些动物是从商业来源获得的（见材料表）。EAE 是在先前发表的报告 12,13,14 之后诱导的。
1. 血清采集
<ol><li>在通过MOG35-55?...

研究自身免疫性脱髓鞘中的体液反应

<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>

在这里，报道了一种简单有效的基于 ELISA 的方案，以准确量化 MS 病理学相关动物模型中的体液反应。该方法最近被用于描述 ataxin-1 蛋白在控制 MOG35-55/C57BL6J EAE 范式12 中自身抗体水平方面的新作用。在这方面，应该考虑许多因素，以便使用这种方法获得一致且具有生物学意义的结果。
首先，收集高质量的血清样本并避免溶血至关重要。在严重溶血的标本中?...

多发性硬化症 （MS） 是一种中枢神经系统 （CNS） 的慢性自身免疫性疾病，其特征是免疫细胞局灶性浸润到脑实质、包裹轴突的髓鞘破裂、神经胶质细胞活化和神经元丢失1。除了致病性 T 细胞的公认作用外，多条证据还强调了 B 细胞参与介导针对中枢神经系统的自身免疫反应。B 细胞在 MS 脑中发生克隆扩增，并且在脱髓鞘病变中检测到针对髓鞘成分的抗体 2,3。最近有记录表明，外周 B 细胞在疾病发作时的选择性激活，表明该免疫细胞区室在疾病起始中也具有假定的作用4。B 细胞耗竭疗法（如抗 CD20 单克隆抗体）的成功进一步证实了异常 B 细胞功能与自身免疫性脱髓鞘之间的机制联系 5,6。从分子的角度来看，B 细胞可以通过自身抗原呈递、促炎细胞因子分泌和自身反应性抗体产生来导致疾病。
已经开发了多种动物模型来概括复杂 MS 表型的特定特征。其中，实验性自身免疫性脑脊髓炎（EAE）是 体内 应用最广泛的范式，依赖于实验动物使用源自髓鞘蛋白的短肽进行免疫，如髓鞘少突胶质细胞糖蛋白（MOG）和髓鞘碱性蛋白（MBP）7。EAE 免疫动物发展出脱髓鞘病理学，在许多方面类似于 MS，包括对脑炎肽8 的强大体液反应。出于这个原因，EAE 研究在解剖 B 细胞和自身抗体在疾病背景下的功能方面发挥了重要作用。例如，研究表明，从 MS 患者中分离出的 MOG 特异性抗体可以加重 EAE 模型9 的临床病程。 值得注意的是，人 MOG 中 42 位的脯氨酸残基被证明对于确定自身抗体致病性至关重要10。最近，已发现 MOG 特异性自身抗体不仅通过介导髓鞘丢失来促进疾病，而且还通过促进中枢神经系统11 内自身反应性 T 细胞的再激活。 
考虑到抗体反应在中枢神经系统自身免疫中的重要性，本文提出了一种基于 ELISA 的方案，以有效测量用 MOG35-55 肽免疫的 C57BL/6J 小鼠 EAE 中自身反应性抗体的血清水平。在方案的第一部分，将描述 通过 心内穿刺收集血清的方法。随后，将详细说明设置 ELISA 测定和获取数据的程序。最后，将讨论数据分析和解释。

<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

实验性自身免疫性脑脊髓炎 （EAE） 是一种疾病模型，在组织病理学和分子水平上概括了自身免疫性疾病多发性硬化症 （MS）。EAE 是通过 皮下注射 短髓鞘肽和特定佐剂对实验动物进行免疫接种来诱导的，以增强免疫反应。与人类对应物一样，EAE小鼠会出现脱髓鞘病变，免疫细胞浸润到中枢神经系统（CNS），神经胶质细胞激活和神经元损伤。一致的证据也支持 B 细胞功能障碍在 MS 和 EAE 病因中的机制作用。B细胞可以作为抗原呈递细胞，也是促炎细胞因子和自身抗体的主要来源。在 EAE 中，产生针对用于诱发疾病的髓鞘肽的抗体。这种自身抗体已被证明可以介导髓鞘丢失或致病性 T 细胞再激活到中枢神经系统中。本文介绍了一种基于 ELISA 的有效方案，用于量化用髓鞘少突胶质细胞糖蛋白 35-55 （MOG35-55） 肽免疫的 C57BL/6J 小鼠血清中的自身抗体。所提出的方法是一种强有力的工具，可用于研究自身免疫性脱髓鞘背景下异常体液反应的特异性和程度。

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.

作者聚焦：在多发性硬化症研究中研究 B 细胞反应的新型检测方法

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

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.

为了证明本 ELISA 测定的稳健性，在免疫 （dpi） 后 20 天，在 C57BL/6J 雌性小鼠队列中分离的血清样品中测试了该方法，在经过验证的 EAE 诱导方案 12,13,14 之后，在完全弗氏佐剂 （CFA） 中。动物在第 0 天和第 2 天还接受了 400 ng 百日咳毒素。来自模拟免疫动物的血清样本，含有所有物质，但没有肽，作为阴性对照。所有 EAE 小鼠?...

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

实验性自身免疫性脑脊髓炎 （EAE） 是一种多发性硬化症 （MS） 动物模型，与人类疾病具有强大的体液自身免疫反应。在这里，我们报告了一种简单而灵活的ELISA方案，用于量化EAE免疫小鼠血清中的自身抗体。

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

因此，我们实验室的重点是了解多发性硬化症的分子基础，多发性硬化症是一种中枢神经系统的免疫疾病，其特征是脱髓鞘、神经炎症和轴突损伤。因此，该协议代表了商业试剂盒的经济且灵活的替代方案。我们可以很容易地进行调整，以测试针对不同EEA肽的B细胞抗体反应。我的实验室致力于进一步了解慢性神经系统疾病（如多发性硬化症）中神经炎症和神经退行性变之间的联系。

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.

科研

免疫与感染

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

通过心内穿刺在实验性自身免疫性脑脊髓炎 （EAE） 免疫小鼠中收集血清

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.