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

研究自身免疫性脱髓鞘中的体液反应

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.

使用 ELISA 定量实验性自身免疫性脑脊髓炎免疫小鼠血清中的自身抗体

首先将冻干的 MOG 35 55 肽重新悬浮于水中。在实验前，在包衣缓冲液中将肽储备液稀释至每毫升 10 微克。现在将 100 微升的最终溶液移液到 96 孔板的孔中。同时用溶解在包被缓冲液中的 100 微升 BSA 包被相同数量的孔。用胶膜密封板以防止蒸发，并将板在 4 摄氏度下孵育过夜。第二天，用 200 微升 PBS 补充 0.5% tween 20 对板进行三次洗涤。随后，每孔加入 100 微升由 3% BSA 组成的 PBS 溶液。将密封板在 37 摄氏度的杂交炉中孵育一小时。孵育后，用每孔 200 微升 PBST 洗涤板 3 次。在封闭溶液中稀释从EAE免疫小鼠血液中获得的每种血清样品，并在MOG 35,55和BSA包被的孔中每孔加入100μL。将相同体积的封闭溶液添加到指定的空白孔中。将密封板在室温下孵育两个小时，并不断摇动。孵育后，从培养箱中取出板，并用每孔 200 微升 PBST 冲洗板三次。在由 0.2% BSA 组成的 PBST 溶液中稀释 HRP 偶联的二抗，并向每个孔中加入 100 μL。将密封板在室温下孵育一小时，保持恒定摇动。用每孔 200 微升 PBST 洗涤板三次。向每个孔中加入 100 微升三、三素、五、五素四甲基联苯胺底物。在黑暗中孵育一到五分钟，监测蓝色的发展。向每个孔中加入 100 微升终止溶液。然后使用设置为 450 纳米波长的酶标仪测量每个孔中的光密度。EAE 样品的光密度值显着高于对照样品，表明免疫球蛋白 G 对 MOG 肽具有强大的反应。

quantification-autoantibodies-serum-experimental-autoimmune

Research

JoVE Journal

Immunology and Infection

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

54 次观看.  East Carolina University. 首先将冻干的 MOG 35 55 肽重新悬浮于水中。在实验前，在包衣缓冲液中将肽储备液稀释至每毫升 10 微克。现在将 100 微升的最终溶液移液到 96 孔板的孔中。同时用溶解在包被缓冲液中的 100 微升 BSA 包被相同数量的孔。用胶膜密封板以防止蒸发，并将板在 4 摄氏度下孵育过夜。第二天，用 200 微升 PBS 补充 0.5% tween 20 对板进行三次洗涤。随后，每孔加入 100 微升由 3% B...

视频: 使用 ELISA 定量实验性自身免疫性脑脊髓炎免疫小鼠血清中的自身抗体

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.

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

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

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.