Lalani Almin I, Zhu Sining, Xie Ping
Department of Cell Biology and Neuroscience and Graduate Program in Cellular and Molecular Pharmacology, Rutgers University.
Department of Cell Biology and Neuroscience, Rutgers University; Rutgers Cancer Institute of New Jersey;
J Vis Exp. 2018 Sep 7(139):57843. doi: 10.3791/57843.
Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.
抗体，也被称为免疫球蛋白（Ig），由分化的B淋巴细胞、成浆细胞/浆细胞分泌，在体液免疫中通过多种机制为抵御入侵病原体提供强大的防御。疫苗接种的一个主要目标是诱导保护性抗原特异性抗体以预防危及生命的感染。胸腺依赖性（TD）抗原和胸腺非依赖性（TI）抗原均可引发强烈的抗原特异性IgM反应，并且在抗原呈递细胞（APC）的帮助下，还可诱导同种型转换抗体（IgG、IgA和IgE）的产生以及记忆B细胞的生成。在此，我们描述了一种使用酶联免疫吸附测定（ELISA）来表征小鼠TD和TI Ig同种型反应的方案。在该方案中，分别通过腹腔内（i.p.）注射用半抗原偶联的模型抗原TNP-KLH（在明矾中）和TNP-多糖（在PBS中）来引发小鼠的TD和TI Ig反应。为了诱导TD记忆反应，在首次用相同抗原/佐剂免疫3周后，给予明矾中的TNP-KLH加强免疫。在免疫前后的不同时间点采集小鼠血清。随后分别使用Ig同种型特异性夹心ELISA和间接ELISA对总血清Ig水平和TNP特异性抗体进行定量。为了正确定量每种Ig同种型的血清浓度，需要对样品进行适当稀释以使其处于标准曲线的线性范围内。使用该方案，我们始终获得了具有高特异性和灵敏度的可靠结果。当与其他互补方法（如流式细胞术、脾B细胞的体外培养和免疫组织化学染色（IHC））结合使用时，该方案将使研究人员能够在给定的实验环境中全面了解抗体反应。
