Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan.
J Immunol Methods. 2012 Dec 14;386(1-2):60-9. doi: 10.1016/j.jim.2012.08.019. Epub 2012 Sep 10.
In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10(-7)M.
体外免疫（IVI）相对于传统免疫具有许多优势。然而，从 IVI 中获得的阳性克隆数量有限，并且衍生克隆的抗体亲和力相对较低。此外，培养中产生的大多数免疫球蛋白是 IgM 而不是 IgG，这限制了其应用。在这里，我们报告了一种使用小鼠脾细胞的改进的 IVI 方案。该方案包括多个抗原刺激循环，随后进行细胞扩增，从而增加了产生抗原特异性 IgG 抗体的浆细胞的频率。发现培养条件，包括细胞密度、刺激物类型和初始细胞制备，对于诱导 IgG 反应很重要。此外，对 IVI 期间表达的基因和细胞因子的分析表明，抗原特异性 B 细胞通过 CD4 阳性辅助 T 细胞特异性激活。作为这一概念的证据，我们的 IVI 方案使我们能够建立针对血蓝蛋白的 IgG 抗体，其解离常数为 10(-7)M 数量级。
