Sedgmen Bradley J, Lofthouse Shari A, Meeusen Els Nt
Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Victoria 3010, Australia.
Immunol Cell Biol. 2003 Aug;81(4):305-10. doi: 10.1046/j.1440-1711.2003.t01-1-01173.x.
Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.
通过酶联免疫斑点法（ELISPOT）对外周血中抗体分泌细胞进行计数已用于人体研究，以检测黏膜组织部位的抗原特异性抗体产生情况。另一种检测和定量抗原特异性抗体反应的方法是培养循环外周血抗体分泌细胞，并通过酶联免疫吸附测定（ELISA）定量培养上清液中的特异性抗体产生量。在本研究中，从皮下免疫的绵羊中分离出抗原特异性外周血淋巴细胞，并优化了体内诱导的抗体分泌细胞在体外产生抗体的参数。在抗原攻击后四天采集的外周血中观察到最大的抗体分泌细胞反应。添加脂多糖和抗绵羊免疫球蛋白对血液抗体分泌细胞的体外抗体分泌没有影响，而商陆有丝分裂原的作用变化很大。然而，发现将抗Ig和重组绵羊白细胞介素-6添加到外周血淋巴细胞培养物中可显著且持续地增强特异性抗体产生。在未刺激的培养物中，产生最大抗体反应的最佳外周血淋巴细胞浓度为每毫升5.0×107个细胞，但在用重组绵羊白细胞介素-6/抗绵羊免疫球蛋白刺激的培养物中，最佳细胞浓度降至约每毫升1.0×107个细胞。在体外，未刺激的培养物通常在第一天达到免疫球蛋白产生高峰。在重组绵羊白细胞介素-6/抗绵羊免疫球蛋白刺激的培养物中，第1天时抗体水平与未刺激的培养物相似，然而，在培养期间水平持续上升，在培养的第4天至第5天达到最大值。这种优化的抗体分泌细胞培养测定法适用于提高灵敏度并减少在绵羊和其他大型动物物种的大规模免疫试验中定量抗原特异性抗体诱导所需的细胞数量。
