Berthon P, Bernard S, Salmon H, Binns R M
INRA, Laboratoire de Pathologie Porcine-Immunologie, Nouzilly, France.
J Immunol Methods. 1990 Aug 7;131(2):173-82. doi: 10.1016/0022-1759(90)90188-2.
A method is described for in vitro studies of viral humoral immune responses in the pig. After oral immunization with transmissible gastroenteritis (TGE) coronavirus, antibody production from primed mesenteric lymph node cells was revealed by an in vitro boost with viral antigen. For the latter the leukocytes were co-cultured with UV-inactivated virus using a variety of different methods of antigenic stimulation. Enumeration of specific antibody-secreting cells (ASC) and titration of secreted anti-virus antibodies were performed with ELISASPOT (using 3-amino 9-ethyl carbazole as the peroxidase chromogen) and ELISA tests respectively, according to the Ig isotype. The results showed a close relationship between ASC numbers and secreted antibody titres. The best in vitro antibody synthesis was observed when the sensitized cells were maintained in contact with virus during the whole culture period. Antibody responses were defined by a kinetic profile characterized by a narrow peak, with a maximum occurring after 4 and 6 days of culture and with the IgA response appearing earlier than the IgG. This methodology, which analyses specific antibody responses at the cellular level, may permit studies on the mechanisms of Ig isotype regulation. Extended to leukocytes from other organs of the immune system, it may also constitute an in vitro model to study antibody responses expressed in different lymphoid tissues of the pig.
本文描述了一种用于猪病毒体液免疫反应体外研究的方法。经口用传染性胃肠炎（TGE）冠状病毒免疫后，通过用病毒抗原进行体外增强刺激，可揭示致敏肠系膜淋巴结细胞产生抗体的情况。对于后者，使用多种不同的抗原刺激方法，将白细胞与紫外线灭活的病毒共培养。根据Ig同种型，分别用ELISASPOT（使用3-氨基-9-乙基咔唑作为过氧化物酶显色剂）和ELISA试验对特异性抗体分泌细胞（ASC）进行计数并滴定分泌的抗病毒抗体。结果显示ASC数量与分泌抗体滴度之间存在密切关系。当致敏细胞在整个培养期间都与病毒保持接触时，观察到最佳的体外抗体合成。抗体反应由一个动力学曲线定义，其特征为一个狭窄的峰值，在培养4天和6天后出现最大值，且IgA反应比IgG反应出现得更早。这种在细胞水平分析特异性抗体反应的方法，可能有助于研究Ig同种型调节机制。扩展到免疫系统其他器官的白细胞，它也可能构成一个体外模型，用于研究猪不同淋巴组织中表达的抗体反应。
