Cain K D, Jones D R, Raison R L
Department of Cell and Molecular Biology, University of Technology, Sydney, Gore Hill, NSW, Australia.
Fish Shellfish Immunol. 2000 Nov;10(8):651-66. doi: 10.1006/fsim.2000.0280.
Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.
在虹鳟鱼（Oncorhynchus mykiss，Walbaum）中，评估了不同抗原递送途径后的黏膜和全身抗体产生情况。一台BIAcore仪器（Pharmacia公司）可通过表面等离子体共振变化直接检测抗体 - 抗原相互作用。这些相互作用可实时测量，无需二级试剂或额外标记。将虹鳟鱼分组，用由异硫氰酸荧光素（FITC）与钥孔戚血蓝蛋白（KLH）偶联而成的半抗原 - 载体抗原或pH 7.2的磷酸盐缓冲盐水（PBS）进行免疫。抗原通过腹腔内（i.p.）注射，添加或不添加弗氏完全佐剂（FCA），或者经肛（p.a.）直接注入胃肠道（GI）。FITC - KLH/FCA组的血清和黏膜抗FITC反应显著更高（P<0.05），清楚地表明添加佐剂可增强黏膜和全身免疫。在未经佐剂腹腔注射和经肛免疫的鱼中，抗原摄取和加工效率低得多，导致血清和黏膜抗体产生水平相对较低。有趣的是，这些组中的黏膜反应在血清反应之前达到峰值，表明黏膜防御可能受到早期刺激。接受FITC - KLH/FCA的鱼的黏膜抗体产生与血清反应更密切相关，表明血清来源的抗体可能转移至黏膜部位。通过随后与流过含有抗FITC抗体的流动池的抗鳟鱼血清IgM单克隆抗体（1.14）反应，确认黏膜和血清反应为免疫球蛋白（Ig）。进一步分析显示，早期黏液抗FITC成分（免疫后4周）与1.14的反应性显著更低（P<0.05）。在还原条件下，通过SDS - PAGE分析，来自未免疫鱼的纯化血清和黏液Ig显示出不同的蛋白条带模式。用1.14进行免疫印迹也显示，对照鱼的黏液Ig反应较弱，而与免疫鱼的黏液Ig反应强烈。这些数据表明，虹鳟鱼早期的黏膜反应可能由特性与血清Ig不同的异质性Ig形式组成。在这种情况下，BIAcore分析作为一种测量抗体反应的手段被证明是有用的，并且有可能成为鱼类免疫学研究中有价值的新工具。
