Kapaklis-Deliyannis G P, Drummer H E, Brown L E, Tannock G A, Jackson D C
Department of Microbiology, University of Melbourne, Parkville, Victoria, Australia.
Electrophoresis. 1993 Sep;14(9):926-36. doi: 10.1002/elps.11501401148.
An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infection of mice with influenza virus or inoculation with noninfectious material with Alhydrogel or complete Freund's adjuvant. The noninfectious material used was beta-propiolactone-inactivated influenza virus and a preparation (HANA) enriched for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). When influenza viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, each of the anti-viral antisera tested exhibited strong binding. Under reducing conditions, however, much weaker binding was observed especially towards the HA1 subunit of HA. This was particularly apparent with antisera raised to virus or HANA in the absence of adjuvant. A panel of monoclonal antibodies directed to HA also bound well to viral HA separated by SDS-PAGE under nonreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is reduced and immobilized on a solid support, it does not display the conformational features essential for the integrity of all epitopes. The immunoblotting procedure was also used to determine the isotype of anti-viral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza-infected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.
采用免疫印迹法来确定特异性，并检测小鼠感染流感病毒或接种用氢氧化铝或完全弗氏佐剂的非感染性物质后产生的抗体的一些特性。所用的非感染性物质是经β-丙内酯灭活的流感病毒以及一种富含表面糖蛋白、血凝素（HA）和神经氨酸酶（NA）的制剂（HANA）。当流感病毒蛋白在非还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离时，所检测的每种抗病毒抗血清都表现出强烈的结合。然而，在还原条件下，观察到的结合要弱得多，尤其是对HA的HA1亚基。在没有佐剂的情况下，针对病毒或HANA产生的抗血清尤其明显。一组针对HA的单克隆抗体在非还原条件下对经SDS-PAGE分离的病毒HA也有良好的结合，但未能识别在还原条件下分离的HA1上的表位。这些结果表明，当HA被还原并固定在固相支持物上时，它不会展现出对所有表位完整性至关重要的构象特征。免疫印迹法还用于确定针对单个病毒蛋白的抗病毒抗体的同种型，并使用针对代表M2蛋白内一个序列的合成肽产生的抗血清，在纯化的流感病毒粒子和流感感染细胞中检测基质蛋白2（M2）。
