Daftarian Pirouz, Chowdhury Raquibul, Ames Philip, Wei Changli, King Alan D, de Rivero Vaccari Juan Pablo, Dillon Lloye, Price Justin, Leung Howard, Ashlock Brittany, Mesri Enrique, Perez Victor, Züchner Stephan, Reiser Jochen, Lemmon Vance, Keane Robert W
Department of Microbiology and Immunology, University of Miami, Miami, Florida, USA.
Hybridoma (Larchmt). 2011 Oct;30(5):409-18. doi: 10.1089/hyb.2010.0120.
In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved.
体内电穿孔已成为DNA免疫的金标准方法。该方法有助于DNA进入细胞，使抗原以天然形式表达并呈递给免疫系统的专职细胞，利用免疫系统的两条途径，具有内置佐剂系统，相对安全且具有成本效益。然而，要实现优化的可重复过程以引发强烈的体液反应并筛选特异性免疫反应存在挑战，特别是当目标是引发体液反应或通过杂交瘤技术产生单克隆抗体时。单克隆抗体的产生需要在淋巴器官中产生大量用于融合的致敏B细胞和CD4 T辅助细胞，传统上这是通过最终静脉内注射抗原来实现的。筛选脾细胞融合后获得的数百个克隆也需要纯化抗原。这些挑战使得DNA疫苗接种依赖于纯化蛋白。在此，我们优化了体内电穿孔、表达抗原的细胞的产生和使用方法以及细胞内蛋白质免疫印迹筛选方法。这些方法实现了：（1）可重复地针对具有不同细胞定位的抗原引发强烈的体液反应；（2）能够筛选抗原，从而无需进行蛋白质/抗原纯化。该过程包括体内电穿孔的优化参数、使用转染细胞进行最终加强免疫以及对细胞进行温和固定/通透处理以进行筛选。通过这个过程，在通过体内电穿孔进行两次疫苗接种（以及最终加强免疫）后，获得了针对细胞核、细胞质和跨膜蛋白的单克隆抗体。
