■单克隆抗体(mAb)在疾病诊断和免疫疗法干预中起关键作用。传统的单克隆抗体生成依赖于主要涉及小鼠的动物免疫程序;然而,体外表达方法的最新进展使大规模生产既适用于工业应用,也适用于科学研究。
■在这项研究中,对两种针对H7亚型禽流感病毒(AIV)的单克隆抗体进行了测序和分析,获得编码重链(HC)和轻链(LC)的DNA序列,并将其克隆到pCHO-1.0表达载体中。然后,将HC和LC表达质粒转染到CHO-S细胞中,以使用具有不同浓度的甲氨蝶呤和嘌呤霉素的两阶段选择方案建立表达这些mAb的稳定细胞系。从细胞培养基中纯化重组抗体,并使用血凝抑制(HI)评估了它们的潜在应用,蛋白质印迹(WB),共聚焦显微镜,和酶联免疫吸附测定(ELISA)。
■结果表明,获得的重组抗体表现出与源自腹水的亲本抗体相似的生物学活性,并且可以用作动物来源的mAb的替代品。对AIVHA蛋白的两种抗体的动力学分析,使用表面等离子体共振(SPR)进行,显示重组抗体和亲本抗体之间的一致性。
■本研究中提供的数据表明,所描述的抗体生产方案可以避免使用实验动物,并更好地符合动物福利法规,为进一步研发基于mAb的诊断产品提供了依据。
UNASSIGNED: Monoclonal antibodies (mAbs) play a pivotal role in disease diagnosis as well as immunotherapy interventions. Traditional monoclonal antibody generation relies on animal immunization procedures predominantly involving mice; however, recent advances in in-vitro expression methodologies have enabled large-scale production suitable for both industrial applications as well as scientific investigations.
UNASSIGNED: In this study, two mAbs against H7 subtype avian influenza viruses (AIV) were sequenced and analyzed, and the DNA sequences encoding heavy chain (HC) and light chain (LC) were obtained and cloned into pCHO-1.0 expression vector. Then, the HC and LC expression plasmids were transfected into CHO-S cells to establish stable cell lines expressing these mAbs using a two-phase selection scheme with different concentrations of methotrexate and puromycin. Recombinant antibodies were purified from the cell culture medium, and their potential applications were evaluated using hemagglutination inhibition (HI), western blotting (WB), confocal microscopy, and enzyme-linked immunosorbent assay (ELISA).
UNASSIGNED: The results indicated that the obtained recombinant antibodies exhibited biological activity similar to that of the parent antibodies derived from ascites and could be used as a replacement for animal-derived mAbs. A kinetic analysis of the two antibodies to the AIV HA protein, conducted using surface plasmon resonance (SPR), showed concordance between the recombinant and parental antibodies.
UNASSIGNED: The data presented in this study suggest that the described antibody production protocol could avoid the use of experimental animals and better conform to animal welfare regulations, and provides a basis for further research and development of mAbs-based diagnostic products.