Bhat Sushant, Bhatia Sandeep, Sood Richa, Bhatnagar Himanshu, Pateriya Atul, Venkatesh G
1 Immunology Section, Indian Veterinary Research Institute , Izatnagar, Bareilly, India .
Monoclon Antib Immunodiagn Immunother. 2013 Dec;32(6):413-8. doi: 10.1089/mab.2013.0053.
The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.
本研究旨在开发抗核蛋白（anti-NP）单克隆抗体（MAbs），用于免疫诊断检测禽流感病毒（AIV）抗原或抗体。克隆于pET载体的AIV核蛋白（NP）基因在大肠杆菌BL 21菌株中表达，产生分子量约为61 kDa的6x-His标签重组NP（rNP）抗原，以可溶性组分形式存在。用抗His HRPO偶联物在细菌细胞裂解物的可溶性组分中检测到rNP抗原，且其在免疫印迹中与参考AIV抗体阳性血清发生反应。rNP用于免疫BALB/c小鼠以产生分泌抗NP单克隆抗体的杂交瘤。在产生的11种抗NP单克隆抗体中，8D2-H5、8D2-H9和6D11-A7为IgM同种型，5D10-C9和5D10-F11为IgG2b型，而3F3-D2、7D2-C9、7D2-G7和7D2-G8为IgG1同种型。在蛋白质印迹分析中，单克隆抗体3F3-D2和7D2-G8与rNP呈高强度阳性反应，与H5N1病毒呈低强度反应。本研究中产生的抗NP单克隆抗体可能对开发基于竞争ELISA或免疫层析试纸条检测的方法用于禽流感的快速诊断具有重要价值。
