Diagnostic Hybrids, Inc., Athens, OH 45701, USA.
J Clin Microbiol. 2010 Aug;48(8):2677-82. doi: 10.1128/JCM.00978-10. Epub 2010 Jun 2.
To develop an immunofluorescence assay for identification of the 2009 H1N1 influenza A virus, we generated a number of monoclonal antibodies (MAbs) by using inactivated H1N1 2009 virus (A/California/07/2009) as the immunogen. Two MAbs that target two different epitopes of the 2009 H1N1 hemagglutinin (HA) were selected to make the D(3) Ultra 2009 H1N1 Influenza A ID kit (2009 H1N1 ID kit; Diagnostic Hybrids, Inc., Athens, OH), which is intended for the identification of the 2009 H1N1 virus by indirect immunofluorescence assay (IFA). The kit does not detect any of 14 seasonal H1N1 or H3N2 prototype influenza virus strains and is also not reactive with seven other major respiratory viruses. Clinical respiratory specimens were evaluated using both the 2009 H1N1 ID kit and the CDC human influenza virus real-time reverse transcription-PCR swine flu panel (CDC rRT-PCR) and showed 100% agreement between the two assays. Four of these clinical specimens, however, were positive by the 2009 H1N1 ID kit but were identified as presumptively positive by the CDC rRT-PCR by virtue of showing threshold cycle (C(T)) values only with universal InfA and swInfA primers, not with swH1 primers. Sequence analysis of the HA genes of these four specimens revealed point mutations in both the primer and probe regions. In addition, unlike the CDC rRT-PCR, the 2009 H1N1 ID kit can differentiate the 2009 H1N1 virus from a swine-derived H1 influenza A virus (A/New Jersey/8/76). The 2009 H1N1 ID kit offers clinical laboratories an alternative to RT-PCR for the identification of the 2009 H1N1 influenza A virus.
为了开发用于鉴定 2009 年 H1N1 甲型流感病毒的免疫荧光检测方法，我们使用灭活的 2009 年 H1N1 病毒（A/加利福尼亚/07/2009）作为免疫原，生成了许多单克隆抗体（MAb）。选择了两种针对 2009 年 H1N1 血凝素（HA）两个不同表位的 MAb 来制作 D(3)Ultra 2009 H1N1 甲型流感 A ID 试剂盒（2009 H1N1 ID 试剂盒；Diagnostic Hybrids，Inc.，雅典，俄亥俄州），该试剂盒旨在通过间接免疫荧光检测法（IFA）鉴定 2009 年 H1N1 病毒。该试剂盒不能检测到任何 14 种季节性 H1N1 或 H3N2 原型流感病毒株，也与其他七种主要呼吸道病毒没有反应。使用 2009 H1N1 ID 试剂盒和 CDC 人类流感病毒实时 RT-PCR 猪流感检测试剂盒（CDC rRT-PCR）对临床呼吸道标本进行了评估，两种检测方法的结果完全一致。然而，在 2009 H1N1 ID 试剂盒中，有四个临床标本呈阳性，但由于仅使用通用 InfA 和 swInfA 引物而不是 swH1 引物显示出阈值循环（C(T)）值，被 CDC rRT-PCR 鉴定为假定阳性。这四个标本的 HA 基因序列分析显示，在引物和探针区域均有突变。此外，与 CDC rRT-PCR 不同，2009 H1N1 ID 试剂盒可以区分 2009 年 H1N1 病毒和源自猪的 H1 流感病毒（A/New Jersey/8/76）。2009 H1N1 ID 试剂盒为临床实验室提供了一种替代 RT-PCR 的方法，用于鉴定 2009 年 H1N1 甲型流感病毒。
