Sensitivity improvement of immunochromatographic strip test for infectious myonecrosis virus detection Pradit Wangman a , Siwaporn Longyant a , Heny Budi Utari b , Saengchan Senapin c,d , Chalinan Pengsuk e , Paisarn Sithigorngul a , Parin Chaivisuthangkura a, ⁎ a Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand b Charoen Pokphand Group of Indonesia, Bogor, West Java, Indonesia c Center of Excellence for Shrimp Molecular Biology and Biotechnology, Mahidol University, Bangkok 10400, Thailand d National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand e Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Bangkok 10110, Thailand abstract article info Article history: Received 28 September 2015 Received in revised form 27 November 2015 Accepted 28 November 2015 Available online 30 November 2015 An immunochromatographic strip test with enhanced sensitivity for the detection of infectious myonecrosis virus (IMNV) was developed using three monoclonal antibodies specific to N- and C-termini fragments of IMNV capsid protein. This strip test can detect IMNV with 20 times higher sensitivity than the previous test strip and approximately 50 times lower than that of one-step RT-PCR. The shrimp sample can be heat-treated to augment the release of antigen from the muscular tissue and ensure the sterilization of the sample. Due to its high specificity, field friendly and rapid result, this test strip is suitable for surveillance of IMNV outbreaks and confirmation of IMNV infection in shrimp farming. Statement of relevance: The IMNV strip test will help farmers to monitor IMNV infection during shrimp culture. The test kit has sensitivity of approximately 50 times lower than that of one-step RT-PCR. © 2015 Elsevier B.V. All rights reserved. Keywords: Immunochromatographic strip test Infectious myonecrosis virus (IMNV) Monoclonal antibody Rapid test 1. Introduction Infectious myonecrosis virus (IMNV) causes disease in a widely cul- tured penaeid shrimp, as first recognized during 2002–2003 on Brazilian farms (Lightner et al., 2004) and later on Indonesian farms (Senapin et al., 2007). IMNV infected shrimp exhibited necrosis of stri- ated muscles and can cause mortality as high as 70% (Andrade et al., 2008). Gross signs of IMNV infection in Penaeus vannamei resulted from necrosis in muscular tissues primarily in the distal abdominal seg- ment, visible as opaque whitish discoloration. Histological examination of infected tissues revealed severe necrosis of muscle, fibrocytic inflam- mation, the appearance of cytoplasmic inclusion and lymphoid organ spheroid (Poulos et al., 2006). Diagnosis of IMNV infection based on clinical signs and histological examination is not adequate, since other pathogens such as P. vannamei nodavirus (PvNV) (Tang et al., 2007), Macrobrachium rosenbergii nodavirus (MrNV) (Senapin et al., 2013) and Vibrio spp. (Longyant et al., 2008) and other abiotic factors such as hypoxia, sudden changes in salinity and temperature can cause muscle whitening namely as noninfectious muscle cramp syndrome (Senapin et al., 2011). Nucleic acid based methods including in situ hybridization (Tang et al., 2005), RT-PCR and nested RT-PCR (Poulos and Lightner, 2006; Senapin et al., 2007), real-time RT-PCR (Andrade et al., 2007; Liu et al., 2013) and RT-loop mediated isothermal amplification (LAMP) com- bined with a lateral flow dipstick (Puthawibool et al., 2009) or LAMP combined with nanogold probe (Arunrut et al., 2013) have been de- scribed for IMNV detection. Although, DNA based methods are highly specific and sensitive methods for detection of IMNV infection, they are not suitable for pond-side detection by farmer and the cost per sam- ple is still quite expensive. The alternative immunological based methods using monoclonal antibody specific to IMNV capsid protein have been developed for detection of IMNV by various immune-assays (Kunanopparat et al., 2011; Seibert et al., 2010). Some of these MAbs were further developed into a rapid immunochromatographic test strip (Chaivisuthangkura et al., 2013). Although, the test strip has lower sensitivity than nucleic based methods, it provides a field friendly, rapid, inexpensive method to detect and confirm the causative agent of shrimp pathogen during the outbreaks with high accuracy and can be used by unskilled personnel (Powell et al., 2006; Sithigorngul et al., 2006, 2007, 2011; Wangman et al., 2012). In this report, new set of higher sensitivity MAbs specific to IMNV capsid proteins was generated and used to develop the rapid strip test with higher sensitivity than the previous one which will provide more accuracy of IMNV detection clos- er to one-step RT-PCR. Aquaculture 453 (2016) 163–168 ⁎ Corresponding author at: Department of Biology, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand. E-mail address: parin@swu.ac.th (P. Chaivisuthangkura). http://dx.doi.org/10.1016/j.aquaculture.2015.11.041 0044-8486/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture