The development of a sensitive and rapid diagnostic test is needed for early detection of avian influenza (AI) H7 subtype. In this study, novel monoclonal antibodies (mAbs) against influenza A H7N9 recombinant hemagglutinin (rHA)1 were developed and applied to a Europium nanoparticle–based rapid fluorescent immunochromatographic strip test (FICT) to improve the sensitivity of the rapid diagnostic system. Two antibodies (2F4 and 6D7) exhibited H7 subtype specificity in a dot-FICT assay by optimization of the conjugate and the pH of the lysis buffer. The subtype specificity was confirmed by an immunofluorescence assay and Western blot analysis. The limit of detection of the FICT employing novel mAbs 31 ng/mL for H7N9 rHA1 and 40 hemagglutination units/mL for H7 subtype virus. Sensitivity was improved 25-fold using Europium as confirmed by comparison of colloidal gold-based rapid diagnostic kit using the 2F4 and 6D7 mAbs.
Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page’s amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.
<b><i>Background:</i></b> When infected with the chikungunya virus (CHIKV), 3% to 28% of CHIKV-infected individuals remain asymptomatic, necessitating the development of improved high-throughput screening methods to overcome the limitations of molecular diagnostics or enzyme-linked immunosorbent assays (ELISAs). <b><i>Objective:</i></b> In this study, two novel monoclonal antibodies (mAbs) targeting envelope 1 (E1) of CHIKV were developed and applied in a fluorescence-linked immunosorbent assay (FLISA) using coumarin-derived dendrimer as the fluorophore. <b><i>Methods:</i></b> The performance of the FLISA was compared with that of ELISA. <b><i>Results:</i></b> Using the two novel mAbs (2B5 and 2C8), FLISA could detect 1 × 10<sup>5</sup> PFU/mL of CHIKV, exhibiting a 2-fold lower limit of detection (LOD) compared to ELISA. The LOD of FICT corresponded to a comparative threshold value of 23.95 and 4 × 10<sup>6</sup> of RNA copy number/µL. In the presence of human sera and blood, virus detection by FLISA was 3-fold better than ELISA, with an LOD of 2 × 10<sup>5</sup> PFU/mL. Sera and blood interfered with the ELISA, resulting in 6 × 10<sup>5</sup> PFU/mL as the LOD. <b><i>Conclusions:</i></b> FLISA using two novel mAbs and coumarin-derived dendrimer is a superior diagnostic assay for detecting CHIKV in human sera and blood, compared to conventional ELISA.
the free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies. The free-living amoebae (FLA) Naegleria spp. and Acanthamoeba spp. are mainly distributed in ponds, rivers, and fresh waters worldwide. Their existing stages are trophozoite and cyst and additional biflagellate form in in case of Naegleria. Trophozoites shows moving, feeding, and proliferation activity. However, cysts are formed in poor environments, such as under abrupt temperature changes, drying, and food depletion, and can survive for long periods 1,2. N. fowleri is a pathogenic agent that causes primary amoebic meningoencephalitis (PAM), which is fatal to humans and laboratory animals 2,3. Acanthamoeba spp. and Balamuthia mandrillaris cause chronic granulomatous amebic encephalitis (GAE) 4-6. Further, A. castellanii and A. polyphaga can infect the eye, resulting in acanthamoeba keratitis (AK) 2,7,8. PAM is mainly associated with activities in amoeba-contaminated water (swimming or water leisure activity), use of Neti-pots in rhinitis treatment, and religious ceremonies in some Asian countries 5,9,10. The amoeba enters through the nasal cavity to invade the mucosal membrane. Subsequently, it moves into the olfactory bulb and meninges via the nasal nerve system, leading to development of meningoencephalitis 11-13. Symptoms of PAM include acute headache, anorexia, nausea, vomiting, high fever (38-40 °C), and limb dysfunction symptoms. It also progresses acutely, with a mortality rate of over 95%. Amphotericin B is mainly used as a therapeutic agent, as a combination treatment by mixing with micronazole, rifampin, and doxycycline; however, only limited therapeutic effects have been demonstrated 1,2,14-16. AK usually occurs after wearing contaminated contact lenses, improper ophthalmic surgery, or corneal injury in various cases. With the popularization of contact lenses and careless lens management, the number of AK patients continues to increase 2,7,17,18. PAM due to N. fowleri occurs annually worldwide. In the United States, 143 cases of PAM occurred from 1962 to...
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