Fluorescent Immunochromatography for Rapid and Sensitive Typing of Seasonal Influenza Viruses

<div><p>Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (<u>fl</u>uorescent <u>i</u>mmuno<u>c</u>hromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.</p></div

Reactivity of FLIC-AB with H5 and H7 subtypes of influenza A viruses.

<p>Taken together, these results indicate that FLIC-AB may be a powerful tool for rapid typing of seasonal influenza A and B viruses, with high sensitivity and specificity.</p><p>Reactivity of FLIC-AB with H5 and H7 subtypes of influenza A viruses.</p

IL-33 induces TRAF6-dependent cytokine production by mast cells.

<p>BMCMCs obtained from B6J-WT mice (A) and B6J-WT and -TLR4^−/− mice (B; left panels) and FLCMCs obtained from B6J-WT and -TRAF6^−/− mice (B; right panels) were cultured in the presence of various concentration of rmIL-33 (A) or in the presence and absence of 100 ng/ml rmIL-33 for 6 h (for TNF measurement) and 24 h (for IL-6 and IL-13 measurement). The levels of IL-6, IL-13 and/or TNF in the culture supernatants were determined by ELISA. Data show the mean + SD (n = 3). *p<0.05 vs. WT.</p

IL-33 enhances LPS-mediated cytokine production by macrophages.

<p>TGC-induced peritoneal macrophages derived from B6J-WT mice (A–D) and B6N-WT and -IL-33^−/− mice (E) were cultured in the presence and absence of 100 ng/ml LPS, with and without 100 ng/ml IL-33, for 9, 24 and/or 48 h. (A, E) The levels of IL-6 in the culture supernatants by ELISA. (B) The percentage of PI-positive cells by flow cytometry. (C) LDH levels in the culture supernatants. (D) The number of IL-33-secreting cells by ELISPOT. Data show the mean +/± SEM (n = 3 [A] or 4 [B–E]). *p<0.05 vs. the indicated group (A) or Medium (B–E), and ^†p<0.05 vs. 24 h (C, D) or WT (E). P+I = PMA+ionomycin.</p

Inhibitory effects of anti-IL-33 mAbs on LPS-mediated macrophage activation by paracrine IL-33 stimulation.

<p>(A, B) TGC-induced peritoneal macrophages derived from B6J-WT mice were cultured in the presence of 100 ng/ml LPS, with and without 40 µg/ml of several anti-ST2 mAbs (A), several anti-IL-33 mAbs (B) or control IgG (A, B) for 24 and 48 h. (C) B6J-WT BMCMCs were cultured in the presence of 1 µg/ml anti-DNP IgE (SPE-7), with and without 40 µg/ml of several anti-IL-33 mAbs or control IgG for 24 and 48 h. The levels of IL-6 or IL-13 in the culture supernatants were measured by ELISA. Data show the mean + SEM ([A] n = 7, [B] n = 8 [C] n = 4). *p<0.05 vs. Rat IgG (A) or Mouse IgG (B).</p

Effects of anti-ST2 mAbs on cytokine production by IL-33-stimulated BMCMCs.

<p>B6J-WT BMCMCs were stimulated with 0–1,000 ng/ml (A) or 100 ng/ml (B) rmIL-33 in the presence of 40 µg/ml of several anti-ST2 mAbs or isotype control rat IgG for 24 h. The levels of IL-6 and IL-13 in the culture supernatants were determined by ELISA. Data show the mean + SEM (n = 3). *p<0.05 vs. rat IgG+IL-33. The expression of ST2 on the cell surface of BALB-WT and ST2^−/− BMCMCs was determined using several distinct anti-ST2 mAb clones. Representative data by flow cytometry are shown (C). Shaded area indicates isotype-matched control IgG staining, and bold line indicates anti-ST2 mAb staining.</p