Development of an immunochromatographic strip for rapid detection of the H9 subtype of avian influenza viruses (H9AIVs)

Laboratory of Animal Microbiology and Immunology, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China

^* To whom correspondence should be addressed. Email: bidingren{at}mail.hzau.edu.cn.

	Abstract

An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry using two McAbs, 4C4 for H9AIV hemagglutinin and 4D4 for nucleoprotein (NP), respectively. The 4C4 was labeled with colloidal gold as a detection reagent, and the 4D4 was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the hemagglutination (HA) and hemagglutination inhibition (HI) test, the strip was specific for detecting H9AIV with the sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for six months or at 4 °C for 12 months did not change their sensitivity and specificity. Evaluation of the strip on experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 post-inoculation, earlier than the appearance of clinical symptoms. Application of the strip for analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms were infected by H9AIV. Further characterization of 10 positive and 30 negative samples randomly selected showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity and stability. This, together with the advantages of rapid detection and easy operation without requirement for special skills and equipments, makes it suitable for the on-site detection and differentiation of H9AIVs from other viruses in poultry.