Development of ELISA Kit for Detecting H5 Subtype Avian Influenza Viruses and Vertical Transmission Test

In this study, we used sandwich ELISA to detect H5 virus and blocking ELISA to detect H5 antibody. The concentrated purified virus (3233/04) and hemagglutinin (HA) recombinant proteins were used for the production of monoclonal antibodies. These monoclonal antibodies were confirmed to be specific to the H5 HA by immunodot blot assay, western blot assay and hemagglutination inhibition test. These monoclonal antibodies were purified and then labeled with horseradish peroxidase to become tracer. The tracer was used for the detection of H5 antibody in serum by blocking ELISA and for the H5 subtype virus by sandwich ELISA. The results showed that sandwich ELISA detected the H5 virus above the titer of 106 EID50/0.1 mL. The specificity and sensitivity for blocking ELISA were 97.36% (222/228) and 98.29% (230/234), respectively.
Little information about AIV vertical transmission is available. This study aimed to prove the existence of vertical transmission of AIV from hens to embryos. We used a H6N1 low pathogenetic AIV Taiwanese isolate (Yilan, Taiwan), 2838K/00, in the vertical transmission test. After challenging the hens with 108.65 EID50/0.1 mL of 2838K/00, and inoculating the specific-pathogen-free embryonated eggs with 1EID50/0.1 mL of 2838K/00, the embryos were hatched. The tracheas, kidneys, and yolk sacs were collected from the hatched chicks for the virus isolation by the allantoic sac route. After two passages, we harvested the allantoic fluid and tested for the detection of NP gene of AIV by RT-PCR. The results showed that we detected the nucleic acid of AIV in the yolk and albumen mixture from unfertilized eggs. But we didn’t detect the AIV in the hatched chicks. According to above results, AIV seems to be no vertical transmission in chickens.