As shown in Table 4, the detection limit of the test varied from 0.5 to 0.125 HA units/200 ul of sample. The detection limit of the commercial kit for influenza A virus detection (Rockeby) was determined to be 200 ul of sample containing at least 1.5 HA titer of virus. Performance of H5 dot ELISA in the detection of variant
H5N1 Indonesia strains in poultry samples relative to RT-PCR The dot ELISA test was further evaluated with poultry JSH-23 samples. The swabs from birds infected with H5N1 virus can secrete virus of titer higher than l08 EID50/ml. Samples were serially diluted 10 times from 10-1 to 10-4 with PBS and tested by the dot ELISA kit to determine the detection limit for swabs. The sensitivity test indicated that the dot ELISA kit
was able to detect the presence of virus at a concentration down to 105 EID50/ml in swabs, suggesting the test can be used for the detection of H5 infection in sick birds. From 150 samples taken from clinically healthy birds, one sample was found to be positive with the test. The same sample ARS-1620 molecular weight was confirmed to be the only positive swab among the 150 samples in RT-PCR with H5 specific primers. 50 tracheal swabs obtained from sick birds were also tested with both dot ELISA and RT-PCR (Table 7). The results with the dot ELISA showed that nine samples were positive for H5 infection. The same ISRIB result was observed from the verification with RT-PCR. Table 7 Results of detection
of H5 virus in random tracheal swabs using the dot ELISA kit and RT-PCR Source of sample (area) Source of animal Clinically condition of animal Number of samples Result of test using Sensitivity (%) Dot ELISA RT-PCR primer H5 Makasar Native chicken Healthy 50 1 1 100 Bogor Layer chicken Healthy 50 1 1 100 Bogor Broiler chicken Healthy 50 eltoprazine 1 1 100 Bogor Chicken and duck Sick 50 9 9 100 As shown in Table 8, specificity test using various H5N1 viruses from several years and areas in Indonesia showed that the ELISA kit is 90% specific compared with RT-PCR using H5 primers, but 100% specific compared to HA2 primer. This indicates that the dot ELISA kit is able to detect H5N1 as long as the virus did not undergo a genetic mutation in their HA genes. Taken together, these findings indicate that the dot ELISA kit is suitable for specific early detection of H5 virus infection in avian species.