Evaluation of Anigen H5 Avian Influenza Virus Antigen Rapid Test Kit
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1 Evaluation of Anigen H5 Avian Influenza Virus Antigen Rapid Test Kit Evaluation period: Mar ~Dec Sung Hwan Woo, Y. J. Lee, J. G. Choi, E.K. Lee, Y.K. Kwon, J. H. Kim, Avian Disease Division, National Veterinary Research and Quarantine Service, Ministry of Agriculture, Korea
2 Background Avian Influenza virus, especially AIV Highly Pathogenic Avian Influenza(HPAI) is spreading fast. It is important to protect the spreading in early stage. To do it, Rapid & Simple Diagnostic Test Method is required. At present, AIV can be detected by PCR assay method. But it takes 5 hours at least and needs some special equipments to do the assay. Anigen Rapid H5 AIV Ag Test Kit can detect the AIV and/or HPAI at the field of a suspect farm and protect AIV spreading in early stage.
3 Evaluation Method Anigen Rapid H5 Avian Influenza Virus Antigen Test Kit : a chromatographic immunoassay for the qualitative, differential detection of avian influenza type A subtype H5 virus antigen (High pathogenic avian influenza virus: HPAI) and other avian influenza Type A virus antigen in avian feces. : Anigen Rapid Avian Influenza Subtype H5 Virus Antigen Test Kit has a letter of 1, 2 as test line 1(avian influenza subtype H5 virus), test line 2 (common avian influenza virus antigen) and C as control line on the surface of the device. Test Method 1. Detection Test by AIV Serotype 2. Specificity Test 3. Comparison Test of other AIV Detection Assay 4. Detection limit Test with fecal specimens of infected poultry
4 Scheme of Anigen Rapid H5 AIV Ag Test Kit Feces/Organs AIV AIV MoAb 12 Gold conjugate 1 AIV H5 MoAb Test line 1 AIV MoAb 2 Test line 2 Control line (C) N/C membrane
5 Detection Test of H5 AIV Ag - Test Line 2 by AIV Serotype Control line Test line 2 Result : AIV subtype : - H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 ve N1 N2 N8 N6 N3 N5 N1 N4 N2 N7 N6 N5 N6 N5 N6 Control
6 Specificity Test of H5 AIV Ag-Test Line 2 with Various Avian viruses serotypes Control line Test line 2 Result : Virus : AIV (ES/03, H5N1) 2. AIV (MS96, H9N2) 3. NDV (Kyojungwon) 4. IBV (KS96) 5. ILTV (Serva) 6. Pneumovirus (TRTV) 7. Reovirus (IVR-1133) 8. IBDV (Winterfield2510) 9. MDV (CVI988HVT) 10. REV (REV-T)
7 Detection Limit Test of H5 AIV Ag-Test Line 1 with AIV H5 serotype (ES/03, H5N1) Control line Test line 1 Result : HA titer : Virus Titer : 2 7 HA unit, EID 50 /0.1ml Detection limit : 0.13HA unit, EID 50 /0.1ml
8 Detection Test of H5 AIV Ag-Test Line 1 by AIV Serotypes Control line H5 Test line 1 Result : AIV subtype : H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 ve N1 N2 N8 N6 N3 N5 N1 N4 N2 N7 N6 N5 N6 N5 N6 Control
9 Specificity Test of H5 AIV Ag-Test Line 2 with Various Avian virus serotypes Control line Test line 2 Result : Virus : AIV (ES/03, H5N1) 2. AIV (MS96, H9N2) 3. NDV (Kyojungwon) 4. IBV (KS96) 5. ILTV (Serva) 6. Pneumovirus (TRTV) 7. Reovirus (IVR-1133) 8. IBDV (Winterfield2510) 9. MDV (CVI988HVT) 10. REV (REV-T)
10 Detection limit test of H5 AIV Ag- Test Line 1 with AIV H5 serotype (ES/03, H5N1) Control line H5 Test line 1 Result : HA titer : Virus Titer : 2 7 HA unit, EID 50 /0.1ml Detection Limit : 1 HA unit, EID 50 /0.1ml
11 Detection Test of H5 AIV Ag Test Kit in Chickens Fecal Specimens infected by Challenge with LPAI 1) (H9N2) virus - Material : SPF chickens(lohmann Valo), 8 heads - Challenge method : 01310(H9N2, EID 50 /0.1 ) by oral inoculation - Specimen collection : Cloacal swab Detection Rate(%) by the Time of collecting specimens 1 DPI 2) 2 DPI 3 DPI 4 DPI 5DPI 7DPI 10DPI Virus Isolation 4/8 (50.0) 5/8 (62.5) 4/8 (50.0) RT-PCR 1/8 (12.5) 3/8 (37.5) 4/8 (50.0) 7/8 (87.5) 7/8 (87.5) 2/8 (25.0) H5 AIV Ag Test Kit- Test line 2 1/8 (12.5) 3/8 (37.5) 4/8 (50.0) 7/8 (87.5) 5/8 (62.5) 3/8 (37.5) 0/8 (0.0) 1) LPAI: Highly Pathogenic Avian Influenza 2) Day Post Inoculation
12 Detection Test of H5 AIV Ag Test Kit in Chicken s diluted Specimens infected by Challenge with HPAIV(ES/03, H5N1) - Material : SPF chicken(lohmann Valo), 8 Heads - Challenge method : ES/03(H5N1, EID 50 /0.1 ), by nasal inoculation - Specimen preparation : Each organs of 10% diluted with PBS 100 Detection Rate(%) Virus Isolation PCR H5 AIV Ag Test Kit- Test line 2 H5 AIV Ag Test Kit- Test line 1 Spleen 7/8 (87.5) Kidney 6/8 (75) Appendix 5/8 (65.5)
13 Detection Rate in the Field Fecal Specimens of HPAI Name of the field(farm) Species Test Method Virus Isolation H5 AIV Ag Kit (No. of Positive/No of Specimen) (No. of Positive/No of Specimen) SKH 1/1 1/1 LCH 1/1 1/1 KYT 0/1 0/1 SWS 3/3 2/3 SDH 3/4 3/4 KMK 4/4 2/4 CMK 7/7 7/7 PKH 4/4 3/4 STB 11/12 10/12 KBK 5/7 5/7 YJ 4/4 4/4 Total By Farms By Layers 10/11(90.9) 43/48(89.6) 10/11(90.9) 38/48(79.1)
14 Detection Rate in the Field Fecal Specimens of Chickens with Clinical Symptoms by HPAI Name of the field(farm) with HPAI Species No of Specimen H5 AIV Ag Test Kit No. of Positive result LKB 3 2 KJT 3 2 SKU 3 1 SWS 3 2 CMK 5 5 STB 6 6 KBK 5 5 Total By Farms By Layers 7/7 23/28(82.1)
15 Comparison of Detection Rate in the Field Chicken s Organic Specimens with HPAI Test Method Name of the field(farm) with HPAI Virus Isolation PCR H5 AIV Ag Test Kit- Test line 2 H5 AIV Ag Test Kit- Test line 1 Kidney Appendix Kidney Appendix Kidney Appendix Kidney Appendix PDK SKH - LCH KYT KDH - LKB Chicken SWS KJT - SKU CMK STB KBK KSB Total By Farm 13/13 13/13 12/13 (92.3) 12/13 (92.3) 13/13 13/13 11/13 (85.4) 11/13 (85.4)
16 Conclusion Anigen Rapid H5 AIV Ag Test Kit could detect all AIV serotypes including LPAI and HPAI by a chromatographic immunoassay method using monoclonal antibodies to AIV. The detection limit of. Anigen Rapid H5 AIV Ag Test Kit is Test line 2 : 0.13 HAU, Test line 1: 1 HAU and Virus titer is Test line 2 : EID 50 /0.1, Test line 1: EID 50 /0.1 The sensitivity of Anigen Rapid H5 AIV Ag Test Kit with specimens of clinical symptoms specific to HPAI is 100% by farms and 82.1% by specimens
17 <AIV Reference Strain List> 1. A/PR/8/34 (H1N1) 2. A/Singapore/1/57 (H2N2) 3. A/Duck/Ukrine/1/63 (H3N8) 4. A/Duck/Czechoslovakia/56 (H4N6) 5. A/Duck/Hong Kong/820/80 (H5N3) 6. A/Shearwater/Australia/1/72 (H6N5) 7. A/Duck/Hong Kong/301/78 (H7N1) 8. A/Turkey/Ontario/6118/68 (H8N4) 9. A/Turkey/Wisconsin/1/66 (H9N2) 10. A/Chick/Germany/N/49 (H10N7) 11. A/Duck/England/56 (H11N6) 12. A/Duck/Alberta/60/76 (H12N5) 13. A/Gull/Maryland/704/77 (H13N6) 14. A/Mallard/Gurjev/263/82 (H14N5) 15. A/Shearwater/West Austalia/2576/79 (H15N9)
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