Deverlopment of Monoclonal Antibody and Antigen Capture ELISA for Detection of H7 Subtype Avian Influenza Virus
|School||Northeast Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Specific monoclonal antibodies H7 subtypes are highly pathogenic avian influenza virus Antigen capture ELISA|
In this study, the highly pathogenic avian influenza virus (Highly pathogenic avian influenza virus, HPAIV) (H7N1) (FPV) A/FPV/Rostock/34 strain as an immunogen, immune 8-week-old BALB / C female mice, three times strengthen the immune spleen cells were fused with SP2 / 0 cells detected by screening, positive cells were cloned by limiting dilution cloning culture, hemagglutination inhibition (HI) test, the last two H7 subtypes of HA-specific monoclonal antibody 6H4 and 7F6 subclass for IgG2a, IgG2b, k light chain chain, has been tested with good specificity. Using purified HPAIV A/FPV/Rostock/34, (FPV) strain (H7N1) as an immunogen and inactivated Houjiarufu's complete adjuvant and incomplete adjuvant made the vaccine repeatedly immune goat anti-H7 subtypes avian influenza hyperimmune goat serum. Goat serum as the capture antibody, H7 subtype-specific monoclonal antibodies for the detection of antibodies, double antibody sandwich ELISA method for detection H7 subtypes are highly pathogenic avian influenza virus. Through optimization of the reaction conditions of each step, to obtain the best working conditions: sheep serum diluted 1:1000, 1:40000 dilution of monoclonal antibody, the enzyme-labeled antibody is a 1:5000 dilution, an anti-reaction time of 1.5h, the enzyme the role of labeled antibody time 1.5h. The EDS76, IBV, IBDV, ILTV, MDV and APV avian viruses known NDV and 15 other HA subtypes of avian influenza standard strain of the virus cross-reactivity test with the establishment of the antigen capture ELISA method, the results show that: the establishment of the H7 subtypes are highly pathogenic avian influenza antigen capture ELISA method and other avian viruses, and other subtypes of avian influenza virus were no significant cross-reactivity and H7 subtypes are highly pathogenic avian influenza virus has a very good specificity. The established antigen capture ELISA method compared with HA 2 times more sensitive, but not as good as the chicken embryo inoculation test sensitive hemagglutination (HA) and egg inoculation test. ELISA using the optimized reaction conditions artificially infected clinical samples detected by the detection of the yin and yang of the spleen, kidney, heart, lung and other samples to finalize the clinical testing as the target organ to the heart, and 0.099 (OD 490 nm ) as yin and yang organs criteria. Test with a variety of reagents and reaction plates assembled into kits, and stored at room temperature, 4 ° C and -20 ° C, on a regular basis to detect the results showed that: the kit is extremely unstable when stored at room temperature, the shelf life is very short, 4 ° C and -20 ° C conditions can be stored for more than 3 months.