| Avian influenza (AI) is a deadly infectious disease,which caused by avian influenza virus. Influenza viruses are segmented, single-stranded negative-sense RNA viruses and classified into type A, B and C. They are divided into different subtypes based on hemagglutinin (HA) and neuraminidase (NA). H9N2 low pathogenic aivain influenza virus has h u man-like virus receptor, so it can get across species and transmit from livestock to h u man,which indicates H9N2 AIV has the potential to be the next pandemic virus and threat to the public healthy seriously. In this study, the H9N2 subtypes anti-HA monoclonal antibody was preparated. Analysis methods of double-antibody sandwich ELISA and colloidal gold immunoassay tests were developed. It shows that the method has the advantages of high sensitivity,good specificity and being easy to operate. The main methods and results are as follows:1. Preparation of H9 subtypes anti-HA monoclonal antibodyThe H9N2 subtype influenza virus (A/Chicken/Tibet/S1/2009) which was separated from a disease Chicken in Tibet was used for antigen to immune 5 weeks BALB/c mouses after amplification and purification, and the immune mouse spleen cells and SP2/0 myeloma cells were taken for fusion.The method of limited dilution is used to clone the positive hybridome filtered by the ELISA and HI. At last,we get six hybridome cell lines for specific sites in the HA of the H9N2 subtype influza virus.The hybridoma cell lines named 5G2?4E8?4D10?5E1?4A9?2E12 have a ascitic hemagglutination inhibition value from 214 to 218 respectively, which can all produce blood clots inhibition reaction to H9N2 subtype influenza virus but be negative to other influenza virus,which suggests it has a good specificity.2. Establishment of ELISA detection methodDouble antibody sandwich ELISA kit for H9 subtype influenza virus with monoclonal antibody 4D10 as coating antigen, HRP4D10 as HRP labeled second antibody, which has advantages of high sensitivity, good specificity and easy to operate. However,H1, H3, H5, H7 influenza virus and NDV, ESDV, IBDV were tested to be negative. The result show that the kit has a good specificity. The detect limitation of the AC-ELISA were 10-2.3TCID50. Negatives/positive threshold was 0.211(OD630). There were 98.9% sensitivity and 98.1% specificity when detected using the isolated virus. And it has a positive rate of 94.3% and the negative rate of 100% for clinical detection. This kit made up for the vacancy of H9 avian flu detection kit. It can detect H9 subtype AIVs rapidly and accuratly, which has a great significance on H9 subtype AIVs asiaassessment, prevention and epidemic investigation.3. Establishment of immune colloidal gold methodThe colloidal gold reduced by citrate sodi ?m was wine red, particle uniform, and had the maxim ?m absorption peak at 520nm. The colloidal gold was labeled with the antibody with the most suitable concentration of 24?g/mL. The polyclonal antibody against H9N2 substype influenza virus(detection line) and goat anti-mouse IgG(control line) were coated respectively on the nitrocellulose membrane (NCM) of the colloidal gold test strip with a concentration of 1.5mg/mL for detection line and 1mg/mL for control. The virus can still be detected by the strip when virus was diluted from 29 to 24. In clinical detection, there was a 94% positive rate using the colloidal gold test strip to detect throat swabs and tissue taken from disease chickens. The Isolated H9 subtype AIVs and the H9 standard antigen were used for test and showed to be positive, while the results of H1, H3, H5 and H7 subtypes standard antigen and the NDV, IBV, EDSV antigen were negative as well. The resusts reveal that our method is specific, fast and easy to interpret and operate, and it can be used for specific diagnosis and epidemic investigation of H9 AIVs and provides a theoretical basis for the clinical diagnosis.4. Clinical detectionHeart, liver, spleen, lung, kidney, brain, throat swab were collected from Sick chicken. Samples were detected by ELISA and colloidal gold. The results showed that the viral titter of different organs increased from day 3 to day 5 and the lung had the highest virus titter. It shows that the ELISA kit and colloidal gold test kit can be used for clinical detection. |
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