| Avian influenza(AI) and New castle disease are the two capital contagious disease in birds caused by avian influenza virus(AIV) and new castle disease virus(NDV) respectively.Both avian influenza and new castle disease can cause tremendous economic losses in poultry industry and since avian influenza virus can infect human beings and cause disease even death,which become a threat for humans and raise great public health concern.Various laboratory methods are currently available for the detection and surveillance of AIV,and include virus isolation,hemagglutination(HA) assay, hemagglutination inhibition(HI) test,enzyme linked immunoabsorbent assay(ELISA) and RT-PCR.However,these assays are laborious and time consuming,difficult to incorporate into automated procedure,and require laboratory operation,skilled technicians,and special equipment/facilities,are limited in spot,real time and rapid application.Immunochromatographic assay is a new immunochromatographic technique in which a cellulose membrane is used as the carrier,and a colloidal gold-labeled antigen or antibody is used as the tracer.It is simple,rapid and cheap,and the result is clear and easy to analyze without any equipment/facilities and skilled technicians.Since it is very suitable for spot,rapid detection and automatic detection,the method has been widely used for rapid detection in human medicine,and more recently,it has been used for rapid detection in veterinary medical analysis.Facing the shortage of the present methods used in detecting pathogens of respiratory disease in poultry,a technology system for rapid detecting pathogens of respiratory disease in poultry was drawn up using the principle and technology of colloidal gold immunochromatographic assay.This study focused on developing and evaluating the method for rapid detection of avian influenza virus and new castle disease virus and the results were reported as follows.1.Preparation of polyclonal antibodies of chicken anti-AIV,chicken anti-NDV and goat anti-mouse IgGAIV(H9N2) and NDV were inoculated into 9 to 10-day embryonating chicken eggs by the allantoic route respectively,then allantoic fluid was harvested and enriched by centrifuging at 30,000r/m.Chickens were immunized with prepared AIV and NDV emulsified with Frund's adjuvant,at the same time,goats were immunized with purified mouse IgG emulsified with Frund's adjuvant.Sera of chicken anti-AIV,chicken anti-NDV and goat anti-mouse IgG were acquired after several inoculations,and the titers of AGID reach 1:64.The depurated antibodies were obtained after these sera were purified by(NH4)2SO4 precipitation and G-200 respectively.2.Preparation monoclonal antibodies used in this studyAfter immunization of BALB/C mice with the purified NDV and GST-NP from AIV respectively,the stimulated splenocytes were routinely fused with SP2/0 myeloma cells to produce hybridomas.After three cycles of cloning,5 stable hybridoma clones producing monoclonal antibodies to AIV and 5 to NDV were obtained.At the same time, Hybridoma cells H55E8 and H94C4 were recovered which were previously prepared in our lab.All the Hybridoma cells were cultured in RPMI-1640 with 5%FCS,harvested and injected into nude mice,and then mouse ascetic fluids were harvested 10 days later. The ELISA titers of 5 monoclonal antibodies for AIV-NP were 1:32000～1:64000 and 5 monoclonal antibodies for NDV-NH were 1:16000～1:64000 with the HI titers of 1:1024～1:16384.The HI titer of monoclonal antibody H55E8 and H94C4 were 1:16384 and 1:65536 respectively.Selected monoclonal antibodies with higher titers were purified from mouse ascetic fluids using sequential precipitation with caprylic acid and ammonium sulfate.3.Preparation of colloidal and immuno-colloidal,goldAfter the preparing conditions were established,colloidal gold of 20nm was prepared with reducing reaction using trisodium citrate.Studies were progressed on the optimal conditions about preparing the colloidal gold conjugates with monoclonal antibodies previously prepared,such as monoclonal antibody 5F4 against neucleoprotein of AIV,monoclonal antibody H94C4 against hemagglutinin of H9AIV,monoclonal antibody H55E8 against hemagglutinin of H5AIV and monoclonal antibody 213 against hemagglutinin-neuramidinase of NDV.After the optimal conditions were developed(the optimal pH is 8.0,the optimal mount of monoclonal antibody is 12μg/mL),colloidal gold-5F4 conjugate,colloidal gold-H94C4 conjugate,colloidal gold- H55E8 conjugate and colloidal gold-213 conjugate were prepared respectively according to the method previously described.4.Development of immunochromatographic strips for rapid detection and differential diagnosis of HS,H9 subtype of avian influenza virusesTwo immunochromatographic strips were developed respectively for rapid detection of the H5,H9 subtype of avian influenza viruses in poultry with sandwich method.The monoclonal antibody 4C4 for H9AIV hemagglutinin,5E8 for H5AIV were labeled with colloidal gold as the detection reagent respectively,and the chicken anti-AIV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.To determine the specificity of the immunochromatographic strip for the detection of H9AIV,allantoic fluids harvested from H9N2 infected eggs at 64 units of HA,allantoic fluids from unmanipulated eggs, together with standard antigens of H5,H7,or H9AIV and other viruses,were characterized using the immunochromatographic strips simultaneously.Clearly,while all of the samples tested showed one strong band on the control line,only allantoic fluid harvested from H9N2 infected eggs and the standard H9 AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect H9AIV,but not other tested AIVs,NDV,IBV,IBDV,ILTV,and FAV,common infectious viruses in poultry.To determine the specificity of the immunochromatographic strip for the detection of H5AIV,allantoic fluids from unmanipulated eggs,together with standard antigens of H5, H7,or H9AIV and other viruses,were characterized using the immunochromatographic strips simultaneously.Clearly,while all of the samples tested showed one strong band on the control line,only the standard H5AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect H5AIV,but not other tested AIVs, or other common infectious viruses in poultry.In comparison with the hemagglutination (HA) and hemagglutination inhibition(HI) test,the sensitivity of the immunochromatographic strip for the detection of H9AIV reached a level of 0.25 units of HA antigens,approximately 4 times higher than HA test,while the immunochromatographic strip for the detection of H5AIV only reached the HA test level. At the same time,another immunochromatographic strip was developed for the differential diagnosis of H5 and H9 subtype of avian influenza virus.The monoclonal antibody 5F4 for neucleoprotein of AIV was labeled with colloidal gold as a detection reagent,and the monoclonal antibody 4C4 for H9AIV and monoclonal antibody 5E8 for H5AIV were blotted on the nitrocellulose membrane with 5mm wide width as test line 1 and test line 2 respectively.Results suggested that the strips were easy to operate and can be applied in rapid detecting and differential diagnosing H5,H9 subtype of AIV with high specificity and sensitivity within 10 minute.5.Development of immunochromatographic strips for rapid detection and differential diagnosis of avian influenza virus and New castle disease virusAn immunochromatographic strip was developed for the detection of AIV in poultry with sandwich method.The monoclonal antibody 5F4 for neucleoprotein of AIV was labeled with colloidal gold as a detection reagent,and the chicken anti-AIV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.To determine the specificity of the immunochromatographic strip,standard antigens of H5,H7,or H9AIV and other viruses were characterized using the immunochromatographic strips simultaneously.Clearly, while all of the samples tested showed one strong band on the control line,only the standard H5,H7 and H9 AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect AIV,but not other tested virus common infected in poultry,such as NDV,IBV,IBDV,ILTV,and FAV,.In comparison with the hemagglutination(HA) and hemagglutination inhibition(HI) test,the sensitivity of the immunochromatographic strip reached a level of 0.25 units of HA antigens,approximately 4 times higher than HA test.At the same time,another immunochromatographic strip was developed for the detection of NDV in poultry with sandwich method.The monoclonal antibody 213 for hemagglutinin neuraminidase of NDV was labeled with colloidal gold as a detection reagent,and the chicken anti-NDV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.One strong band showed on control line when the standard H5,H7,H9 AIV antigens and other virus common infected in poultry were tested by the strip,while an additional band showed on test line when detecting NDV.Results suggested that the strip can specifically detect NDV with high sensitivity at 4 times higher than HA.Based on the previous work,an immunochromatographic strip was developed for the differential diagnosis of NDV in poultry.The colloidal gold-5F4 conjugate and colloidal gold-213 were mixed as the detection reagent,and the chicken anti-NDV IgG and chicken anti-AIV IgG were blotted on the nitrocellulose membrane with 5mm wide width as test line 1 and test line 2 respectively.Results suggested that the strip can be applied in differential diagnosing NDV and AIV with high specificity and sensitivity within 10 minute.6.Preparation and application of kits for rapid detection of general type and H9 subtype avian influenza virusTwo kits were developed for rapid detection of avian influenza virus and H9 subtype avian influenza virus based on rapid detecting immunochromatographic assay previously developed.According to the method described above,experiments were progressed to evaluate the specificity,sensitivity,reproducibility and stability.Storage of the kits at room temperature for six months or at 4℃for 12 months did not change their sensitivity and specificity.Evaluation of the kits on experimental tracheal and cloacal swab samples collected from HgN2-infected chickens revealed that both of the two rapid test kits detected the HgN2 viruses on day 3 post-inoculation,earlier than the appearance of clinical symptoms.Application of the kits for detection of AIV and H9AIV for analysis of 311 samples collected from potentially infected chickens,the positive ratio was 39.2%and 35%with a coincidence at 100%and 92%respectively with the ELISA kits previously prepared in our lab.Further characterization samples randomly selected showed that no single sample was false positive or negative,as determined by the standard virus isolation and HI assays.Therefore,the two rapid test kits for the detection of AIV and H9AIV both have high specificity,sensitivity and stability.This,together with the advantages of rapid detection and easy operation without requirement for special skills and equipments,makes it suitable for the on-site detection of AIV and differentiation of H9AIV from other viruses in poultry.
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