Development Of Colloidal Gold Strip For Rapid Detection Of Antibody Against Serotype ? Riemerella Anatipestifer

The Riemerella anatipestifer infection is caused by Riemerella anatipestifer(RA),which induced a commom,acute and contagious disease of ducks,geese and other poultry.It is one of the most seriously infectious diseases in duck industry due to bringing about tremendous economic losses to the culturist.There are reports about applications of duck Riemerella anatipestifer inactivated vaccine.The IgY antibody is generally found in birds,reptiles and amphibians,which is one of the important immunoglobulin molecules.IVE-2 is specificly induced in vivo during the RA infection.The purpose of this research lies in the preparation of monoclonal antibodies against duck IgY Fc fragment and establishing a diagnosis method,through which we can distinguish the serum of ducks between infection and inactivated vaccine of RA.1.The production of monoclonal antibodies against duck IgY Fc fragment CH2The cloning of duck IgY Fc fragment CH2 was gained by the template of pMD-18T-CH1-CH2-CH3-CH4,which encodes 288bp.The sequencing analysis of the recombinant plasmids pMD-18T-CH2 was consistent with the known sequence.Constructed prokaryotic expression vector pET-28a and pGEX-KG with the gene of IgY Fc fragment CH2 to get pET-28a-CH2 and pGEX-KG-CH2 plasmids.The expression and purification of the fusion protein HIS-CH2,GST-CH2 were detected by SDS-PAGE,17 KDa and 37 KDa.The purified fusion protein GST-CH2 was used for the immunization of the Balb/c mice.Those with high potency were used for the productionThe hybridoma strain of 2F and 6F were filtered after cell fusion which can produce the antibodies of anti-immunogen stab lely.The titer of 2F was up to 1:1.024×106,and the potency of 6F was 1 5.12×105.The titer of the purified 2F was 1:3.2×104,and the other was 1:1.6×104-The result of western blot of 2F monoclonal antibody showed that the intact duck IgY and chicken IgY can bind the 2F monoclonal antibody.But the result of ELISA showed 2F or 6F monoclonal antibody couldn't detect the intact duck IgY and chicken IgY in the serum.It showed that the 2F monoclonal antibody could detect the IgY in other poultry's serum2.The production of monoclonal antibodies against duck IgY Fc fragment CH3CH4The purified fusion protein GST-CH3CH4 was used for the immunization of the Balb/c mice.Those with high potency are used for the production.The hybridoma strain of 2C5 and 10A1 were filtered after cell fusion which can produce the antibodies of anti-immunogen stablely.The titer of 2C5 was up to 1:1.024 × 106,and the potency of 10A1 was 1:5.12×105.The titer of puried 2C5 up to 1:5.12×105,and the other was 12.56×105 The result of western blot and ELISA showed positive results of the reaction with the serums of duck,chicken,goose,pigeon and ostrich.While the results with the serums of pig,rabbit and cattle were negative.These results indicated that the monoclonal antibodies against duck IgY Fc fragment CH3CH4 could be used not only for the decetion of IgY in duck serum,also applicable to IgY in other poultry's serum.3.Development and application of the colloidal gold immunochromatograph strip for rapid detection of antibody against RABy the amplification of the recombinant plasmids pMD-18T-CH1-CH2-CH3-CH4,we obtained the ive-2 fragament which encodes 1062 bp.The prokaryotic expression vector pGEX-KG was constructed with the gene ive-2 to get pGEX-KG-ive-2.The expression and purification of the fusion protein GST-IVE-2 were detected by SDS-PAGE,66 KDa.The 20nm colloidal was preparated with sodium citrate reduction method.The monoclonal antibody against IgY Fc fragment CH3CH4 was conjugated with colloidal gold to serve as tracer,artificial antigen of IVE-2 protain used as test line and goat anti-mouse IgG used as control line for the preparation of colloidal gold immunochromatographic strip for rapidly detecting RA and identified its sensitivity,specificity and so on.We explored the ideal conditions for preparing the colloidal gold immunochromatographic strip.The result showed that the ideal McAb concentration were 4?g monoclonal antibody and 6?g monoclonal antibody per milliliter colloidal gold,the best conjugate pH were 10 pL K2CO3 per milliliter colloidal gold,respectively,the best coating antigen concentration was 1.5mg/mL,the best conjugation used for the strip was gold-monoclonal antibody 10A1 and the spot rate was 15 ?L/cm,the coating solution prepared the PB,the sample diluent selected the PBS(pH7.4).The result of the strip properties showed that the strip had good specificity and acceptable sensitivity.Tested by the strip,only the serum of ducks with natural infection were detected positive,the serum of inactivated immunological or other antigen read negative.The IVE-2 antibodies were detected as earlier as the fifth day post-infection through ELISA,but the seventh day post-infection through the strip.The results showed the colloidal gold immunochromatographic strip could be used to distinguish the serum of ducks with natural infection and the serum of inactivated immunological.