Research Of Indirect Sandwich ELISA For Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG) is a primary pathogen causing chronic respiratory disease in chicken and infectious sinusitis in turkey.For many years,people pay great attention to the prevention of MG's infection in many country,but there is not any effective method yet now.At present,the main methods for MG- diagnosis in lab are the isolation and identification of pathogen/immunologic method/molecular biological method,the isolation and identification of pathogen is the most reliable method for MG-diagnosis, and it is the classic method,too.but this approach needs more time/complicated manipulation and large workload,so it is limited in the spot application.The primary immunologic method for MG-diagnosis are serum plate agglutination(SPA)/hemagglutination inhibition(HI)/ELISA/fluorescent antibody technique(FAC) and so on.These approaches are convenient,but most of them can just detect antibody,and they can't be used for diagnosis of pristine infection and inapparent infection.Besides,These approaches have nonspecific reaction and cross reactivity,too.so they can't make a fast/presice diagnosis for MG infection.Along with the development of molecular biology,the rapid detective technology for antigen have made a large improvement,molecular biological methods can be used for detecting antigen of the etiologic agents,and they can be used for diagnosis of MG pristine infection and inapparent infection,but these approaches need expensive instruments and professional people,so they are hard to extend in basic level.In view of all the reasons above,the diagnosis of MG infection is stll quite a hard project in lab.Facing the shortage of the present methods used in detecting pathogens of MG, This study apply monoclonal antibody technique to product mouse anti MG McAb,and combined with the principle of indirect sandwich ELISA to develop a new method for rapid detection of MG antigen and the results were reported as follows:1 Preparation of rabbit anti-MG polyclonal antibodiesInoculate purified MG-HS strain onto FM-4 solid medium and cultivate it in 37℃/CO2 incubator for about 7～10d,then observe colonial morphology with microscope(40×).There are tipical fried egg-shaped colony.cultivate the purified MG-HS strain with bulk and centrifuge it,then wash the sedimentum four times, finally,suspend the sedimentum with Sterile PBS(0.01M/L,pH7.2) and measure protein concentration,the protein concentration is 18mg/ml.To emulsify it with Freund's adjuvant and immune the rabbit,we obtained rabbit anti MG serum,its' AGID(agarose gel immunodiffusion) titer is 1:32.To Purify The serum with saturated ammonium sulfate and G-200,we obtained purified rabbit anti MG IgG,then evaluate the purity of the IgG with SDS-PAGE,There are two straps with the size of 55kD和25kD,they are exactly the H-chain and L-chain of IgG.It established a good corporeal foundation for detection of MG antigen with indirect sandwich ELISA.2 Preparation of mouse anti-MG monoclonal antibodiesTo adjust the protein concentration of MG-HS strain into 2mg/ml,then emulsify it with Freund's adjuvant and immune the BALB/cn mouse.By using the hybridoma technique,screened by indirect ELISA,two positive hybridoma cell lines secreting monoclonal antibodies(McAbs) against MG proteins were established:2B7and 6G9.The number of chromosome of the two hybridoma cell lines are 86.6 and 86.9,respectively.To product ascites with the two hybridoma cell lines,indirect ELISA titers of the nascites are 1:5.12×10~5(2B7)and 1:2.56×10~5(6G9).To Purify the ascites with caprylic acid and saturated ammonium sulfate, we obtained purified mouse anti MG IgG,then evaluate the purity of the IgG with SDS-PAGE,there are two straps with the size of 55kDand 25kD,they are exactly the H-chain and L-chain of IgG.To evaluate the biologic activity and specificity of the purified IgG with indirect ELISA,the result indicate that the mouse anti MG monoclonal antibodies we have made have good affinity and don't have cross reactivity with pastometer/Escherichia coli/salmonella/Mycoplasma synoviae/NDV/ IBV.3 Development of indirect sandwich ELISA for MG antigenWith the purified rabbit anti-MG polyclonal antibodies(Ab1)/mouse anti-MG monoclonal antibodies(Ab2) and goat anti mouse IgG-HRP,A indirect sandwich ELISA for detection of MG antigen was developed.The result of Checkerboard titration test indicate that the optimum working concentration of Ab1 and Ab2 are 1μg/mL and 5μg/mL,respectively.To detect 30 pieces of MG-negative samples by the indirect sandwich ELISA to decide critical value between negative samples and positive samples,finally,according to the OD450 value,we adopted 0.2 as the critical value To detect MG-positive samples with different MG-concentration by the indirect sandiwich ELISA to evaluate the sensitivity of this method,the result indicate that the indirect sandiwich ELISA we developed can detect the MG-positive samples with only 5×10~1cfu/ml.To estimate the specificity of the indirect sandiwich ELISA for MG antigen With pastometer/Escherichia coli/salmonella/Mycoplasma synoviae/NDV/IBV,the result indicate that this method don't have cross-reactivity with all of them.The coefficient variation of the same sample within one plate is between 0.41 %～6.72%and it is between 0.88%～10.72%among plates.the result indicate that the indirect sandiwich ELISA we developed have a good repeatability...