Preparation And Research On Monoclonal Antibody Against Mycoplasma PG₃ Of Cotagious Caprine Pleuropneumonia

Objective The antigen was obtained from splitting by freezing thawing Mycoplasma mycoides subsp,Capri.PG₃ which were grown in improved Hartleys solid medium.Through immuning animal,developping indirect ELISA,cell fusion,screening positive clones and subcloning, positive monoclone cell lines were gained.The high titer and specificity monoclone cell lines were obtained by ascites prepared,titer detected and specificity identified. They were prepared for studying on Mycoplasma mycoides subsp,Capri surface antigenic structure and screening protective antigens. By coating purified monoclonal antibody and using purified goat anti-PG₃ antibody as the sandwich antibody,the sandwich ELISA was developped,which could provide a simple quickly sensitivity and specificity method for Cotagious Caprine Pleuropneumonia diagnosis.Method Mycoplasma mycoides subsp,Capri.PG₃ was revivaled and grown in improved Hartleys medium according to the condition of our lab and literature reported in deomestic or intemational;Thalline was elutinged from solid medium,and removed precipitation by brachytely centrifugalization.Through high speed centrifugation of clear supematant liquid,the purifed thalline was obtained. Repeated ablution three times using PBS,and splitted thalline by freezing thawing at-20℃several times and removed precipitation.The clear supernatant liquid was the membrane protein and detected protein level.The membrane protein was emulsified with Freund's adjuvant, immuned 330μg on BALB/c mice everytime,total four times.Determining the blood serum antibody titer and developping indirect ELISA method.Cell fusion was acted when the blood serum antibody titer rise to 1:5000.Selected the high blood serum antibody titer mice,the spleen cell was taken and fused with SP2/0 bone myeloma cell by 50%PEG1500.Positive clones were got by indirect ELISA screening and subcloned to get monoclone cell lines by limit dilution.Monoclones were enlarged and frozen,at the same time prepared ascitices.Detected specificity and titre of ascitices.The monoclonal antibody isotype was identified by the antibody isotype kit.Through coating purified monoclonal antibody and using purified goat anti-PG₃ antibody as the sandwich antibody,the sandwich ELISA method was developped while detected the specificity sensitivity and initial application.Result Mycoplasma mycoides subsp,Capri.PG₃ was successfully revivaled and grown in improved Hartleys medium.The membrane protein level was 1.65mg/mL;After four times immuned,the mice serum antibody titer achieved above 1:6400.After cell fusion with 50%PEG1500,we got 364 fusion cell clones,the fusion rate was about 75.8%.Through indirect ELISA screening,31 positive clones were obtained,positive rate achieved 8.5%.After twice subcloning we got four positive monoclone cell lines which were 1C10,1E2,2E8,2F12.According to preparing ascites,detecting titer and specificity, 1E2 was got ultimum which antibody isotype was IgG₃,κand purity was detected by SDS-PAGE.The coating density of sandwich ELISA was 1:200 dilution(20μg/mL),goat anti-PG₃ antibody was 1:400 dilution,the lower limitation of detection was 2μg/mL.It couldn't react with Mycoplasma hyopneumoniae,Mycoplasma MS,Escherichia coli, Salmonella, Staphylococcus aureus,Streptococcus and horse serum. 12 nasal cavity swabs and serum of goats with clinical symptoms were detected by indricet ELISA and sandwich ELISA,the former (sandwich ELISA)positive rate was 58.3%,the latter was 66.7%;5 of the typical clinical symptom goats,all of them were positive by sandwich ELISA,positive rate was 100%,but only 3 were positive by indirect ELISA which positive rate was 60%.7 of just appearanced dry cough goats positive were 3,positive rate was 28.6% by sandwich ELISA,while 7 were positive and positive rate was 71.4% by indirect ELISA.Conclusion The PG₃ membrane protein was obtained by grown in improved Hartleys solid medium and freezing thawing.Through immuning animal,cell fusion,screening positive clones by indirect ELISA and subcloning,four positive monoclone cell lines which could secretoried antibody were gained.While one of them was used in developing sandwich ELISA method which possessing high sensitivity and specificity.Comparing with indirect ELISA,sandwich ELISA method was suitted in diagnosis and detection on pristine Cotagious Caprme Pleuropneumonia,while indirect ELISA was suitted in epidemiological investigation.