1. Development Of Mycoplasma Hyorhinis Immune Colloidal Gold Strip 2. The Preparation And Identification Of The Monoclonal Antibodies To Streptobacillus Moniliformis

Gold immunochromatography assay (GICA) has been used widely in epidemiology investigations and clinical diagnosis especially infection diseases because of its specificity, sensitivity, convenience and rapidness. The aim of this study is to establish sandwich GICAs to detect M. hyorhinis due to low level antibody in serum and the fact that they exist in human's cavity. The most suitable double antibody was selected to be used in sandwich GICAs. The results are as follow:Firstly, GICA materials has been prepared and screened primarily. Colloidal gold was prepared by reduction of HAuCl₄ with trisodium citrate. Different size Colloidal gold particles have been prepared by using different volumes of trisodium citrate. Their diameters and particle distribution uniformity were detected through visible spectroscopy. One of them was selected to be labeled with one of the double antibody, another was used as coat antibody. All the GICA materials were screened based on their chromatography ability and application value, the buffers and the work concentration of antibodies were also determined in methodology.Further more, the qualifications and conditions of sandwich GICA were selected in the second time and they were optimized for the development of sensitivity, specificity and stability.Finally, the applications of the GICAs were evaluated in sensitivity, specificity and stability. Threshold value of the GICAs was 1.72×10³CFU/mL and 500ng/100μL of bacterial protein, without cross reaction with 11 bacteria and 11 mycoplasmas which are common in human and laboratory animals. The process would take about 5 minutes for positive result and 10 minutes for lightly positive result. Positive result could be repeated using either same or different groups of the GICAs. The positive result could be seen as before above the threshold value even the GICAs were stored at 4℃more than six months later.In a word, we have successfully developed the GICAs firstly that can be used for epidemiology studies and clinic diagnosis of M. hyorhinis infections for its excellence in sensitivity, specificity and stability. Streptobacillus moniliformis (SM) is the pathogenic bacteria both in human and rat. At present it is absent of rapid serological reagent to detect the disease caused by SM. the conventional culture method is time costing and the bacteria is easy to be induced to L-form, PCR is cockamamie and be limited. So we prepared the monoclonal antibodies (McAb) anti SM using routine cell fusion technique for the further development of rapid serodiagnostic reagent to detect SM.Firstly, the hybridoma cell strains was prepared as follows: Titers of ascetic fluid were up to 1: 10⁵ by immunizing BALB/c mice with bacteria protein for one basic and 3 trengthen immunity, then get the spleen cells and confuse them with the SP2/0 cell strain. The cells were Clone Repeat 3 times using methyl cellulose semisolid culture and 12 antibody-positive strains were screened finally by ELISA.Secondly, the McAb is identified as follows: The 12 hybridoma cell strains were confirmed by chromosome count. The McAb' subtypes were belong to IgG1,IgG2a,IgG2b and IgM by ELISA. McAb 5G4 had a clear reactivity to the bacteria membrane antigen by IFA, their specificity was primarily tested by ELISA. The Western-blot shows that the molecular weight of the antigens that the McAb 5G4,6D10,5B9 recognized were located at 32×10³,66×10³ and 60-67×10³ and they are common strains of SM of different origin, which indicates that they may be used in serodiagnostic reagent as the antibodies anti genus antigen of SM. But their sensitivity and some problems such as mix subtypes of Ig must be researched furthermore when used as serodiagnostic reagent.