| Complement C3d is one of the degradation products of C3 which plays a key role in the complement-mediated immune defence, and occupies a central position within the complement cascade system. Therefore, C3d indicated the complement system activation which determine the state of an illness. In some infectious diseases and autoimmune diseases, detecting the plasma C3d level will help diagnosing, judging the patient's condition and treat effect. In the last over twenty years, Research about C3d was the hot spot all around the world. The foreign workers not only studied fundamentally its production, molecular structure, biological characters, function, and so on, but also investigated the C3d level in some patients with infectious or autoimmune,diseases which was mainly domestic research about C3d. At present, there is no commercially available purified C3d. In order to apply C3d assay in clinical laboratory and help further research about C3d, we utilized the conjugate of inulin and C3b to prepare the monoclonal antibody (mAb) and polyclonal antibody against C3d and develop immunogold to detect C3d by the strip chromatographic assay. The strips with different detection limitation were used to test the samples from the normal adults and patients with mild, moderate and severe hepatitis B. All the results were discussed. ObjectivesTo prepare a monoclonal antibody (mAb) against C3d and develop immunogold chromatographic assay for the purpose of detecting complement fragment C3d simply and rapidly, which helps judging the degree of the complement system activation.Methods1. Preparation of monoclonal antibody against C3d Inulin-C3b obtained by the way that washed inulin activated fresh human serum was utilized as antigen to immune Balb/c mice, in order to develop hybridoma. The microplates coated by goat anti-human C3 capturing C3d from old serum to screen and select the required hybridoma which produced mAb against C3d. When the antibody against C3d in the mice serum was positive, the spleen cells of the mice were took out and hybridized with SP2/0 myeloma cells. The hybridoma was cultivated selectively in HAT culture medium. The positive hybridoma was judged and selected by examining the supernatant from its medium. Then it was amplified and subcloned till all the clones from the single hybridoma were positive. Thus, the hybridoma series excreting mAb against C3d was created.2. Evaluation of the specificity of the mAb After being purified by protein G column, the complex of mAb IgG and C3d was analyzed by SDS-PAGE. According to their molecular weight, the specificity of the mAb was found out and tested.3. Conjugation of mAb with colloidal gold The purified mAb IgG was clialysed in low-salt solution. Then the mAb with optimal amount was conjugated with colloidal gold under about pH 8.0. And the immunogold was centrifuged and concentrated.4. Creation of immunogold chromatographic assay to detect C3d The C3d mAb IgG and goat anti-mouse IgG were immobilized on the nitrocellulose membrane, which formed detection line and control line respectively. Then the membrane was blocked by bovine serum albumin. The sample absorption material, the glass membrane where immunogold lay, the nitrocellulose membrane and water absorption material were pasted on a plastic plate which was finally cut to 2 mm-wide and 80mm-long. By way of mixing the immunogold withdifferent mounts of mAb, the detection limitation of the strips was modulated and controlled. Then they were used to detect the C3d level of the samples from the normal adults and patients with mild, moderate and severe hepatitis B. Results1. On the map of SDS-PAGE, there were three bands whose molecular weight accorded with heavy chain of the mAb IgG, C3d and light chain respectively.2. The detection limitation of the strip was 6 ~ 10 ng/ml (strip I ), however, when the immunogold was mixed with 0.1ug/ml free mAb, it rose to 10 ~ 15ng/ml (strip II ). By strip I, 83.3% (25/30) of th... |
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