Monoclonal antibody for detecting and treating senile dementia
A monoclonal antibody, age-related technology, applied in the direction of antibodies, microbial-based methods, measurement devices, etc., can solve the problem of reducing the ability of cells to resist oxidative stress, and achieve high-affinity effects
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Embodiment 1A
[0050] Synthesis of embodiment 1Aβ42 peptide and its fragments
[0051] According to the public sequence of Aβ42 peptide (Genebank: AB066441.2; PMID: 8248178), the full-length Aβ42 and its fragments (including N-terminal fragment, C-terminal fragment and intermediate fragment) were artificially synthesized. The full-length Aβ42 and its fragments were provided by Jill Synthesized by Biochemical (Shanghai) Co., Ltd.
Embodiment 2
[0052] The preparation of embodiment 2 monoclonal antibody
[0053] 2.1 Immunization of mice
[0054] Full-length Aβ42 can be directly immunized, and other fragments are coupled with KLH to immunize mice separately. The immunization method is as follows:
[0055] Initial immunization: 50ug of antigen plus an equal volume of Fluorine's complete adjuvant emulsified until dripping water can be used for intraperitoneal injection of mice, and the injection volume is 500ul / mouse two weeks later. The second immunization: 50ug antigen plus an equal volume of Fluorine's incomplete adjuvant emulsified until dripping water can be used for intraperitoneal injection of mice, the injection volume is 500ul / mouse. After two weeks, the tail vein blood of the mice was collected and centrifuged to obtain the serum to measure the titer (detected by ELISA method). Mice with a titer of more than 10,000 were boosted with 25ug of antigen three days before fusion, and the injection volume was 200ul / ...
Embodiment 3
[0058] Embodiment 3 Dot blot and Western blot experiments
[0059] 3.1Dot blot experiment
[0060] Spot 10ng of synthetic Aβ42 peptide (synthesized by Jill Biochemical Company) on PVDF membrane, dry at 60°C, block with 5% skimmed milk powder for 1h, then put it into the diluents of different dilutions of the antibody prepared in Example 2, and incubate at room temperature 1h, after washing 3 times with TBST, incubate with 1:2000-fold diluted HRP goat anti-mouse antibody for 30min at room temperature, wash 3 times, add ECL luminescent reagent, and expose to X-ray film in a dark room. Scan the light sheet to record the results of the DOT Blot experiment.
[0061] 3.2Western blot experiment
[0062] 3.2.1 Trieine SDS PAGE electrophoresis
[0063]Since the Aβ42 peptide has only 42 amino acid residues and a relative molecular weight of 4200, conventional SDS-PAGE cannot make clear bands. In this experiment we used Tricine SDS PAGE electrophoresis. Gel preparation with all reag...
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