Humanized anti-aβ monoclonal antibody and its application
A monoclonal antibody, humanized technology, applied in the direction of antibodies, applications, chemical instruments and methods, etc., can solve problems such as peroxidative damage, achieve the effect of promoting phagocytosis and improving cognitive learning and memory ability
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Embodiment 1A
[0167] The preparation of embodiment 1Aβ antigen and positive control antibody
[0168] 1. Preparation of Aβ monomer and polymer mixture
[0169] Aβ 1-42 , Aβ 1-16 , Aβ 14-29 The peptide was synthesized by Gill Biochemical (Shanghai) Co., Ltd. Aβ 1-42 The amino acid sequence of the polypeptide is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID: 35), Aβ 1-16 The amino acid sequence of the polypeptide is: DAEFRHDSGYEVHHQK (SEQ ID: 36), Aβ 14-29 The amino acid sequence of the polypeptide is: HQKLVFFAEDVGSNKGA (SEQID: 37).
[0170] Aβ 1-42 Monomer (Aβ monomer for short) preparation method: to 1mg Aβ 1-42 Add 1ml of hexafluoroisopropanol (HFIP) to the dry peptide powder, vortex for 1min, and sonicate in a water bath for 1-5min until completely dissolved; place in a shaking incubator at 37°C and 200rpm for 1.5h; use a vacuum rotary dryer to evaporate completely Hexafluoroisopropanol; to dry Aβ 1-42 Add 192 μl of anhydrous dimethyl sulfoxide (DMSO) to the polypeptide to...
Embodiment 2
[0187] Example 2 Preparation of anti-Aβ monoclonal hybridoma
[0188] 1. Immunization of BALB / c mice
[0189] Aβ 1-42 The polypeptide antigen and complete Freund's adjuvant were vortexed according to the dosage, and after complete emulsification, 6-week-old BALB / c female mice were immunized for the first time. Each mouse was intraperitoneally injected with 200 μg antigen, and 3 groups of mice were immunized with 5 mice in each group. Two weeks after the first immunization, the mice were immunized intraperitoneally for the second time, using Freund's incomplete adjuvant, and the dose of immunizing antigen was the same as the first immunization. Thereafter, the mice were intraperitoneally immunized twice a month with the same dose of adjuvant and antigen as the second immunization.
[0190] After the first immunization, a small amount of orbital blood was collected from the mice every six weeks and the serum titer was tested. After the serum titer was detected by indirect ELI...
Embodiment 3
[0229] Example 3 Detection of Anti-Aβ Monoclonal Antibody Inhibiting Aβ Polymerization
[0230] Dissolve Aβ dry powder to 1 mg / ml with 8.2% DMSO / DPBS solution (DMSO: sigma; DPBS: Hyclone), dilute Aβ solution to 33 μg / ml with DPBS, anti-Aβ monoclonal antibody 066-4.6.8, 066-4.18.2, 066-4.22.1, 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2 diluted to 450μg / ml (IC100), Dilute ThT(sigma) to 20 μM with ultrapure water. Take 50 μl of antibody dilution and add it to a 96-well black plate (corning), then add 50 μl of Aβ dilution to it, and finally add 100 μl of ThT, incubate at room temperature in the dark for 24 hours, and detect the fluorescence intensity with a multifunctional microplate reader (Ex / Em=440 / 485). The abscissa represents different sample addition groups, and the ordinate represents the relative fluorescence intensity. The results are shown in Figure 3. In Figure 3 (A), when the relative fluorescence intensity of the IgG group was 1.0, the rela...
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