Monoclonal antibody against amyloid beta protein and use thereof
A technology of monoclonal antibody and β protein, applied in the direction of antibody, anti-animal/human immunoglobulin, immunoglobulin, etc.
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Embodiment I
[0174] Production of monoclonal antibodies 8F5 and 8C5
[0175] Balb / c mice were subjected to sub-q with 50 micrograms of Aβ(1-42) globulomers (in CFA (Sigma)) as described in Barghorn et al., 2005, J Neurochem, 95, 834-847. Immunized and boosted twice at one-month intervals. The spleen was collected, and the splenocytes were fused with mouse myeloma SP2 / 0 cells at a ratio of 5:1 by PEG procedure. Fused cells were 2x10 5 Cells / ml, 200ml / well were inoculated in the diazoserine / hypoxanthine selection medium in 96-well plates. Cells were allowed to grow to form visible colonies and supernatants were assayed for A[beta] oligomer reactivity by direct ELISA assay. Anti-Aβ oligomer antibody-secreting hybridomas were subcloned by limiting dilution until antibody expression stabilized.
Embodiment II
[0177] 8F5 and 8C5 bind A beta (1-40) and A beta Globulomers of (1-42) are preferred over the monomers that bind them products
[0178] To test the selectivity of 8F5, two different solubilized Aβ(1-42) monomer preparations were used, and freshly prepared Aβ(1-40) was used as a surrogate for the monomer. Two types of experiments were performed. In the first experiment, globulomer derived but conformer non-specific MAb6G1 was derivatized by sandwich ELISA (see S. Barghorn et al. J. Neurochemistry, 95:834 (2005) ) as a capture antibody to test the specificity of 8F5 for Aβ globulomers. Biotinylated 8F5 was used as a secondary and conformer selective antibody. This experiment is described in Example 2.1 below.
[0179] In a second experiment (described in Example 2.2 below), oligomer selectivity for A[beta](1-42) monomers and for A[beta](1-40) monomers was analyzed by dot blot immunoassay. In this experiment, 8F5 was shown to be resistant to Aβ(1-42) globulomers (comp...
Embodiment 21
[0180] Example 2.1: Oligomer selectivity of monoclonal antibodies 8F5 and 8C5
[0181] a) Preparation of Aβ(1-42) globulomers:
[0182] 9 mg Aβ(1-42)Fa.Bachem were dissolved in 1.5 ml HFIP (1.1.1.3.3.3 hexafluoro-2-propanol) and incubated at 37°C for 1,5 h. The resulting solution was evaporated in a SpeedVac and suspended in 396 [mu]l DMSO (5 mM A[beta] stock solution). The samples were sonicated for 20 seconds in an ultrasonic water bath, shaken for 10 minutes, and stored overnight at -20°C.
[0183] Dilute the sample with 4.5ml PBS (20mM NaH 2 PO 4 ; 140mM NaCl; pH7, 4) was diluted, and 0.5ml of 2% SDS aqueous solution (0.2% SDS content) was added. The resulting mixture was incubated at 37 °C for 7 h with 16 ml H 2 O diluted and incubated for another 16 hours at 37 °C. Then, the Aβ(1-42) globulomer solution was centrifuged at 3000 g for 20 minutes. The supernatant was concentrated to 0.5ml with 30K Dacentriprep. The concentrate was heated to 5 mM NaH at 6 °C 2 PO ...
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