Fluoroquinolone medicine cluster specific immunoaffinity chromatography glue and preparation method thereof
A technology of fluoroquinolones and chromatographic chromatography, which is applied in the field of fluoroquinolone drug cluster-specific immunoaffinity chromatography gel and its preparation, can solve the problems of few preparation reports, limited detection and strong technical support, and achieve The effect of high sensitivity and strong versatility
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[0061] In the preparation of the immunoaffinity chromatography gel / column of the present invention, the required high-purity monoclonal antibody protein can be obtained in the following manner:
[0062] Hybridoma cell line - 11F10 induced ascites in vivo was used as a sample, purified by protein G resin to obtain the eluted part of the collection, each added 1 / 10 volume of 1M Tris-Cl (pH8.5) to neutralize, and the protein was provided by Beyontian Company The protein concentration determined by the content detection kit is greater than or equal to 2mg / ml and the purity of the antibody identified by SDS-PAGE electrophoresis is greater than or equal to 99% (there are only two protein bands of 50kd and 25kd in the protein electrophoresis map) as qualified monoclonal antibody protein pure products, and the above qualified Collect liquid samples, reverse dialysis with 0.01M PBS (pH 7.4) for 24 hours, and then use 0.1M NaHCO 3 / 0.5M NaCl (pH 8.3) reverse dialysis for 24h. The sample...
Embodiment 1
[0066] Example 1: Acquisition of ascites samples containing monoclonal antibodies according to the present invention
[0067] 1. Recovery and proliferation of hybridoma cell line -11F10
[0068] Take the cryopreservation tube of the hybridoma cell line-11F10 from the liquid nitrogen tank, melt it rapidly in a water bath at 37°C, centrifuge at 600r / min for 5min, discard the supernatant, add 15% FBS / RPMI-1640 culture medium to suspend the cells, and supplement each After adding the above-mentioned culture solution to 5ml, plant it in a 50ml cell culture bottle and culture it in a carbon dioxide incubator. After the cells grow to a density of 30%, the medium is replaced halfway. Passage once every 2-3 days according to 1:3-4.
[0069] 2. In vivo induction of monoclonal antibody protein and ascites acquisition
[0070] 7-10 days before planting hybridoma cells in vivo, 12 male BALB / c mice aged 8-10 weeks were injected intraperitoneally with 0.5ml / mouse of pristane, and carefully...
Embodiment 2
[0074] Embodiment 2: Purification of the monoclonal antibody protein of the present invention
[0075] The ascites was diluted 4× with 0.01M PBS (pH 8.0) pre-cooled in an ice-water bath and used as a preparation solution for later use. Take 1ml of Protein G Resin for every 50mg of total protein in the ascites, fill the gel in a small column for chromatography (10ml), equilibrate the column with pre-cooled 0.01M PBS (pH 8.0) of 50× column bed volume, and keep the column at room temperature The preparation solution was filtered five times through the Protein G Resin column (natural flow rate), so that the monoclonal antibody protein in the ascites could fully bind to the Protein G Resin specifically, and then pre-cooled 0.01M PBS (pH 8.0) with 50× column bed volume to fully Wash the column (natural flow rate) to remove foreign proteins stuck in the column.
[0076] Add 100 μl 1M Tris-Cl (pH 9.6) to each 1.5ml EP tube in advance, add ice water to pre-cool 0.1M Glycine-HCl (pH 2....
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