Antibody fragment of DNA polymerase, antibody and application thereof
A technology of antibody fragments and polymerases, applied in the fields of application, anti-enzyme immunoglobulin, biochemical equipment and methods, etc., can solve problems such as complicated use process and application limitations, and achieve high affinity effect
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Embodiment 2
[0085] Example 2 cell fusion
[0086] 1. Preparation of Myeloma Cells
[0087] In this example, SP2 / 0Ag14 myeloma cells were used. The cells were cultured to a good growth state and good shape. Before fusion, the cells were cultured to the logarithmic growth phase, and suspended to prepare a suspension with a certain cell density.
[0088] 2. Preparation of Splenocytes
[0089] Several mice with the highest titers were selected and sacrificed. Fix the dissecting board, take out the spleen, put it in fresh medium, remove the adhesive tissue, grind the spleen, filter it with a 200-mesh drying net, add red blood cell lysate to lyse for 10 minutes after washing, add fresh medium to dilute, centrifuge to remove the supernatant, and re- Dissolve in the medium, measure the cell density, and dilute the cells to a certain concentration.
[0090] 3. Cell Fusion
[0091] Mix the above SP2 / 0Ag14 cells and splenocytes at a ratio of 1:10, then add polyethylene glycol 2000 (PEG2000) for ...
Embodiment 3
[0092] Embodiment 3 hybridoma subcloning screening
[0093] After the hybridoma cells in the 96-well plate in Example 2 were cultured for 2 weeks, the supernatant was taken for ELISA to identify the positive value of the antibody in the supernatant. Positive cell lines were screened for the second round of limited dilution subcloning. ELISA was then used to identify and screen positive cell lines. The ELISA screening process is as follows:
[0094] (1) Coat 100 μl of antigen (DNA polymerase concentration 1 μg / ml) on the enzyme strip overnight. Add 200 μl PBST and shake for 10 min, wash 3 times, and wash with PBS 3 times.
[0095] (2) Add 200 μl of 1% BSA-PBS and block at 37° C. for 2 hours. PBST 200μl, 10min, wash 3 times, PBS 200μl, 10min, wash 3 times.
[0096] (3) Add 100 μl of the diluted supernatant, and incubate at 37° C. for 2 hours. PBST 200μl, 10min, wash 3 times, PBS 200μl, 10min, wash 3 times.
[0097] (4) Add 100 μl of diluted commercial horseradish peroxida...
Embodiment 4
[0104] Example 4 Monoclonal Antibody Preparation and Neutralizing Activity Screening
[0105] The above-mentioned positive hybridoma cell lines were expanded and cultured. The supernatant was collected, centrifuged at 8000rpm for 15min, filtered through a 0.22μm filter membrane, purified with a protein A (protein A) column, and eluted with an eluent to obtain a purified monoclonal antibody. Monoclonal antibodies were screened using polymerase polymerization activity assays. Finally, the monoclonal antibody capable of inhibiting the polymerization activity of DNA polymerase was obtained through screening. The screening experiment of monoclonal antibody inhibiting polymerase activity is as follows:
[0106] (1) Mix the monoclonal antibody and DNA polymerase at a molecular ratio of 1:1, and incubate at room temperature for 2 hours.
[0107] (2) react according to the system of table 3:
[0108] table 3
[0109]
[0110] React at 37°C for 2 hours.
[0111] The double-stra...
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