Monoclone antibody against HnRNP A2/B1 antigen, its preparation and use
A monoclonal antibody, antibody technology, applied in the biological field, can solve problems such as restricted expression
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Embodiment 1
[0037] Embodiment 1 obtains monoclonal antibody
[0038] 1) Immunize mice and obtain spleen cell stock solution
[0039] The human lung cancer cell line GLC82 was intraperitoneally injected with 20 million cells into Balb / C mice, and then injected with the same number of cells for booster immunization 4 times. 24 hours after the last injection, separate the spleen of the mouse and prepare the spleen cell stock solution: cut the abdominal cavity of the mouse in an ultra-clean bench, take out the spleen, and place it in a sieve in a plate containing 20ml of serum-free RPMI-1640 culture solution . Use two elbow droppers, one to fix the spleen, the other to scrape the spleen, squeeze out the splenocytes to make a splenocyte suspension, transfer it into a centrifuge tube (generally counting more than 200 million is better), and centrifuge at 1200rpm for 10 minutes. Aspirate the supernatant, add 10 ml of serum-free RPMI-1640 medium (Hyclone) to suspend the cells, and centrifuge at...
Embodiment 2
[0049] The identification of embodiment 2 monoclonal antibody type
[0050] The type and subtype of the monoclonal antibody obtained in Example 1 were identified.
[0051] The ImmunoPure® Monoclonal antibody isotyping kit from Pierce was used to detect the supernatant of the CGMCC0854 hybridoma cell culture, and it was found that the monoclonal antibody belonged to the IgG1 / κ chain type.
Embodiment 3
[0052] Example 3 Immunohistochemical Identification Test
[0053]Paraffin sections were dewaxed in xylene and dehydrated in alcohol. This was followed by incubation with 3% hydrogen peroxide-methanol for 20 minutes. Rinse with PBS buffer, then microwave repair in pH 6.0 citrate buffer, and rinse with PBS. The ascites to be tested was diluted with three dilution ratios of 1:100; 1:1000; 1:10000, and the diluted ascites was dropped on the slices. Incubate at 37°C for 1 hour. Anti-IgG polyperoxidase was added dropwise and incubated at 37°C for 20 minutes, then rinsed with PBS. Afterwards, DAB was used for color development, counterstained with hematoxylin, transparent, and neutral gum to seal the slides. Observe under a microscope and make a judgment on the result. Take photos of positive results. The results are shown in Figure 1. The results showed that "HnRNP A2 / B1" monoclonal antibody had obvious intracytoplasmic antigen distribution in cancer tissues, but had no speci...
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