Kit for detecting free colorectal cancer cell markers in blood
A colorectal cancer cell and kit technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as difficult acceptance by ordinary patients, inaccurate judgments, and inaccurate detection methods.
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Embodiment 1
[0030] Example 1 Preparation of Magnetic Beads
[0031]Preparation of antibody immunomagnetic beads: the antibodies used were anti-BerEP4 monoclonal antibody, anti-cytokeratin monoclonal antibody and anti-CK20 monoclonal antibody. This kit uses the above three antibodies to be labeled separately and then mixed for use.
[0032] Methods: Dynabeads Antibody Coupling Kit produced by Invitrogen was used. The labeling method was carried out in strict accordance with the manufacturer's instructions.
Embodiment 2
[0033] Example 2 Separation of colorectal cancer cells
[0034] 1. Sample collection:
[0035] Collect 5-7.5 ml of peripheral blood from patients with colorectal cancer, anticoagulate with EDTA, and use it within 4 hours (or within 48 hours after storage at 4 degrees).
[0036] 2. Select magnetic beads:
[0037] 2.1 Handling of magnetic beads:
[0038] · Mix the magnetic beads evenly, use a pipette to blow gently, and do not use a vortexer;
[0039] Use PBS buffer solution to wash, and do not touch the microspheres when washing;
[0040] · Pipette more magnetic beads than the required sample volume into a 1.5 ml centrifuge tube;
[0041] Put on the magnet for 1 minute, discard the supernatant;
[0042] · Wash 3 times with 1ml PBS to remove preservatives;
[0043] · Remove the supernatant;
[0044] · Aspirate 100ul microspheres containing PBS with the same volume as the original one.
[0045] 2.2 Selection of tumor cells
[0046] Put 5ml of peripheral blood into a 15...
Embodiment 3
[0065] Example 3 Detection of colorectal cancer cell tumor markers
[0066] 3.1 Kit preparation
[0067] · Keep the test tube at room temperature;
[0068] Put the RNase-free water in the kit at room temperature;
[0069] · Place the centrifuge tube above the tube containing the supernatant on ice;
[0070] · Prepare magnetic beads.
[0071] 3.2 Preparation of oligonucleotides
[0072] Take an appropriate amount of magnetic beads with oligonucleotide Oligo(dT) (it is recommended not to mix with a suspension apparatus, but to suspend by hand);
[0073] · Transfer to 1.5ml tube;
[0074] · Wash 2 times with Lysis / Binding Buffer.
[0075] 3.3 Binding mRNA to magnetic beads
[0076] Add 20 μl of magnetic beads to each cell lysis sample;
[0077] • Incubate for 10 minutes.
[0078] 3.4 Purification of mRNA
[0079] · Wash twice with buffer solution;
[0080] · Wash the magnetic beads twice with buffer;
[0081] · Wash again with 100 μl Tris-HCl buffer;
[0082] · Sus...
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