Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit
A technology of pyruvate kinase and epitope, which is applied in the field of human tumor M2 type pyruvate kinase epitope polypeptide, can solve the problems of inapplicability to early detection of tumors, and achieve the effect of strong antigenicity
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Embodiment 1
[0031] Example 1: Preparation of two M2-PK epitope polypeptide fragments.
[0032] The preparation method uses chemical synthesis method: using the American ABI431A automatic peptide synthesizer, the epitope peptide is synthesized by the solid phase method. The purity of the peptide was assessed by thin-layer chromatography and high-performance liquid chromatography, and the concentration of the antigenic peptide was determined.
[0033] The two polypeptide fragments are synthesized by a solid-phase method. The main idea of solid-phase peptide synthesis is: first connect the carboxyl group of the carboxyl-terminal amino acid of the peptide chain to be synthesized with an insoluble polymer compound (resin) in the form of a covalent bond, and then combine it with the amino acid on the solid-phase carrier. As an amino component, the peptide chain is extended by removing the amino protecting group and reacting with excess activated carboxyl components. Such steps can be repeat...
Embodiment 2
[0088] Example 2: The M2-PK epitope polypeptide fragment obtained in Example 1 was linked to a carrier protein and used as an antigen to immunize animals to prepare specific polyclonal antibodies.
[0089] (-) M2-PK epitope polypeptide fragment 1 polyclonal antibody preparation:
[0090] 1. Antigen preparation: M2-PK polypeptide was linked with carrier protein KLH (hemocyanin) by carbodiimide method to prepare antigen.
[0091] 2. Preparation of antiserum from animals: Take the antigen and 1ml of Freund's complete adjuvant (basic immunization) or Freund's incomplete adjuvant (boost immunization) and fully emulsify it to immunize Balb / c mice, inject intraperitoneally, and each dose is 50- 200μg / ml, intradermally injected at 20 points, and immunized 7-8 times in total. The tenth day after the last immunization, ear blood was taken to measure the antibody titer.
[0092] 3. Determination of antibody titer: The antibody titer was determined by ELISA method, and the result showed ...
Embodiment 3
[0104] Example 3: Preparation of M2-PK ELISA kit.
[0105] The first and second steps of preparing the kit are to prepare M2-PK epitope polypeptide fragments and polyclonal antibodies, and the third step is to use the above-prepared M2-PK polyclonal antibodies for the preparation of diagnostic kits. In this example, the two The fragment 1 polyclonal antibody in the M2-PK polyclonal antibody was used as the coating antibody in the preparation box, and the fragment 2 polyclonal antibody was used as the binding antibody.
[0106] The preparation and operation of the M2-PK ELISA kit are as follows:
[0107] 1. Preparation of various buffers and reagents:
[0108] A. Coating buffer: 0.050M, CB (carbonate buffer) at pH 9.6
[0109] Na 2 CO 3 (MW 105.99) 16.0 grams
[0110] NaHCO 3 (MW 84.01) 29.0 grams
[0111] Dissolve in distilled water to 1000ml.
[0112] B. Sample / wash buffer: PBS-Tween 20 at pH 7.2
[0113] Na2HPO4.12H2O 58g
[0114] KH2PO4 4 g
[0115] NaCl 100g
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