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

Active Publication Date: 2007-01-03
深圳市安群生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the clinical application of a single tumor marker is mainly for

Method used

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  • Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit
  • Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit
  • Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention is one kind of human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnostic kit. The human tumor M2-type pyruvate kinase antigen determinant polypeptide has one of two polypeptide segments as shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence list. The human tumor M2-type pyruvate kinase antibody is prepared with the human tumor M2-type pyruvate kinase antigen determinant polypeptide of the present inventor. The human tumor M2-type pyruvate kinase antibody may be used in preparing extracorporeal diagnosis kit for human tumor M2-type pyruvate kinase.

Description

technical field [0001] The invention relates to a human tumor M2 type pyruvate kinase antigenic determinant polypeptide, a corresponding polyclonal antibody and the application thereof in preparing a human tumor M2 type pyruvate kinase in vitro diagnostic kit. Background technique [0002] At present, the incidence of cancer continues to increase, and it has developed into one of the three major clinical causes of death. The number of patients who die of cancer every year in the world is second only to cardiovascular patients. Problems related to cancer diagnosis and treatment have become a major focus of medical research in the past two decades. Modern science and technology have proved that the basic cure of malignant tumors such as liver cancer and lung cancer depends almost entirely on early detection, early diagnosis and early surgical treatment. The cure rate of early cancer can reach 80%. However, the early diagnosis of cancer is difficult, because on the one hand, ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/40C07K16/18A61K38/10A61K39/44A61P35/00G01N33/574
CPCY02P20/582
Inventor 朱建安
Owner 深圳市安群生物工程有限公司
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