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Antibody combination established aiming at tumor marker and ELISA method thereof

A tumor marker and marker technology, applied in anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, biological testing, etc., can solve the problem of low stability, high Production cost and other issues, to achieve the effect of low production cost, small size and strong activity

Inactive Publication Date: 2017-02-22
广州瑞博奥生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the key material in the antibody chip - the detection antibody is affected by the volume, low stability, high production cost and the difference between batches and the steric hindrance caused by its own large molecular weight, the antibody is used in the diagnostic field including the antibody chip. Applications are often limited

Method used

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  • Antibody combination established aiming at tumor marker and ELISA method thereof
  • Antibody combination established aiming at tumor marker and ELISA method thereof
  • Antibody combination established aiming at tumor marker and ELISA method thereof

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Embodiment 1

[0038] The invention discloses five better antibody analogs of the tumor marker S100B as S100B-A10, S100B-A12, S100B-B11, S100B-D10 and S100B-D12; the antibody analogs of the tumor marker GP73 are GP73-A5, GP73-B4, GP73-F6, and GP73-G4; antibody analogs of the tumor marker CK8 are CK8-F5 and CK8-12.

[0039] The preparation of antibody analogs, the process is as follows:

[0040] First, the biotin-labeled target protein was coated on a streptavidin-labeled magnetic bead microtiter plate, and then added phage (stock capacity 3×10 10 );

[0041] After 1 hour of reaction, unbound phages are still in the solution and can be removed by washing, and then the specifically bound phages are eluted;

[0042] The eluted phages are propagated and amplified after infecting the host cells, and the next round of elution is carried out;

[0043] In this way, after 3 to 5 rounds of "adsorption-elution-amplification", useful genes can be isolated from more than one million phage clones.

[...

Embodiment 2

[0053] For the reported antibody analogs, the present invention first studies their activity to identify whether they have better binding properties to the corresponding antigens.

[0054] The direct ELISA method is used, and the antibody analog needs to be biotinylated before the direct ELISA assay. Then coat the corresponding antigen gradient on the enzyme-linked plate, incubate overnight at 4°C and block at room temperature for 1.5 h; after blocking, add biotin-labeled antibody analogs and incubate at room temperature for 2 h; then add streptavidin Labeled horseradish peroxidase (streptavidin-HRP), incubate at room temperature for 45 min; finally add TMB to develop color, after the color development is completed, add stop solution to stop until yellow, and use a microplate reader to perform OD 450 According to the obtained data, observe whether there is a corresponding gradient decrease with the decrease of the antigen concentration, if so, it means that there is binding or...

Embodiment 3

[0057] The sandwich ELISA method screens the antibody pairs of the corresponding targets. The method of screening antibody pairs is sandwich ELISA:

[0058] Scheme 1: Coat the antibody analogue on a NUNC microwell plate, incubate and block, then add the target antigen and incubate for 1 hour;

[0059] Subsequent operations, such as phage ELISA, add antibody analogs isolated from phage, then add HRP-labeled anti-phage antibodies, and finally develop color through TMB. Antibody analogs that can be paired will react blue, while analogs that cannot be paired and the control group did not develop color.

[0060] Scheme 2: Use your own monoclonal antibody against the corresponding target as the coating antibody, coat it on the microtiter plate, and incubate overnight at 4°C;

[0061] After blocking with blocking solution at room temperature for 2 hours, add the corresponding target (antigen) in serial dilution, and incubate at room temperature for 2 hours; after washing, add the ab...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibody combination established aiming at a tumor marker and an ELISA method thereof. Compared with an antibody, an antibody analogue has the advantages of having good solubleness, good tissue penetration, good heat stability, good enzyme stability, low production cost and the like. Therefore, the invention discloses the antibody combination established aiming at the tumor marker, wherein the combination comprises stable and specific a pairing antibody analogue, and is expected to replace conventional antibody and be applied in a high-throughout technology (antibody chip) in screening antibody analogue libraries of various kinds to conduct development of antibody analogue bonded reagents.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody combination established for tumor markers and an ELISA method thereof. Background technique [0002] The most important features of malignant tumors are local invasion and distant metastasis, and they are the main cause of death. It is formed by the continuous proliferation and multiplication of a cell transformation, which can develop, infiltrate into local tissues, and metastasize to distant parts. This kind of tumor that has spread to other parts is already a highly malignant grade, and the possibility of its cure is already very small. At present, the diagnosis and treatment of tumors in developed countries are mostly in the early stage, and some corresponding tumor markers are mandatory items. Therefore, tumor-specific markers are the key to early diagnosis of tumors, personalized medicine, and tumor prediction and prevention. However, there are few specific markers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28G01N33/68G01N33/574
Inventor 黄若磐罗数红达伦·汤姆林森迈克尔·麦克菲尔森克里斯汀·蒂德吕志强
Owner 广州瑞博奥生物技术有限公司
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