Preparation of monoclone antibody of anti-HnRNPB1 antigen and uses thereof

A monoclonal antibody and antigen technology, applied in the field of immunity, to achieve the effects of safety assurance, convenient material collection, and increased sensitivity

Inactive Publication Date: 2009-07-29
欧平 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In summary, HnRNP B1 is closely related to lung cancer. If the transcription and translation process of normal cells it regulates loses normal control, it will lead to the occurrence of cancer.

Method used

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  • Preparation of monoclone antibody of anti-HnRNPB1 antigen and uses thereof
  • Preparation of monoclone antibody of anti-HnRNPB1 antigen and uses thereof
  • Preparation of monoclone antibody of anti-HnRNPB1 antigen and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining HnRNP B1 monoclonal antibody

[0027] 1) Procedure for immunizing mice with specific HnRNP B1 antigen

[0028] Soluble antigens have weak immunogenicity, and Freund's complete adjuvant is used in the immunization process. Mix the HnRNP B1 antigen and Freund's complete adjuvant in equal volumes, fully oscillate, mix and emulsify, and grind into a water-in-oil chylus (put a drop on the water surface and it is not easy to spread immediately, and the droplet shape indicates that the water-in-oil has been achieved. State), to fully mix the precipitated mycobacteria. The specific operation is as follows:

[0029] a. Initial immunization: HnRNP B1 antigen 1-50 μg plus Freund's complete adjuvant subcutaneously injected into mice at multiple points (generally 0.5-1ml, 0.1ml / point);

[0030] b. The second immunization: 1 week later, the dose was the same as above, and the mice were injected subcutaneously with Freund's incomplete adjuvant at multiple point...

Embodiment 2

[0084] Example 2: Purification of anti-HnRNP B1 monoclonal antibody by affinity chromatography with Protein G-Sepharose

[0085] 1) Gel preparation

[0086] a. Use Binding buffer (20mmol / L, PH 7.0PBS) to dissolve Protein G-Sepharose dry powder at a ratio of 3.5ml: 1g to form a gel and store aseptically at 4°C;

[0087] b. The gel returns to room temperature when used.

[0088] 2) Column packing

[0089] a. Use the Binding buffer to flush out the gas around the cylinder and the pores at the bottom, close the outlet when there is a little liquid in the outlet pipe, and keep 1 / 3 of the gel volume buffer in the chromatography column;

[0090] b. Pour the gel into the chromatography column along the glass rod to avoid air bubbles as much as possible;

[0091] c. Make up the remaining pores with Binding buffer, seal the inlet, connect the peristaltic pump, and start the pump (the speed should be controlled at 1-2ml / min, not too fast, less than 75cm / h);

[0092] d. Fully balance ...

Embodiment 3

[0100] Example 3: Anti-HnRNP B1 Monoclonal Antibody Titer Determination——Using ELISA Detection Method

[0101] Dilute the HnRNP B1 antigen with the coating solution (0.05mol / L, pH 9.6CB buffer) to a concentration of 10μg / ml and add it to the microplate, 100μl / well, and then incubate the microplate in a 37°C incubator for 2h , or put in a 4°C refrigerator overnight; take out the coated plate from the refrigerator, pour off the coating solution, pat dry the liquid in the wells with absorbent paper, add 1% BSA to each well, 120 μl / well, and incubate in a 37°C incubator for 2 hours Shake off the liquid in the well, wash with PBS-T 4 times, pat dry with absorbent paper, and store at 4°C; add 4× diluted anti-HnRNPB1 monoclonal antibody at different dilutions, 100 μl / well, and add PBS as a negative control ; Incubate in a 37°C incubator for 2h, then wash with PBS-T 4 times, 3min each time, and pat dry on absorbent paper; add goat anti-mouse-HRP secondary antibody (concentration recom...

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Abstract

The invention provides a preparation method of a monoclonal antibody specifically combined with HnRNP B1 antigenic determinant in early human lung cancer tissues and precancerous lesion tissues. Artificially synthesized 18-peptide and hemocyanin are coupled as an antigen immune Balb / c mouse, and the splenocyte thereof is then used and fused with mouse myeloma cells to prepare hybridoma; myeloma cell strains are sieved with functional detection; the myeloma cells can be used for preparing anti-HnRNP B1 monoclonal antibodies in large scale by an in vivo or in vitro method; the HnRNP B1 monoclonal antibodies are purified by a saturated ammonium sulfate precipitation method and Protein G-Sepharose affinity chromatography. The invention further provides the application of the antibody in a kit for detection of early human lung cancer.

Description

technical field [0001] The present invention relates to the technical field of immunization, in particular to the preparation of a monoclonal antibody, the monoclonal antibody prepared by the method is effective against the protein-heterogeneous ribonucleoprotein B1 (HnRNP) specifically expressed in early lung cancer tissue and precancerous lesion tissue cells. B1) The antigen has specific reactivity. Background technique [0002] At present, the morbidity and mortality of lung cancer rank first among cancers in the world. On average, one person dies of lung cancer every 30 seconds. Among 10 patients diagnosed with lung cancer, only one person can survive for more than 5 years. The World Health Organization (WHO) reported in 2000: In 1997, a total of 7.065 million people died of malignant tumors in the world, accounting for 12.6% of the death toll. Among them, lung cancer accounted for 19% of malignant tumor deaths, ranking first in the cause of malignant tumor death. In m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/18G01N33/577
Inventor 欧平李为民魏大鹏
Owner 欧平
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