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Preparation method and application of anti-human leukemia inhibitory factor monoclonal antibody

A monoclonal antibody and inhibitory factor technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, antibodies, etc., to achieve enhanced therapeutic effect, improved therapeutic effect, and strong affinity

Active Publication Date: 2021-08-10
BEIJING HAOGUYUANFANG BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, screening monoclonal antibodies for disease treatment is a very complex process

Method used

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  • Preparation method and application of anti-human leukemia inhibitory factor monoclonal antibody
  • Preparation method and application of anti-human leukemia inhibitory factor monoclonal antibody
  • Preparation method and application of anti-human leukemia inhibitory factor monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation method of recombinant human leukemia inhibitory factor is:

[0053] S1: Mix Tris-HCL, NaCl, and EDTA, and the concentrations of the three in the mixture reach 20mM, 60mM, and 1mM respectively, then add Triton X-100 to make the concentration reach 0.5%, and obtain a lysate for later use.

[0054] S2: Inoculate Escherichia coli containing the expression vector of human leukemia inhibitory factor into LB medium, culture overnight at 37°C and 200 rpm, then inoculate in freshly prepared LB medium at a volume ratio of 1:50, and incubate at 37°C 200 rpm shaking culture until the bacterial OD600nm detection result reaches 0.6, add IPTG with a final concentration of 0.5mM to induce, then reduce the temperature of the bacterial culture solution to 16°C, and change the bacterial culture temperature to 16°C at the same time, continue for 200 Harvest the bacteria after shaking and culturing for 20 hours; after weighing the harvested bacteria, add the lysate according...

Embodiment 2

[0059] The immunization method of experimental animals is as follows:

[0060] Apply 80 μg of recombinant human leukemia inhibitory factor protein mixed with an equal volume of complete Freund's adjuvant to immunize 8-week-old female BALB / c mice. After the first immunization, boost immunization every 2 weeks. After a total of 3 immunizations, apply ELISA The method detects that the titer of mouse serum reaches 1:10 5 At that time, the mouse with the highest serum titer was selected, and the mice were given a booster immunization once. After 3 days, the mice were sacrificed, and the spleen was aseptically removed and ground on a plate to make a spleen single cell suspension for later use.

Embodiment 3

[0062] The preparation method of hybridoma cells is as follows:

[0063] Mouse SP2 / 0 myeloma cells were revived while immunizing mice. For cell fusion, take the SP2 / 0 myeloma cells in the logarithmic growth phase, wash them twice with incomplete RPMI-1640 medium, resuspend and induce fusion with the above-mentioned spleen single cell suspension with 50% PEG4000. Selective culture was carried out in RPMI1640 medium for 1 week, the supernatant was drawn, and the antibody secretion was detected by indirect ELISA method. 2 μg / mL antigen was coated with 100 μL / well of the volume (human leukemia inhibitory factor protein) on a 96-well plate, and the washing solution After washing three times, add 150 μL / well blocking solution to block at room temperature for 2 hours, and pat dry after washing. Add the supernatant of the cells to be tested, dilute to 8 concentrations according to a 2-fold gradient, incubate at 37°C for 1 hour, wash the plate and pat dry, take horseradish peroxidase-...

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Abstract

The invention relates to the field of biotechnology, in particular to a preparation method and application of an anti-human leukemia inhibitory factor monoclonal antibody. The present invention provides a mouse hybridoma cell whose preservation number is CGMCC No.19161, and provides a monoclonal antibody produced by the mouse hybridoma cell whose preservation number is CGMCC No.19161. Human leukemia inhibitory factor monoclonal antibody has good biological activity, can effectively bind human leukemia inhibitory factor, block the binding of human leukemia inhibitory factor and its receptor, and has obvious inhibitory effect on the growth of tumor cells transplanted in animals. Or used in combination with other antitumor drugs in the treatment of tumors, and has the application prospect of preparing various tumor drugs. At the same time, the preparation method can greatly improve the screening efficiency for obtaining antibodies with high therapeutic value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of an anti-human leukemia inhibitory factor monoclonal antibody. Background technique [0002] Human leukemia inhibitory factor (Leukemia Inhibitory Factor, LIF) is a multifunctional cellular inflammatory factor with a wide range of biological activities secreted by the body, which can be produced by various tissues. LIF consists of 180 amino acids and belongs to the IL-6 cytokine family. Because LIF was originally found to act as a differentiation inducer and a proliferation inhibitor of the mouse myeloid leukemia cell line M1 macrophages, it was named leukemia inhibitory factor. [0003] In recent years, studies have found that the increase of LIF expression level is closely related to the occurrence and development of various malignant tumors. LIF can promote tumor cell proliferation, epithelial-mesenchymal transition and tumor cell metastasis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N5/20G01N33/68G01N33/577A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/244C07K2317/73C07K2317/76G01N33/577G01N33/6869G01N2333/5415
Inventor 杜伯雨宋伟赵宏吴帆樊旭王艳红郗雪艳
Owner BEIJING HAOGUYUANFANG BIOMEDICAL TECH CO LTD
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