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

A technology of monoclonal antibodies and inhibitors, applied in biochemical equipment and methods, anti-animal/human immunoglobulins, antibodies, etc.

Active Publication Date: 2020-07-07
BEIJING HAOGUYUANFANG BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, screening monoclonal antibodies for

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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

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Experimental program
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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 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 inoculate 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; weigh the harvested bacteria, add lysate at a volume ratio of 1:10 a...

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 boosted once. After 3 days, the mice were sacrificed, and the spleen was aseptically taken and ground on a plate to make a spleen single-cell suspension for subsequent 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. Select and culture in RPMI1640 medium for 1 week, draw the supernatant, and use indirect ELISA method to detect antibody secretion, coat 96-well plate with 2 μg / mL antigen according to the volume (human leukemia inhibitory factor protein) 100 μL / well, wash 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-labeled goat anti-mouse IgG as the sec...

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Abstract

The present invention relates to the technical field of biology, and particularly relates to a preparation method and application of an anti-human leukemia inhibitory factor monoclonal antibody. The present invention provides a mouse hybridoma cell with a collection number of CGMCC No.19161, and provides a monoclonal antibody generated by the mouse hybridoma cell with the collection number of CGMCC No.19161. The anti-human leukemia inhibitory factor monoclonal antibody has the high biological activity, can be effectively combined with a human leukemia inhibitory factor, closes combination of the human leukemia inhibitory factor and a receptor thereof, has the obvious inhibition effect for growth of tumor cells transplanted in an animal body, can be applied to tumor treatment separately orin combination with other anti-tumor drugs, and has the application prospect of preparing various tumor drugs. Meanwhile, the preparation method can greatly improve the screening efficiency of obtaining the antibody with the 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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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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