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Anti-human B7-H1 monoclonal antibody preparation method and application of antibody to immunohistochemical detection

A B7-H1, monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, immunoglobulin, etc., to improve the diagnostic accuracy , the effect of improving the accuracy

Inactive Publication Date: 2018-05-29
SUZHOU BRIGHT SCISTAR BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of clinical trials have raised new questions for immunotherapy at the control point: patients need to detect the expression of PD-1 and PD-L1 and the evaluation criteria for determining the expression level before medication
At present, there is no diagnostic reagent in China with high sensitivity, good specificity and the same detection effect as the two PD-L1 companion antibodies approved by the FDA. Appropriate and predictive patient response to specific drugs is critical

Method used

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  • Anti-human B7-H1 monoclonal antibody preparation method and application of antibody to immunohistochemical detection
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  • Anti-human B7-H1 monoclonal antibody preparation method and application of antibody to immunohistochemical detection

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Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Obtaining of Hybridomas Producing B7-H1 Monoclonal Antibody

[0046] (1) Immune mice

[0047] Mice were immunized with fusion protein or transgenic cells, immunized four times with an interval of 21 days each time, and the orbital blood titer of the mice was measured 7-10 days after the fourth immunization.

[0048] (2) Cell culture

[0049] 1. One day before fusion, take one BALB / c mouse aged 6-7 weeks and place it in 75% ethanol solution for 2 minutes.

[0050] 2. The mouse spleen was aseptically taken out, placed in a 200-mesh stainless steel screen, and ground to obtain a single cell suspension. Wash twice with 1640 basal medium (1400rpm, 5min) for later use. Use 15% FBS 1640 medium to adjust the cell concentration to 2×10 5 / ml, drop into 96-well culture plate, 100μL per well, 37℃, 5%CO 2 cultured in an incubator.

[0051] 3. Cultivate overnight, and observe under a low-power microscope the next day. And spread subcloned cells 150ul.

[0052] (3) ...

Embodiment 2

[0066] Example 2 Sequencing of B7-H1 Antibody Heavy and Light Chain Variable Regions

[0067] 1. Extract the cDNA of hybridoma cells: extract RNA from the hybridoma cell line, and use RT-PCR technology to reverse-transcribe the obtained RNA into cDNA; use specially designed upstream and downstream primers to PCR clone the hybridoma cell chain variable region (mVH) and light chain variable region (mVL);

[0068] 2. mVH and mVL were respectively connected to the cloning vector (pJET cloning vector), and the connection product was transformed into competent bacteria DH5a. Since the pJET vector carries the ampicillin (Amp+) resistance gene, the transformed bacteria can be applied to the Amp-resistant LB solid culture medium, 37 ° overnight culture;

[0069] 3. The bacteria to be plated grow scattered colonies, select colonies with clear edges and good growth, and further sequence and identify them;

[0070] 4. According to the sequencing results, the candidate heavy and light ch...

Embodiment 3

[0079] Example 3 Karyotype Analysis of Hybridoma Cells

[0080]Take well-grown cells and add colchicine to make the final concentration 0.04~0.08μl / ml. After culturing for 2 hours, collect the cells (1000rpm, 10min) in a centrifuge tube, add 0.5ml of 37℃ pre-warmed 0.075mol / L KCl solution drop by drop, then add 5~10ml, blow gently with a pipette evenly, 37℃ Incubate for 20min. Add 1ml of freshly prepared fixative solution (3 parts of methanol plus 1 part of glacial acetic acid, prepared before use) to the tube, centrifuge at 1000rpm for 10min and discard the supernatant. Then add 8~10ml of fixative, pipette evenly, fix for 15~20min, centrifuge, and discard the supernatant. Then add 5ml of fixative and fix for 30min. Discard the supernatant by centrifugation, then add 1.5ml fixative, and pipette evenly. Take a glass slide frozen at -10°C, add 1-2 drops of cell suspension, and stain with fresh Giemsa solution (1 part of Giemsa stock solution plus 9 parts of 0.075mol / L, pH6.8...

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Abstract

The invention discloses an anti-human B7-H1 (PD-L1) monoclonal antibody, an application of the antibody to immunohistochemical detection and an antibody heavy chain and light chain variable region identification method, and relates to an anti-human B7-H1 monoclonal antibody preparation method and an application of the monoclonal antibody to immunohistochemical sample detection and tumor tissue B7-H1 analysis. A heavy chain variable region (mVH) and a light chain variable region (mVL) are extracted from hybridoma cells secreting the anti-human B7-H1 monoclonal antibody, and B7-H1 heavy and light chain variable sequences are verified by sequencing. A eukaryotic cell strain expression antibody is implemented on the basis, the obtained antibody has good binding capacity according to verification, specific staining for tumor specimens is performed by immunologic tissue chemical detection, and the staining effects and specificity of the self-developed B7-H1 monoclonal antibody and a commercial B7-H1 antibody for the same tissue specimen are compared.

Description

technical field [0001] The invention relates to the detection application of antibody immunohistochemistry, and the verification of the sequence of the antibody heavy chain variable region and light chain variable region, belonging to the field of biotechnology, in particular to a preparation method of anti-human B7-H1 monoclonal antibody and its Application in immunohistochemical detection. Background technique [0002] The tumor microenvironment is an important location for the interaction between tumor cells and the host immune system. In-depth understanding of immune cells in the microenvironment and the mechanism of tumor immunity is the key to improving the body's tumor immunity and establishing effective cancer immunotherapy. The microenvironment formed by tumor cells and the human immune system promotes tumor immune escape, which eventually leads to tumor recurrence and metastasis. A group of negative co-stimulatory molecules involved in the downregulation and susp...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/70G01N33/577
CPCC07K16/2827C07K2317/51C12N15/70G01N33/577
Inventor 张学光
Owner SUZHOU BRIGHT SCISTAR BIOTECH CO LTD
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