Soluble B7-H4 quantitative detection kit and application thereof

A B7-H4, quantitative detection technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of lack of detection methods

Pending Publication Date: 2021-04-20
SUZHOU BRIGHT SCISTAR ANTIBODY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, due to the lack of effective detection methods, there have been no research reports on soluble B7-H4 (sB7-H4) at home and abroad

Method used

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  • Soluble B7-H4 quantitative detection kit and application thereof
  • Soluble B7-H4 quantitative detection kit and application thereof
  • Soluble B7-H4 quantitative detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of anti-human B7-H4 monoclonal antibody

[0038] (1) Reagents and materials: RPMI1640 medium (Gibco, USA) containing 10% calf serum (FCS, Hyclone, USA); HAT, HT selection medium (Sigma, USA); Freund's adjuvant (Freund'adjuvant , Sigma, USA); cell fusion agent polyethylene glycol (PEG1500); Protein G affinity layer suction column (Pharmacia, Sweden); Pristane (Sigma, USA). Cell culture flasks, plates (Nunc, Denmark); CO 2 Incubator, centrifuge (Jouan, France), inverted microscope (O1ympus, Japan), flow cytometer (Coulter, USA); human B7-H4 gene cell line CHO / B7-H4 (constructed by myself). All cell lines were tested and found no mycoplasma contamination; 6-8 week-old female BALB / c mice were from Shanghai Experimental Animal Center.

[0039] (2) Cell culture: Using RPMI1640 medium containing 10% FCS, CHO / B7-H4 and CHO / Mock gene transfected cell lines are regularly pressurized with the resistance screening drug Zeocin (500 μg / ml) to maintain the tar...

Embodiment 2

[0049] Example 2: Identification of biological characteristics of anti-human B7-H4 monoclonal antibodies 8D4 and 7E1

[0050] (1) Subtype and sequence identification of the monoclonal antibody: Take hybridoma cell culture supernatant and dilute at 1:50 and ascitic fluid at 1:20000. Take 0.2ml of the diluent and add it to a small test tube containing the subclass qualitative reagent, place it at room temperature for 1min, wait for it to dissolve naturally, mix gently, gently insert the subclass qualitative test strip into the tube, after 5-10min, the test paper The blue protein band corresponding to the name of the mAb heavy chain appears on one side, which is the mouse Ig subclass to which the antibody belongs; while the blue protein band corresponding to the name of the mAb light chain appears on the other side. That is, the light chain type of the mAb; the monoclonal antibody was sequenced, and the heavy chain of the mouse anti-human B7-H4 monoclonal antibody 8D4 was determi...

Embodiment 3

[0063] Example 3: A kit capable of quantitatively detecting human soluble B7-H4

[0064] (1) Kit composition:

[0065] 1) Coating antibody: mouse anti-human B7-H4 monoclonal antibody (8D4), 30 μg / tube, 1 tube;

[0066] 2) Standard protein: B7-H4Ig protein (R&D), 25ng / tube, 1 tube;

[0067] 3) Detection antibody: mouse anti-human B7-H4 monoclonal antibody (Biotin-7E1), 10 μg / tube, 1 tube;

[0068] 4) Streptavidin-HRP (Sigma-Aldrich), 1 μl / / tube, 1 tube;

[0069] 5) TMB (Sigma-Aldrich), 10ml;

[0070] 6) BSA (Shanghai Sangong), 2g / 100ml, 30ml;

[0071] 7) ELISA plate (Costar), 1 piece;

[0072] 8) Lotion: 10×PBS, 100ml; Tween-20, 0.5ml;

[0073] 9) Stop solution: 2M H 2 SO4, 5ml;

[0074] (2) See Table 1 for storage conditions:

[0075] Table 1

[0076]

[0077]

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Abstract

The invention discloses a coated antibody which is a mouse anti-human B7-H4 monoclonal antibody 8D4, the heavy chain of the coated antibody is an amino acid sequence shown as SEQ ID NO: 1, and the light chain of the coated antibody is an amino acid sequence shown as SEQ ID NO: 2; a detection antibody is a mouse anti-human B7-H4 monoclonal antibody 7E1, the heavy chain of the detection antibody is an amino acid sequence shown as SEQ ID NO: 3, and the light chain of the detection antibody is an amino acid sequence shown as SEQ ID NO: 4. The expression level of soluble B7-H4 protein in human serum, plasma and tissue fluid can be quantitatively analyzed.

Description

technical field [0001] The invention relates to the field of quantitative detection, in particular to a soluble B7-H4 quantitative detection kit and application thereof. Background technique [0002] In 1970, it was first proposed and confirmed by Brestcher and Cohn on the basis of the dual signal theory of T cell activation and the molecular function of CD28 / B7 that the activation of T lymphocytes requires dual signals, that is, T cells obtain the first signal through the combination of TCR and MHC-antigen peptide. Signals need to obtain stimulation other than antigenic signals, that is, second signals or co-stimulatory signals. Molecules that mediate the second signal are collectively referred to as co-stimulatory molecules or co-stimulatory molecules. Studies have shown that co-stimulatory molecules and their regulatory networks play an extremely important regulatory role in the effective initiation, moderate effect and timely suspension of immune responses. With the co...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/543
CPCY02A50/30
Inventor 张学光丁思思
Owner SUZHOU BRIGHT SCISTAR ANTIBODY BIOTECHNOLOGY CO LTD
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