Human soluble B7-DC quantitative detection kit

A B7-DC, mouse anti-human technology, applied in the field of white system, can solve problems such as lack of detection methods, and achieve the effect of good specificity and good sensitivity

Active Publication Date: 2011-11-23
苏州市第五人民医院 +1
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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-DC (sB7-DC) at home and abroad.

Method used

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  • Human soluble B7-DC quantitative detection kit
  • Human soluble B7-DC quantitative detection kit
  • Human soluble B7-DC quantitative detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Reagents and materials: Calf serum Hyclone company (USA); add calf serum 100ml, L-glutamine 0.15g, NaHCO 3 2.0g, sodium pyruvate 0.11g, glucose 3.6g, HEPES 4.766g, 2-mercaptoethanol 10.0ml; HAT, HT selection medium Use 50 times concentration of HAT, HT selection medium (Sigma, USA), use RPMI1640 or DMEM complete medium was diluted to the working concentration; Freund'adjuvant (Freund'adjuvant, Sigma, USA); cell fusion agent polyethylene glycol (PEG1500); Protein G affinity layer suction column (Pharmacia, Sweden); Pristane (Sigma ,United States). Cell culture flasks and plates (Nunc, Denmark); CO2 incubator, centrifuge (Jouan, France), inverted microscope (O1ympus, Japan), flow cytometry (Coulter, USA); human B7-DC gene cell line L929 / B7-DC (built by me). All cell lines were tested and found no mycoplasma contamination; 6-8 weeks old female Balb / c mice (Shanghai Experimental Animal Center).

[0042] (2) Cultivation of cell lines: Using RPMI1640 medium conta...

Embodiment 2

[0061] A kit capable of quantitatively detecting soluble B7-DC, comprising the following components:

[0062] (1) Coating antibody: mouse anti-human B7-DC monoclonal antibody (8F2), 30μg / tube, 1 tube;

[0063] (2) Standard protein: B7-DC Ig protein (R&D), 25ng / tube, 1 tube;

[0064] (3) Detection antibody: mouse anti-human B7-DC monoclonal antibody (Biotin-10D6), 10 μg / tube, 1 tube;

[0065] (4) Streptavidin-HRP (Sigma-Aldrich), 1μl / / tube, 1 tube;

[0066] (5) TMB (Sigma-Aldrich), 10ml;

[0067] (6) BSA (Shanghai Sangong), 2g / 100ml, 30ml;

[0068] (7) ELISA plate (Costar), 1 piece;

[0069] (8) Washing solution: 10×PBS, 100 ml; Tween-20, 0.5 ml;

[0070] (9) Stop solution: 2M H 2 SO 4 , 5 ml;

[0071] Storage conditions:

[0072]

Embodiment 3

[0074] Analysis of the specificity of the human soluble B7-DC ELISA kit described in Example 2:

[0075]B7.1Ig, OX40Ig, B7-DCIg and B7-H4Ig were serially diluted to different concentrations. Anti-human B7-DC monoclonal antibody (8F2) was adjusted to 3 μg / ml with carbonate buffer (0.01M CBS, pH9.3) to coat the ELISA detection plate, overnight at 4°C. Wash 3 times with PBS (containing 0.1% Tween 20), and block with 2% BSA for 1 h at room temperature. After washing with PBS for 3 times, the above-mentioned diluted commercial protein was added, reacted at room temperature for 2 hours, and washed with PBS for 3 times. Then, add biotin-labeled monoclonal antibody biotin-10D6 (1 μg / ml, 100 μl / well), continue to react at room temperature for 1 hour, wash with PBS for 3 times, add Streptavidin-HRP (1:3000, 100 μl / well), and react for 1 hour at room temperature , washed 6-8 times with PBS, then added HRP reaction substrate TMB (100μl / well), reacted at room temperature for 10-15min, an...

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PUM

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Abstract

The invention discloses a kit capable of quantitatively detecting soluble B7-DC. The kit comprises a horse radish peroxidase marker, tetramethyl benzidine serving as a reaction substrate, bovine serum albumin, an elisa plate, a wash solution and a stop solution, and is characterized by further comprising a coated antibody, standard proteins and a detection antibody, wherein the coated antibody isa mouse anti-human B7-DC monoclonal antibody 8F2; and the detection antibody is a mouse anti-human B7-DC monoclonal antibody 10D6. The kit capable of quantitatively detecting soluble B7-DC has high specificity, can be used for accurately and quantitatively analyzing the concentrations of soluble B7-DC protein factors in liquids such as human cell culturing supernatant, blood serum, blood plasma, hydrothorax and the like, and can be applied to clinical differential diagnosis and treatment effects of asthma.

Description

technical field [0001] The invention relates to a kit capable of quantitatively detecting human soluble B7-DC, in particular to a kit for quantitative detection prepared by using two strains of specific mouse anti-human B7-DC monoclonal antibodies 8F2 and 10D6 and B7-DCIg fusion protein An enzyme-linked immunoassay kit for analyzing soluble B7-DC factor, the quantitative detection system can be applied in the fields of differential diagnosis and curative effect judgment of asthma. Background technique [0002] Many receptor / ligand interactions are known to be involved in the induction, establishment and regulation of antigen-specific immune responses. In order to effectively activate a T cell response, at least two signals are usually required. In addition to the T cell antigen receptor (TCR) recognizing the MHC-antigen complex on the antigen-presenting cell (APC) to provide the first signal, the antigen-specific signal, T cells need to interact with costimulatory molecules...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 陈永井张学光王勤白利雄施敏骅柏发蕊
Owner 苏州市第五人民医院
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