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Soluble B7-H1 quantitative detection kit

A technology of B7-H1 and ZH11, applied in the field of soluble B7-H1 quantitative detection kits, can solve the problems of lack of detection methods and achieve good linear relationship, good sensitivity and good specificity

Inactive Publication Date: 2011-11-23
SUZHOU UNIV
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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-H1 (sB7-H1) at home and abroad

Method used

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  • Soluble B7-H1 quantitative detection kit
  • Soluble B7-H1 quantitative detection kit
  • Soluble B7-H1 quantitative detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Reagents and materials: Calf serum Hyclone company (USA); add calf serum 100ml, L-glutamine 0.15g, NaHCO3 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 ,U.S). Cell culture flasks and plates (Nunc, Denmark); CO2 incubator, centrifuge (Jouan, France), inverted microscope (O1ympus, Japan), flow cytometer (Coulter, USA); transfected human B7-H1 gene cell line L929 / B7-H1 (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).

[0049] (2) Cultivation of cell lines: Using RPMI1640 medium conta...

Embodiment 2

[0069] A kit capable of quantitatively detecting soluble B7-H1, comprising the following components:

[0070] (1) Coating antibody: mouse anti-human B7-H1 monoclonal antibody (ZH11) as described in Example 1, 30 μg / tube, 1 tube;

[0071] (2) Standard protein: B7-H1Ig protein (R&D), 25ng / tube, 1 tube;

[0072] (3) Detection antibody: mouse anti-human B7-H1 monoclonal antibody (Biotin-10D7) as described in Example 1, 10 μg / tube, 1 tube;

[0073] (4) Horseradish peroxidase-labeled Streptavidin-HRP (Sigma-Aldrich), 1 μl / / tube, 1 tube;

[0074] (5) Reaction substrate: tetramethylbenzidine TMB (Sigma-Aldrich), 10ml;

[0075] (6) Bovine Serum Albumin BSA (Shanghai Sangong), 2g / 100ml, 30ml;

[0076] (7). ELISA plate (Costar), 1 piece;

[0077] (8). Washing solution: 10×PBS, 100 ml; Tween-20, 0.5 ml;

[0078] (9). Stop solution: 2M H 2 SO 4 , 5 ml;

[0079] Storage conditions:

[0080]

[0081] Specificity analysis of the above kits:

[0082] 1. Prepare the coating antibo...

Embodiment 3

[0094] Embodiment 3: The kit described in Embodiment 2 can be applied to the quantitative analysis of the concentration of soluble B7-H1 protein factors in liquids such as human cell culture supernatant, serum, plasma and pleural effusion.

[0095] The methods of sample processing are as follows:

[0096] 1. Cell culture supernatant: 2500 rpm×10 min, store at -20°C, avoid repeated freezing and thawing.

[0097] 2. Serum samples: 2500 rpm×10 min, store at -20°C, avoid repeated freezing and thawing.

[0098] 3. Plasma samples: 2500 rpm×10 min, store at -20°C, avoid repeated freezing and thawing.

[0099] Quantitative detection operation steps:

[0100] 1. Prepare the coating antibody ZH11 with 0.01M CBS (pH9.6) buffer solution to make a coating antibody working solution with a concentration of 3 μg / ml, add 100 μl / well to the ELISA plate, and let it stand overnight at 4°C;

[0101] 2. Discard the coating solution, wash the plate 3 times with 0.01M PB; add 1% BSA 200μl / well, bl...

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Abstract

The invention discloses a kit capable of quantitatively detecting soluble B7-H1, which comprises a horseradish peroxidase label, tetramethylbenzidine serving as a reaction substrate, bovine serum albumin, an elisa plate, washing liquor, stop solution, a coating antibody, a standard protein and a detection antibody, wherein the coating antibody is a mouse anti-human B7-H1 monoclonal antibody and the nucleotide sequence of the heavy chain of the coating antibody is represented by a nucleotide sequence SEQ ID No.3 in the sequence list, and the nucleotide sequence of the light chain of the coating antibody is represented by a nucleotide sequence SEQ ID No.4 in the sequence table. The kit capable of quantitatively detecting soluble B7-H1 has high specificity and can be used for accurately quantitative analysis on soluble B7-H1 protein factor concentration in liquid for human cell culture supernate, serum, plasma, hydrothorax and the like.

Description

technical field [0001] The invention relates to a kit capable of quantitatively detecting soluble B7-H1, in particular to the development of two strains of specific anti-human B7-H1 monoclonal antibodies ZH11, 10D7 and B7-H1Ig fusion protein for quantitative detection and analysis of soluble B7-H1 The method of the enzyme-linked immunoassay kit for factors, the quantitative detection system can be applied in the field of differential diagnosis and prognosis judgment of lung cancer. Background technique [0002] A number of 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. Among them, 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, it is also necessary to obtain th...

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 陈永井王勤张学光施敏骅胡振华
Owner SUZHOU UNIV
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