CN111435137A - Quantitative detection kit for DcR3 - Google Patents
Quantitative detection kit for DcR3 Download PDFInfo
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- CN111435137A CN111435137A CN201910032448.4A CN201910032448A CN111435137A CN 111435137 A CN111435137 A CN 111435137A CN 201910032448 A CN201910032448 A CN 201910032448A CN 111435137 A CN111435137 A CN 111435137A
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
Compared with the conventional DcR3E L ISA detection kit, the quantitative detection kit of DcR3 has the advantages of high detection sensitivity and low detection limit, can be directly used for detecting the content of DcR3 in human serum, and can be directly used for screening, prognosis judgment and treatment guidance of tumors.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly discloses a quantitative detection kit for DcR 3.
Background
Research finds that the concentration of DcR3 in serum and tissues of a tumor patient is positively correlated with the malignancy, and the level of R3 in serum of a patient after detection can be used as an important index (Liu Rui Zheng and Zhang et al 2009) for the complete tumor excision (DcR 3 is closely related to tumor metastasis and is an ideal tumor marker (L i, Xie et al 2017) for diagnosing the cancer metastasis, so that an E L tumor diagnosis kit based on the DcR3 tumor marker is developed, and is favorable for screening, prognosis judgment and treatment guidance of the cancer.
The E L ISA kit for detecting the content of DcR3 generally adopts a double-antibody sandwich method, and the basic principle of the kit is that a certain amount of coating antibody is fixed on the surface of a polystyrene microporous plate by a physical adsorption method, an irrelevant protein carrier is added to seal unbound sites, then a sample to be detected is added, a detection antibody is added, a second antibody is labeled by enzyme and developed by a substrate, and the shade of the color in the microporous plate is positively correlated with the concentration of an object to be detected.
Several human DcR3E L ISA detection kit products are currently on the market, including R & D Systems, Invitrogen, Abcam, and Boster, but the sensitivity of the DcR3E L ISA detection kit on the market is not high enough, and the detection limit is not low enough, and the antibody used by the E L ISA kit of some companies is polyclonal antibody, which can not ensure high quality uniformity among samples of each batch.
Disclosure of Invention
In view of the above, the present invention aims to provide a quantitative detection kit for DcR3, which solves the problems of low sensitivity and high detection line in the prior art.
A quantitative detection kit for DcR3 comprises a DcR3 monoclonal antibody, a biotin-labeled secondary antibody and streptavidin-peroxidase.
In the kit, the avidin is connected with a biotinylated antibody and an enzyme combined with biotin as a bridge to form an avidin-biotin-peroxidase complex (ABC complex), and an antigen in a sample to be detected is combined with a primary antibody, a biotinylated secondary antibody and the ABC complex in sequence during detection to finally form a complex with a lattice-like structure, so that a reaction signal of the antigen and the antibody is amplified to increase the sensitivity.
Preferably, the DcR3 monoclonal antibody is a murine-derived DcR3 monoclonal antibody, a rabbit-derived DcR3 monoclonal antibody, or an ovine-derived DcR3 monoclonal antibody.
Preferably, the DcR3 monoclonal antibody is a monoclonal antibody produced by monoclonal hybridoma 7a51H2 or IB 7.
Preferably, the DcR3 monoclonal antibody is diluted to a working concentration of 4. mu.g/ml with Phosphate Buffered Saline (PBS).
Preferably, the biotin-labeled secondary antibody is a biotin-labeled murine anti-human DcR3 monoclonal antibody.
Furthermore, the quantitative detection kit for DcR3 also comprises a color development solution TMB, PBS and 1M H2SO4Skim milk powder, PBS containing 0.05% Tween-20. .
The invention also provides a quantitative detection method of DcR3, which comprises the following steps:
(1) taking a DcR3 monoclonal antibody as a primary anti-coating enzyme label plate, and sealing with a sealing solution after washing;
(2) after washing, adding a sample to be tested, and incubating for 2h at room temperature;
(3) adding a biotin-labeled secondary antibody after washing, and incubating for 2h at room temperature;
(4) washing, adding streptavidin-peroxidase, and incubating for 20min in dark;
(5) and adding color development liquid TMB after washing, incubating for 20min in a dark place, terminating the reaction, and reading the OD value at 450nm by using an enzyme-labeling instrument.
Compared with the conventional DcR3E L ISA detection kit, the quantitative detection kit of DcR3 has the advantages of high detection sensitivity and low detection limit, can be directly used for detecting the content of DcR3 in human serum, and can be directly used for screening, prognosis judgment and treatment guidance of tumors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the SDS-PAGE electrophoresis of the purified DcR3 antibody of example 1;
FIG. 2 shows a high performance liquid chromatogram of the purified DcR3 antibody of example 1.
Detailed Description
The invention discloses a quantitative detection method of DcR 3. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1 preparation of human DcR3 monoclonal antibody
The preparation method comprises the steps of immunizing female BA L B/c mice (6 weeks old) with fusion protein of human full-length DcR3 and IgG1Fc domains (provided by Beijing Yinqiao Shenzhou science and technology Co., Ltd.) as an antigen, carrying out first immunization by using Freund's complete adjuvant to emulsify the antigen, carrying out subcutaneous 5-6-point injection by using Freund's incomplete adjuvant to emulsify the antigen at the beginning of second immunization, carrying out subcutaneous 5-6-point injection by using 100 mu g of antigen injected into each mouse 10 days after 3 rd immunization, collecting a small amount of blood from the tail of the mouse to carry out serum titer E L ISA detection, selecting the mouse with high antibody titer (>1:100000) to strengthen 4 th immunization, carrying out intraperitoneal injection of the antigen protein, carrying out injection by using 100 mu g of the mouse 3-5 days after 4 th immunization, killing the mouse to take spleen cells of the mouse to fuse with SP2/0 cells, carrying out culture by HAT culture medium to obtain stable DcR3 antibody secreting hybridoma cells, carrying out filtration by E L ISA screening, carrying out limited liquid nitrogen dilution, and carrying out cloning to obtain hybridoma strain 3651 and amplifying by using liquid nitrogen.
Ascites antibody preparation, namely injecting 0.5ml of Freund's incomplete adjuvant into a female BA L B/c mouse (8 weeks old) in an intraperitoneal mode, injecting hybridoma cells in a logarithmic growth phase into the intraperitoneal mode after 3-5 days, and injecting (1-5) × 10 into each mouse5Cells (0.5ml), mice were sacrificed 11 days after injection of hybridoma cells to obtain ascites fluid, centrifuged at 3000rpm at 4 ℃ for 10min, the precipitate was removed, the ascites fluid was diluted with 10-fold volume of 1 × PB solution, mixed well and filtered through a 0.45 μm filter, the ascites fluid was affinity-purified by Protein G (Protein GSepharose 4 Fast Flow, GE Healthcare) to obtain purified DcR3 antibody Protein, the antibody concentration was measured by BCA method, the purified antibody was subjected to SDS-PAGE (sample loading 5.4 μ G) electrophoresis, stained with Coomassie Brilliant blue, and the purity of the purified antibody was measured by high performance liquid chromatography, as shown in FIG. 2.
Example 2 human DcR3 avidin-biotin-peroxidase Complex E L ISA detection kit
The kit comprises a mouse anti-human DcR3-7A51H2 antibody, a mouse anti-human DcR3-IB7-Biotin, Streptavidin-HRP (200 times), a chromogenic solution TMB, skimmed milk powder and 1M H2SO4PBS, PBS containing 0.05% Tween-20.
Example 3 human DcR3 avidin-biotin-peroxidase Complex E L ISA assay (ABC-E L ISA assay)
(1) Coated primary antibody (mouse antibody human DcR3-7A51H2)
Murine anti-human DcR3-7A51H2 was diluted with Phosphate Buffered Saline (PBS) to a working concentration of 4. mu.g/ml and added to a 96-well plate at 100. mu.l/well immediately, at 4 ℃ overnight.
(2) PBS-T (PBS containing 0.05% Tween-20) wash, 400. mu.l/well, 5 washes.
(3) 5% (w/v) skimmed milk powder blocking solution, 300. mu.l/well, incubation at 37 ℃ for at least 1 h.
(4) 0.05% (v/v) PBS-T wash, 400. mu.l/well, 5 washes.
(5) Addition of the Standard antigen DcR3
The standard antigen DcR3 was diluted in series of concentrations to draw a standard curve. The standard antigen DcR3 was diluted to an initial concentration of 12000pg/ml, using PBS-T as a diluent. The samples were diluted to a series of concentrations (12000pg/ml, 6000pg/ml, 3000pg/ml, 1500pg/ml, 750pg/ml, 375pg/ml, 188pg/ml, 94pg/ml, 47pg/ml, 24pg/ml, 12pg/ml) by 2-fold dilution, 100. mu.l/well. Incubation was performed for 2h at room temperature using PBS-T as a blank control.
(6) PBS-T wash, 400. mu.l/well, 5 washes.
(7) According to the EZ-L ink NHS-Biotin Reagents kit instructions, the secondary antibody (mouse anti human DcR3-IB7-Biotin) was prepared in advance, the secondary antibody required ice incubation for at least 2h or room temperature incubation for 30min, then 100 u l/well to 96 well plate, room temperature incubation for 2 h.
(8) PBS-T wash, 400. mu.l/well, 5 washes.
(9) Streptavidin-HRP (200 fold) was diluted with PBS-T to working concentration, 100. mu.l/well and incubated for 20min in the absence of light.
(10) PBS-T wash, 400. mu.l/well, 5 washes.
(11) Adding color development liquid TMB, 100 mul/well, incubating for 20min in dark, or terminating the reaction according to the color depth of the reaction, wherein the reaction can be terminated in advance if the color of the blank control changes.
(12) Add 1M H2SO4The stop solution, 100. mu.l/well, was gently mixed and immediately detected for OD450 absorbance, and if the difference between the readings is large, the data can be calibrated by OD570, and a standard curve can be drawn by using the data from OD450 to OD 570.
(13) And drawing a standard curve according to the experimental data and judging the concentration of DcR3 in the sample.
The result shows that the linear range of the E L ISA detection method is 11-12000pg/m L, the stability is good (CV < 5%), and the detection limit is 11pg/m L.
Claims (6)
1. A quantitative detection kit for DcR3 comprises a DcR3 monoclonal antibody, a biotin-labeled secondary antibody and streptavidin-peroxidase.
2. The quantitative detection kit of claim 1, wherein the DcR3 monoclonal antibody is a murine DcR3 monoclonal antibody, a rabbit DcR3 monoclonal antibody, or an ovine DcR3 monoclonal antibody.
3. The quantitative determination kit according to claim 1, wherein the DcR3 monoclonal antibody is a monoclonal antibody produced by monoclonal hybridoma 7A51H2 or IB 7.
4. The quantitative detection kit of claim 1, wherein said DcR3 monoclonal antibody is diluted to a working concentration of 4 μ g/ml with Phosphate Buffered Saline (PBS).
5. The quantitative detection kit according to claim 1, wherein the biotin-labeled secondary antibody is a biotin-labeled murine anti-human DcR3 monoclonal antibody.
6. The quantitative determination kit according to any one of claims 1 to 5, further comprising a color-developing solution TMB, PBS, 1MH2SO4Skim milk powder, PBS containing 0.05% Tween-20.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112213481A (en) * | 2020-08-13 | 2021-01-12 | 茅台学院 | Hapten directly coated bisphenol A ABC enzyme-linked immunosorbent assay method based on monoclonal antibody |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408880A (en) * | 2001-09-30 | 2003-04-09 | 华星生物科技股份有限公司 | Detecting and trapping receptor 3 monoclonal antibody, hybrid tumor for producing said antibody and its use |
| CN1752104A (en) * | 2004-09-21 | 2006-03-29 | 北京市肿瘤防治研究所 | Monoclonal antibody of synapse protein gamma and its application |
| US20080026411A1 (en) * | 2003-09-04 | 2008-01-31 | Jiangping Wu | Diagnostic and prognostic methods and compositions of matter for cell proliferative diseases |
| CN101135686A (en) * | 2007-06-13 | 2008-03-05 | 吉林大学 | Double antibody sandwich ELISA method for detecting decoy receptor 3 |
| KR20140056501A (en) * | 2012-10-26 | 2014-05-12 | 서울대학교산학협력단 | Novel biomarker indicative of inflammatory arthritis and its uses |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408880A (en) * | 2001-09-30 | 2003-04-09 | 华星生物科技股份有限公司 | Detecting and trapping receptor 3 monoclonal antibody, hybrid tumor for producing said antibody and its use |
| US20080026411A1 (en) * | 2003-09-04 | 2008-01-31 | Jiangping Wu | Diagnostic and prognostic methods and compositions of matter for cell proliferative diseases |
| CN1752104A (en) * | 2004-09-21 | 2006-03-29 | 北京市肿瘤防治研究所 | Monoclonal antibody of synapse protein gamma and its application |
| CN101135686A (en) * | 2007-06-13 | 2008-03-05 | 吉林大学 | Double antibody sandwich ELISA method for detecting decoy receptor 3 |
| KR20140056501A (en) * | 2012-10-26 | 2014-05-12 | 서울대학교산학협력단 | Novel biomarker indicative of inflammatory arthritis and its uses |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112213481A (en) * | 2020-08-13 | 2021-01-12 | 茅台学院 | Hapten directly coated bisphenol A ABC enzyme-linked immunosorbent assay method based on monoclonal antibody |
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