CN103675273A - Chemiluminiscence detection kit for canine distemper virus antigen - Google Patents
Chemiluminiscence detection kit for canine distemper virus antigen Download PDFInfo
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- CN103675273A CN103675273A CN201310562376.7A CN201310562376A CN103675273A CN 103675273 A CN103675273 A CN 103675273A CN 201310562376 A CN201310562376 A CN 201310562376A CN 103675273 A CN103675273 A CN 103675273A
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/13—Canine distemper virus
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Abstract
The invention provides a chemiluminiscence detection kit for a canine distemper virus antigen. The chemiluminiscence detection kit comprises a coated plate and a reagent which are arranged in a box body, wherein the reagent comprises a horseradish peroxidase marked goat anti-mouse antibody, a coating solution, a sealing solution, a washing solution and chemiluminiscence liquid; the coated plate is a light-emitting plate coated with a canine distemper virus antibody; the diluting degree of the canine distemper virus antibody is 1:(700-1000); the diluting degree of the horseradish peroxidase marked goat anti-mouse antibody is 1:2500. The chemiluminiscence detection kit for the canine distemper virus antigen has the characteristics of low cost, convenience, rapidness and low testing condition requirements; the detection efficiency is improved and the detection accuracy is also guaranteed; the chemiluminiscence detection kit can be used for export inspection, food export inspection and field rapid detection of an animal breeding industry.
Description
Technical field
The present invention relates to virus immunity and the kit that detects use, specifically a kind of CDV antigen chemiluminescence detection kit.
Background technology
Canine distemper (CD) is by caused acute, the height contagious disease of CDV (CDV) infected dogs.CDV is that infectiousness is extremely strong, can cause dog class and some other carnivore ill even dead.The pup of 3～6 months more easily infects canine distemper than adult dogs.CDV often can cause dog class dead, so occurring that similar symptom is early stage, detect and be necessary with CDV antigen detection kit.
At present, the traditional detection method of CDV has Serology test and aetology detection method.Serology test is mainly hemagglutination test, enzyme linked immunosorbent assay and immune colloid gold detection method, but sensitivity is not high, specificity is not strong.Aetology detection method is commonly used the separated virus of cultivating of the continuous cell lines such as F81, MDCK at present, although virus separation method can detect an infective virion in theory, make a definite diagnosis the infection of CDV, but also because the factors such as complex operation, the cost of isolated viral is high and make a definite diagnosis slowly do not drop into clinical use.
Along with molecular biological development, existing multiple PCR method, for the detection of CDV, though improve to some extent aspect sensitivity, owing to needing supporting the use of exact instrument, equally also cannot meet the needs that detect with terrain at present.
Summary of the invention
For solving existing CDV, detect that the sensitivity existing is not high, cost is high makes a definite diagnosis the problems such as slow, the invention provides a kind of CDV antigen chemiluminescence detection kit.
The present invention solves the problems of the technologies described above the technical scheme of employing to be: a kind of CDV antigen chemiluminescence detection kit, comprise the coated plate and the reagent that are arranged in box body, described reagent comprises the antibody of the sheep anti mouse of horseradish peroxidase mark, coated solution, lock solution, cleansing solution and chemical luminescence for liquid, described coated plate is the luminous plaque that is coated with CDV antibody, the dilutability of described CDV antibody is 1:700-1000, and the antibody dilution of the sheep anti mouse of horseradish peroxidase mark is 1:2500;
Described luminous plaque is the opaque polystyrene 96 hole Chemiluminescent plates of milky;
The coated of described CDV antibody is CDV antibody to be placed in to the coated solution of 0.1mol/L, and at the temperature of 4 ℃, the coated 12h of reaction, then adopts lock solution to be sealed in the hole on luminous plaque;
Described coated solution is sodium carbonate and the sodium bicarbonate carbonate buffer solution of making soluble in water, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that adds the BSA of 10g to make in every 100ml water, then adds successively wherein skimmed milk power, 1% gelatin and 0.05% the NaN3 of BSA solution weight 5%.
The antibody of the sheep anti mouse of described horseradish peroxidase mark is that the dilution of horseradish peroxidase-sheep anti-mouse igg stoste obtains.
Described chemical luminescence for liquid comprises substrate A and substrate B, substrate A is that 0.01M luminol is dissolved into 0.01M PH=8.8 Tris damping fluid, substrate B is to add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01M PH=8.8 Tris damping fluid to iodophenol, described luminol is luminous substrate, to iodophenol, is luminescence enhancer.
Described cleansing solution is that to contain mass concentration be the 0.1mol/L phosphate buffer that 0.05% Tween-20, pH value are 7.5.
Beneficial effect: the present invention has with low cost, easily and fast and the low feature of Test Condition Requirements, not only improved detection efficiency, and guaranteed the degree of accuracy detecting, can be used for export inspection, Food Safety Analysis and animal farming industry field quick detection.
Accompanying drawing explanation
Fig. 1 is the experimental result table of the specific embodiment of the invention.
Embodiment
A kind of CDV antigen chemiluminescence detection kit, comprise the coated plate and the reagent that are arranged in box body, described reagent comprises the antibody of the sheep anti mouse of horseradish peroxidase mark, coated solution, lock solution, cleansing solution and chemical luminescence for liquid, described coated plate is the luminous plaque that is coated with CDV antibody, the dilutability of described CDV antibody is 1:700-1000, and the antibody dilution of the sheep anti mouse of horseradish peroxidase mark is 1:2500;
Described luminous plaque is the opaque polystyrene 96 hole Chemiluminescent plates of milky;
The coated of described CDV antibody is CDV antibody to be placed in to the coated solution of 0.1mol/L, and at the temperature of 4 ℃, the coated 12h of reaction, then adopts lock solution to be sealed in the hole on luminous plaque;
Described coated solution is sodium carbonate and the sodium bicarbonate carbonate buffer solution of making soluble in water, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that adds the BSA of 10g to make in every 100ml water, then adds successively wherein skimmed milk power, 1% gelatin and 0.05% the NaN3 of BSA solution weight 5%.
The antibody of the sheep anti mouse of described horseradish peroxidase mark is that the dilution of horseradish peroxidase-sheep anti-mouse igg stoste obtains.
Described chemical luminescence for liquid comprises substrate A and substrate B, substrate A is that 0.01M luminol is dissolved into 0.01M PH=8.8 Tris damping fluid, substrate B is to add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01M PH=8.8 Tris damping fluid to iodophenol, described luminol is luminous substrate, to iodophenol, is luminescence enhancer.
Described cleansing solution is that to contain mass concentration be the 0.1mol/L phosphate buffer that 0.05% Tween-20, pH value are 7.5.
The step of utilizing the present invention to detect is as follows:
(1) join washing lotion: 50ml cleansing solution (20 *) is added to purified water and be diluted to 1000ml, fully shake up rear standby;
(2) numbering: should establish height Quality Control reference substance hole numbering on coated plate during test, all the other are sample well to be checked;
(3) well dilution: add sample diluting liquid with sample injector, every hole 90ul; Quality Control reference substance hole does not add sample diluting liquid;
(4) application of sample: add corresponding Quality Control reference substance 100ul respectively at Quality Control reference substance hole, and add sample 10ul to be checked in each sample well to be checked, 37 ± 2 ℃ of incubations 30 minutes;
(5) washing: the middle liquid that portals that inclines, every hole adds wash solution 250 μ L, washs 3 times, pats dry;
(6) enzyme-added mark sheep anti-mouse antibody solution: every hole adds enzyme mark sheep anti-mouse antibody solution 100 μ L, 37 ± 2 ℃ of incubations 30 minutes;
(7) washing: the middle liquid that portals that inclines, every hole adds wash solution 250 μ L, washs 3 times, pats dry;
(8) add luminous solution: every hole adds luminous solution 100 μ L;
(9) detect: the luminous intensity of measuring every hole with chemical illumination immunity analysis instrument.
(10) with ten samples, detect:
Result is as shown in Figure 1: cut-off=2.1*4752.5(negative control)
Cut-off > 1.0 is positive.
Claims (4)
1. a CDV antigen chemiluminescence detection kit, comprise the coated plate and the reagent that are arranged in box body, described reagent comprises the antibody of the sheep anti mouse of horseradish peroxidase mark, coated solution, lock solution, cleansing solution and chemical luminescence for liquid, it is characterized in that: described coated plate is the luminous plaque that is coated with CDV antibody, the dilutability of described CDV antibody is 1:700-1000, and the antibody dilution of the sheep anti mouse of horseradish peroxidase mark is 1:2500;
Described luminous plaque is the opaque polystyrene 96 hole Chemiluminescent plates of milky;
The coated of described CDV antibody is CDV antibody to be placed in to the coated solution of 0.1mol/L, and at the temperature of 4 ℃, the coated 12h of reaction, then adopts lock solution to be sealed in the hole on luminous plaque;
Described coated solution is sodium carbonate and the sodium bicarbonate carbonate buffer solution of making soluble in water, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that adds the BSA of 10g to make in every 100ml water, then adds successively wherein skimmed milk power, 1% gelatin and 0.05% the NaN3 of BSA solution weight 5%.
2. a kind of CDV antigen chemiluminescence detection kit according to claim 1, is characterized in that: the antibody of the sheep anti mouse of described horseradish peroxidase mark is that the dilution of horseradish peroxidase-sheep anti-mouse igg stoste obtains.
3. a kind of CDV antigen chemiluminescence detection kit according to claim 1, it is characterized in that: described chemical luminescence for liquid comprises substrate A and substrate B, substrate A is that 0.01M luminol is dissolved into 0.01M PH=8.8 Tris damping fluid, substrate B is to add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01M PH=8.8 Tris damping fluid to iodophenol, described luminol is luminous substrate, to iodophenol, is luminescence enhancer.
4. a kind of CDV antigen chemiluminescence detection kit according to claim 1, is characterized in that: described cleansing solution is that to contain mass concentration be the 0.1mol/L phosphate buffer that 0.05% Tween-20, pH value are 7.5.
Priority Applications (1)
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| CN201310562376.7A CN103675273B (en) | 2013-11-12 | 2013-11-12 | A kind of CDV antigen chemiluminescence detection kit |
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| CN201310562376.7A CN103675273B (en) | 2013-11-12 | 2013-11-12 | A kind of CDV antigen chemiluminescence detection kit |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007010240A2 (en) * | 2005-07-18 | 2007-01-25 | European Cardiovascular Genetics Foundation | Method for high-throughput screening |
| CN201053964Y (en) * | 2007-05-21 | 2008-04-30 | 四川省迈克科技有限责任公司 | Vaccine protective antibody quick detection reagent kit |
| CN103018450A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Chloramphenicol chemiluminescence enzyme-linked immunodetection kit |
| CN103163125A (en) * | 2013-04-05 | 2013-06-19 | 湖南康润药业有限公司 | Preparation method and clinical application of HCV (hepatitis C virus) core antigen novel quantitative detection kit |
-
2013
- 2013-11-12 CN CN201310562376.7A patent/CN103675273B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007010240A2 (en) * | 2005-07-18 | 2007-01-25 | European Cardiovascular Genetics Foundation | Method for high-throughput screening |
| CN201053964Y (en) * | 2007-05-21 | 2008-04-30 | 四川省迈克科技有限责任公司 | Vaccine protective antibody quick detection reagent kit |
| CN103018450A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Chloramphenicol chemiluminescence enzyme-linked immunodetection kit |
| CN103163125A (en) * | 2013-04-05 | 2013-06-19 | 湖南康润药业有限公司 | Preparation method and clinical application of HCV (hepatitis C virus) core antigen novel quantitative detection kit |
Non-Patent Citations (2)
| Title |
|---|
| 孙婧等: "犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立", 《中国预防兽医学报》, vol. 35, no. 8, 31 August 2013 (2013-08-31), pages 635 - 639 * |
| 张鸿等: "犬瘟热病毒单抗夹心ELISA检测方法的建立", 《扬州大学学报（农业与生命科学版）》, vol. 32, no. 4, 31 December 2011 (2011-12-31), pages 15 - 18 * |
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Inventor after: Wang Shanpu Inventor after: Li Jingzhao Inventor after: Hu Qinghong Inventor after: Wang Qingyun Inventor after: Wu Lei Inventor after: Sun Jie Inventor after: Zhang Xiaofei Inventor after: Geng Yujing Inventor before: Wang Shanpu Inventor before: Wu Tingcai Inventor before: Wang Xingbing Inventor before: Wang Xiaojun Inventor before: Wang Yudong |
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Effective date of registration: 20160722 Address after: 471000 Luoyang, Luolong, science and Technology Park, west of Peony Road, No. N3, No. Patentee after: LUOYANG LAIPSON INFORMATION TECHNOLOGY CO., LTD. Patentee after: Luoyang Modern Biotechnology Research Institute Co., Ltd. Address before: 471000 Luoyang, Luolong, science and Technology Park, west of Peony Road, No. N3, No. Patentee before: LUOYANG LAIPSON INFORMATION TECHNOLOGY CO., LTD. |
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Inventor after: Wang Shanpu Inventor after: Li Jingzhao Inventor after: Hu Qingjiang Inventor after: Wang Qingyun Inventor after: Wu Lei Inventor after: Sun Jie Inventor after: Zhang Xiaofei Inventor after: Geng Yujing Inventor before: Wang Shanpu Inventor before: Li Jingzhao Inventor before: Hu Qinghong Inventor before: Wang Qingyun Inventor before: Wu Lei Inventor before: Sun Jie Inventor before: Zhang Xiaofei Inventor before: Geng Yujing |
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