CN103675273B - A kind of CDV antigen chemiluminescence detection kit - Google Patents
A kind of CDV antigen chemiluminescence detection kit Download PDFInfo
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- CN103675273B CN103675273B CN201310562376.7A CN201310562376A CN103675273B CN 103675273 B CN103675273 B CN 103675273B CN 201310562376 A CN201310562376 A CN 201310562376A CN 103675273 B CN103675273 B CN 103675273B
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- G—PHYSICS
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- 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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- 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
A kind of CDV antigen chemiluminescence detection kit, comprise and be arranged on bag in box body by plate and reagent, described reagent comprise horseradish peroxidase mark sheep anti mouse antibody, wrap by solution, lock solution, cleansing solution and chemical luminescence for liquid, described bag is the luminous plaque being coated with CDV antibody by plate, 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.The present invention have with low cost, easily and fast and the low feature of Test Condition Requirements, not only increase detection efficiency, and also ensure that the degree of accuracy of detection, can be used for export inspection, Food Safety Analysis and animal farming industry field quick detection.
Description
Technical field
The present invention relates to virus immunity and the kit detected, a kind of CDV antigen chemiluminescence detection kit specifically.
Background technology
Canine distemper (CD) is acute, high degree in contact sexually transmitted disease caused by CDV (CDV) infected dogs.CDV is that infectiousness is extremely strong, can cause dog class and the ill even death of some other carnivore.The pup of 3 ~ 6 months is easier than adult dogs infects canine distemper.CDV often can cause dog class dead, so early stage in the similar symptom of appearance, carry out detection be necessary with CDV antigen detection kit.
At present, the traditional detection method of CDV has Serology test and pathogeny 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.Pathogeny detection method is commonly used the continuous cell lines such as F81, MDCK at present and is separated cultivation virus, although virus isolation procedure can detect an infective virion in theory, make a definite diagnosis the infection of CDV, but also because the complex operation of isolated viral, cost are high and the factor such as slowly of making a definite diagnosis does not drop into Clinical practice.
Along with molecular biological development, existing multiple PCR method is used for the detection of CDV at present, though improve to some extent in sensitivity, owing to needing supporting the use of exact instrument, and the needs that equally also cannot meet and now detect.
Summary of the invention
Make a definite diagnosis the problems such as slow for the sensitivity solving existing CDV detection existence is not high, cost is high, the invention provides a kind of CDV antigen chemiluminescence detection kit.
The present invention is the technical scheme solving the problems of the technologies described above employing: a kind of CDV antigen chemiluminescence detection kit, comprise and be arranged on bag in box body by plate and reagent, described reagent comprise horseradish peroxidase mark sheep anti mouse antibody, wrap by solution, lock solution, cleansing solution and chemical luminescence for liquid, described bag is the luminous plaque being coated with CDV antibody by plate, 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 milky opaque polystyrene 96 hole Chemiluminescent plate;
The bag of described CDV antibody is that CDV antibody is placed in the bag of 0.1mol/L by solution, and at the temperature of 4 DEG C, reaction bag is by 12h, then adopts lock solution to be sealed in the hole on luminous plaque;
Described bag is sodium carbonate and the sodium bicarbonate carbonate buffer solution made soluble in water by solution, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that the BSA adding 10g in every 100ml water makes, then add successively wherein the skimmed milk power of BSA solution weight 5%, the gelatin of 1% and 0.05% NaN3.
The antibody of the sheep anti mouse of described horseradish peroxidase mark is that horseradish peroxidase-sheep anti-mouse igg stoste dilution obtains.
Described chemical luminescence for liquid comprises substrate A and substrate B, substrate A is that 0.01M luminol is dissolved into 0.01MPH=8.8Tris damping fluid, substrate B is add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01MPH=8.8Tris damping fluid to iodophenol, described luminol is luminous substrate, is luminescence enhancer to iodophenol.
Described cleansing solution be containing mass concentration be 0.05% Tween-20, pH value be 7.5 0.1mol/L phosphate buffer.
Beneficial effect: the present invention has with low cost, easily and fast and the low feature of Test Condition Requirements, not only increase detection efficiency, and also ensure that the degree of accuracy of detection, 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 and be arranged on bag in box body by plate and reagent, described reagent comprise horseradish peroxidase mark sheep anti mouse antibody, wrap by solution, lock solution, cleansing solution and chemical luminescence for liquid, described bag is the luminous plaque being coated with CDV antibody by plate, 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 milky opaque polystyrene 96 hole Chemiluminescent plate;
The bag of described CDV antibody is that CDV antibody is placed in the bag of 0.1mol/L by solution, and at the temperature of 4 DEG C, reaction bag is by 12h, then adopts lock solution to be sealed in the hole on luminous plaque;
Described bag is sodium carbonate and the sodium bicarbonate carbonate buffer solution made soluble in water by solution, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that the BSA adding 10g in every 100ml water makes, then add successively wherein the skimmed milk power of BSA solution weight 5%, the gelatin of 1% and 0.05% NaN3.
The antibody of the sheep anti mouse of described horseradish peroxidase mark is that horseradish peroxidase-sheep anti-mouse igg stoste dilution obtains.
Described chemical luminescence for liquid comprises substrate A and substrate B, substrate A is that 0.01M luminol is dissolved into 0.01MPH=8.8Tris damping fluid, substrate B is add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01MPH=8.8Tris damping fluid to iodophenol, described luminol is luminous substrate, is luminescence enhancer to iodophenol.
Described cleansing solution be containing mass concentration be 0.05% Tween-20, pH value be 7.5 0.1mol/L phosphate buffer.
The step utilizing the present invention to carry out detecting is as follows:
(1) join washing lotion: 50ml cleansing solution (20 ×) is added purified water and is diluted to 1000ml, fully shake up rear for subsequent use;
(2) number: should plate be established height Quality Control contrast sample wells at bag and number during test, all the other be measuring samples hole;
(3) well dilution: add sample diluting liquid with sample injector, every hole 90ul; Quality Control contrast sample wells does not add sample diluting liquid;
(4) application of sample: add corresponding Quality Control reference substance 100ul respectively at Quality Control contrast sample wells, and add measuring samples 10ul in each measuring samples hole, 37 ± 2 DEG C of incubations 30 minutes;
(5) wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat 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 DEG C of incubations 30 minutes;
(7) wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
(8) luminescent solution is added: every hole adds luminescent solution 100 μ L;
(9) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
(10) detect with ten samples:
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 and be arranged on bag in box body by plate and reagent, described reagent comprise horseradish peroxidase mark sheep anti mouse antibody, wrap by solution, lock solution, cleansing solution and chemical luminescence for liquid, it is characterized in that: described bag is the luminous plaque being coated with CDV antibody by plate, 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 milky opaque polystyrene 96 hole Chemiluminescent plate;
The bag of described CDV antibody is that CDV antibody is placed in the bag of 0.1mol/L by solution, and at the temperature of 4 DEG C, reaction bag is by 12h, then adopts lock solution to be sealed in the hole on luminous plaque;
Described bag is sodium carbonate and the sodium bicarbonate carbonate buffer solution made soluble in water by solution, and the concentration of carbonate is 0.1mol/L;
Described lock solution is the BSA solution that the BSA adding 10g in every 100ml water makes, then add successively wherein the skimmed milk power of BSA solution weight 5%, the gelatin of 1% and 0.05% NaN3;
The detection method of mentioned reagent box, comprises the following steps:
1) join washing lotion: 50ml20 × cleansing solution is added purified water and is diluted to 1000ml, fully shake up rear for subsequent use;
2) number: should plate be established height Quality Control contrast sample wells at bag and number during test, all the other be measuring samples hole;
3) well dilution: add sample diluting liquid with sample injector, every hole 90ul; Quality Control contrast sample wells does not add sample diluting liquid;
4) application of sample: add corresponding Quality Control reference substance 100ul respectively at Quality Control contrast sample wells, and add measuring samples 10ul in each measuring samples hole, 37 ± 2 DEG C of incubations 30 minutes;
5) wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat 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 DEG C of incubations 30 minutes;
7) wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
8) luminescent solution is added: every hole adds luminescent solution 100 μ L;
9) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;
10) detect with ten samples.
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 horseradish peroxidase-sheep anti-mouse igg stoste dilution 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 0.01MpH=8.8Tris damping fluid, substrate B is add 3/10000 hydrogen peroxide after 0.001M dissolves 0.01MpH=8.8Tris damping fluid to iodophenol, described luminol is luminous substrate, is luminescence enhancer to iodophenol.
4. a kind of CDV antigen chemiluminescence detection kit according to claim 1, is characterized in that: described cleansing solution be containing mass concentration be 0.05% Tween-20, pH value be 7.5 0.1mol/L phosphate buffer.
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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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| CN103675273B true CN103675273B (en) | 2015-12-09 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0514678D0 (en) * | 2005-07-18 | 2005-08-24 | Europ Cardiovascular Genetics | Method for high-throughput screening |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| 犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立;孙婧等;《中国预防兽医学报》;20130831;第35卷(第8期);635-639 * |
| 犬瘟热病毒单抗夹心ELISA检测方法的建立;张鸿等;《扬州大学学报（农业与生命科学版）》;20111231;第32卷(第4期);15-18 * |
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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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