CN103630688A - Chemiluminiscence detection kit for canine parvovirus - Google Patents

Chemiluminiscence detection kit for canine parvovirus Download PDF

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Publication number
CN103630688A
CN103630688A CN201310556137.0A CN201310556137A CN103630688A CN 103630688 A CN103630688 A CN 103630688A CN 201310556137 A CN201310556137 A CN 201310556137A CN 103630688 A CN103630688 A CN 103630688A
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canine parvovirus
kit
solution
detection
liquid
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CN103630688B (en
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王善普
李秀梅
李长印
王会会
张莎莎
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Luoyang Modern Biotechnology Research Institute Co., Ltd.
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LUOYANG LAIPSON INFORMATION TECHNOLOGY Co Ltd
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus

Abstract

The invention provides a chemiluminiscence detection kit for canine parvovirus and relates to a kit for detecting the canine parvovirus. The kit comprises a kit body, wherein the kit body is internally provided with a polystyrene 96-pore micro-pore plate coated with a 10 micrograms/milliliter canine parvovirus coating antibody, a canine parvovirus antigen quality control comparison product, a goat-anti-mouse antibody solution marked by horseradish peroxidase, a canine parvovirus standard solution, a sample diluted solution, a concentrated washing solution and a chemiluminiscence solution. According to the kit, the sensitivity can be up to 3ng/mL; the kit has the characteristics of high specificity, wide detection range, simple operation method, easiness in observing of a reaction result and the like; the detection efficiency is improved and the detection accuracy is guaranteed; the chemiluminiscence detection kit for the canine parvovirus can be used for export inspection, food safety inspection and field rapid detection of animal breeding industries.

Description

A kind of canine parvovirus chemiluminescence detection kit
Technical field
The present invention relates to immunology detection field, relate to specifically a kind of canine parvovirus chemiluminescence detection kit.
Background technology
Canine parvovirus (CPV) is the virus that U.S. Eugster found from the dog ight soil of the courageous and upright enteritis in affected part in 1978, and this virus is that Parvoviridae parvovirus belongs to, sub-thread wire DNA virus.Canine parvovirus to be to cause the dog incidence of disease high, the important Causative virus that infectiousness is strong, mortality ratio high has become canid clinical onset.Canine parvovirus has become China and has supported one of infectious disease that dog industry is the most serious, and this disease had both affected the life and health of domestic pets, is endangering again the development that dog industry, Fur Animal Feeding industry and conservation of wildlife industry are supported by China.
At present, the traditional detection method of canine parvovirus 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 canine parvovirus, 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 and meet basic unit and use.Along with molecular biological development, existing multiple PCR method, for the detection of canine parvovirus, though improve to some extent aspect sensitivity, owing to needing supporting the use of exact instrument, equally also cannot meet the needs of existing detection at present.
Summary of the invention
The object of this invention is to provide and a kind ofly have that highly sensitive, high specific, method of operating are simple, reaction result is easy to observe, be highly suitable for the chemical luminescence reagent kit of Site Detection canine parvovirus.
To achieve these goals, technical scheme of the present invention is: a kind of canine parvovirus chemiluminescence detection kit, comprise box body, in box body, be provided with the sheep anti-mouse antibody solution, canine parvovirus standard solution, sample diluting liquid, concentrated cleaning solution and the chemical luminescence for liquid that are coated with polystyrene 96 hole microwell plates that concentration is the canine parvovirus coated antibody of 10ug/mL, canine parvovirus antigen Quality Control reference substance, horseradish peroxidase mark.
Described canine parvovirus antigen Quality Control reference substance comprises the low Quality Control reference substance of 30-80IU/L and the high Quality Control reference substance of 200-350IU/L.
Described canine parvovirus standard solution has 6 kinds, and concentration is respectively: 16ng/mL, 22ng/mL, 35ng/mL, 40ng/mL, 55ng/mL and 65ng/mL.
Described sample diluting liquid is by 8g NaCl, 0.2g KH 2p0 4, 22.9g Na 2hP0 412H 20,0.2g KC1 and casein are dissolved in 1000mL distilled water and are prepared from, and wherein casein concentration is 1%.
Described concentrated cleaning solution is by 80g NaCl, 2.0g KH 2p0 4, 229.0g Na 2hP0 412H 20,2.0g KC1 and Tween-20 are dissolved in 1000mL distilled water and are prepared from, and wherein the concentration of Tween-20 is 5%.
Described chemical luminescence for liquid is divided into Chemoluminescent substrate A and two kinds of liquid of Chemoluminescent substrate B, Chemoluminescent substrate A for the preparation of Tris damping fluid containing 2mmol/L luminol and the mixed liquor of 0.2mmol/L to iodophenol, Chemoluminescent substrate B is the mixed liquor containing 7.5mmol/L hydrogen peroxide with the preparation of Tris damping fluid.
The sheep anti-mouse antibody solution of described horseradish peroxidase mark is horseradish peroxidase-sheep anti-mouse igg stoste.
The coated process of described canine parvovirus coated antibody is that canine parvovirus monoclonal antibody is dissolved in to the solution that forms 10ug/mL in sodium carbonate-sodium bicarbonate buffer liquid of 0.1mol/L, above-mentioned solution is joined in each hole of polystyrene 96 hole microwell plates, and at 4 ℃ standing 12h, then above-mentioned solution inclines, with cleansing solution wash each hole 3 times, pat dry, then to each hole, add the confining liquid in 150ul/ hole, and hatch 2h at 37 ℃, liquid in hole inclines, cleansing solution washing 3 times, pat dry, finally microwell plate is used to aluminium foil bag vacuum seal, and preserve at 4 ℃.Wherein, the dilutability of sodium carbonate-sodium bicarbonate buffer liquid of described canine parvovirus monoclonal antibody and 0.1mol/L is 1:800, and described confining liquid is to be dissolved in 100ml water by 10g BSA, then adds 5% skimmed milk power, 1% gelatin, 0.05% NaN 3make.
Beneficial effect: the highly sensitive 3ng/mL that reaches of kit of the present invention, and have that high specific, wide sensing range, method of operating are simple, reaction result is easy to the features such as observation, 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.
Embodiment
A kind of canine parvovirus chemiluminescence detection kit, comprise box body, in box body, be provided with the sheep anti-mouse antibody solution, canine parvovirus standard solution, sample diluting liquid, concentrated cleaning solution and the chemical luminescence for liquid that are coated with polystyrene 96 hole microwell plates that concentration is the canine parvovirus coated antibody of 10ug/mL, canine parvovirus antigen Quality Control reference substance, horseradish peroxidase mark.
Described canine parvovirus antigen Quality Control reference substance comprises the low Quality Control reference substance of 30-80IU/L and the high Quality Control reference substance of 200-350IU/L.
Described canine parvovirus standard solution has 6 kinds, and concentration is respectively: 16ng/mL, 22ng/mL, 35ng/mL, 40ng/mL, 55ng/mL and 65ng/mL.
Described sample diluting liquid is by 8g NaCl, 0.2g KH 2p0 4, 22.9g Na 2hP0 412H 20,0.2g KC1 and casein are dissolved in 1000mL distilled water and are prepared from, and wherein casein concentration is 1%.
Described concentrated cleaning solution is by 80g NaCl, 2.0g KH 2p0 4, 229.0g Na 2hP0 412H 20,2.0g KC1 and Tween-20 are dissolved in 1000mL distilled water and are prepared from, and wherein the concentration of Tween-20 is 5%.
Described chemical luminescence for liquid is divided into two kinds of Chemoluminescent substrate A and Chemoluminescent substrate B, Chemoluminescent substrate A for the preparation of Tris damping fluid containing 2mmol/L luminol and the mixed liquor of 0.2mmol/L to iodophenol, Chemoluminescent substrate B is the mixed liquor containing 7.5mmol/L hydrogen peroxide with the preparation of Tris damping fluid.
The sheep anti-mouse antibody solution of described horseradish peroxidase mark is horseradish peroxidase-sheep anti-mouse igg stoste.
The coated process of described canine parvovirus coated antibody is that canine parvovirus monoclonal antibody is dissolved in to the solution that forms 10ug/mL in sodium carbonate-sodium bicarbonate buffer liquid of 0.1mol/L, above-mentioned solution is joined in each hole of polystyrene 96 hole microwell plates, and at 4 ℃ standing 12h, then above-mentioned solution inclines, with cleansing solution wash each hole 3 times, pat dry, then to each hole, add the confining liquid in 150ul/ hole, and hatch 2h at 37 ℃, liquid in hole inclines, cleansing solution washing 3 times, pat dry, finally microwell plate is used to aluminium foil bag vacuum seal, and preserve at 4 ℃.Wherein, the dilutability of sodium carbonate-sodium bicarbonate buffer liquid of described canine parvovirus monoclonal antibody and 0.1mol/L is 1:800, and described confining liquid is to be dissolved in 100ml water by 10g BSA, then adds 5% skimmed milk power, 1% gelatin, 0.05% NaN 3make.
The method of applying kit detection canine parvovirus of the present invention comprises the steps:
(1) join washing lotion: by 20 times of distilled water dilutings for concentrated cleaning solution, make the cleansing solution of working concentration;
(2) numbering: on the described polystyrene 96 hole microwell plates that were coated with canine parvovirus coated antibody, establish high Quality Control reference substance hole, low Quality Control reference substance hole, canine parvovirus standard solution hole and sample well to be checked, and numbering;
(3) well dilution: add sample diluting liquid to sample well to be checked, every hole 90ul;
(4) application of sample: add corresponding Quality Control reference substance 100ul to high Quality Control reference substance hole and low Quality Control reference substance hole respectively, in canine parvovirus standard solution hole, add each standard solution 100ul/ hole, and to each sample well to be checked, add sample 10ul to be checked, incubation 30 minutes at 37 ± 2 ℃ then;
(5) washing: incline and the liquid in each hole, then add the cleansing solution 250 μ L of step (1) to each hole, wash 3 times, pat dry;
(6) enzyme-added mark sheep anti-mouse antibody solution: the dilution that the cleansing solution of the sheep anti-mouse antibody solution of horseradish peroxidase mark and step (1) is mixed with to 1:2500 working concentration, then to each hole, add the above-mentioned dilution of 100 μ L, then at 37 ± 2 ℃ incubation 30 minutes;
(7) washing: incline and the liquid in each hole, the cleansing solution 250 μ L that every hole adds step (1), wash 3 times, pat dry;
(8) add chemical luminescence for liquid: to each hole, all successively add each 50 μ L of Chemoluminescent substrate A and Chemoluminescent substrate B;
(9) detect: the luminous intensity of measuring each hole with chemical illumination immunity analysis instrument;
(10) result judgement: the concentration logarithm with canine parvovirus standard solution is done horizontal ordinate, the luminous value logarithm of standard solution is done ordinate, do typical curve, according to the luminous value of each sample to be checked, can calculate from typical curve the concentration of sample to be checked.
The testing result of the specificity of kit of the present invention, sensitivity and precision is as follows:
1, specific test result
Figure 888973DEST_PATH_IMAGE001
The luminous value testing result of high and low Quality Control reference substance meets standard.
2, accuracy test findings
Figure 2013105561370100002DEST_PATH_IMAGE002
Accuracy: CV%=8%.
3, sensitivity test result
Figure 868431DEST_PATH_IMAGE003
The sensitivity of this kit is 3ng/mL as calculated.

Claims (1)

1. a canine parvovirus chemiluminescence detection kit, comprise box body, it is characterized in that: in box body, be provided with the sheep anti-mouse antibody solution, canine parvovirus standard solution, sample diluting liquid, concentrated cleaning solution and the chemical luminescence for liquid that are coated with polystyrene 96 hole microwell plates that concentration is the canine parvovirus coated antibody of 10ug/mL, canine parvovirus antigen Quality Control reference substance, horseradish peroxidase mark;
Described canine parvovirus standard solution has 6 kinds, and concentration is respectively: 16ng/mL, 22ng/mL, 35ng/mL, 40ng/mL, 55ng/mL and 65ng/mL;
Described sample diluting liquid is by 8g NaCl, 0.2g KH 2p0 4, 22.9g Na 2hP0 412H 20,0.2g KC1 and casein are dissolved in 1000mL distilled water and are prepared from, and wherein casein concentration is 1%;
Described concentrated cleaning solution is by 80g NaCl, 2.0g KH 2p0 4, 229.0g Na 2hP0 412H 20,2.0g KC1 and Tween-20 are dissolved in 1000mL distilled water and are prepared from, and wherein the concentration of Tween-20 is 5%;
Described chemical luminescence for liquid is divided into Chemoluminescent substrate A and two kinds of liquid of Chemoluminescent substrate B, Chemoluminescent substrate A for the preparation of Tris damping fluid containing 2mmol/L luminol and the mixed liquor of 0.2mmol/L to iodophenol, Chemoluminescent substrate B is the mixed liquor containing 7.5mmol/L hydrogen peroxide with the preparation of Tris damping fluid.
CN201310556137.0A 2013-11-11 2013-11-11 A kind of canine parvovirus chemiluminescence detection kit Active CN103630688B (en)

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Cited By (1)

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CN109444411A (en) * 2018-10-26 2019-03-08 成都普利泰生物科技有限公司 A kind of canine parvovirus antibody chemical luminescence immue quantitative detection reagent box

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Publication number Priority date Publication date Assignee Title
CN109444411A (en) * 2018-10-26 2019-03-08 成都普利泰生物科技有限公司 A kind of canine parvovirus antibody chemical luminescence immue quantitative detection reagent box

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Inventor after: Li Xiumei

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