CN109342725A - A kind of canine distemper, canine parvovirus detection kit - Google Patents
A kind of canine distemper, canine parvovirus detection kit Download PDFInfo
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- CN109342725A CN109342725A CN201811450672.7A CN201811450672A CN109342725A CN 109342725 A CN109342725 A CN 109342725A CN 201811450672 A CN201811450672 A CN 201811450672A CN 109342725 A CN109342725 A CN 109342725A
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- monoclonal antibody
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
-
- 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
Abstract
The invention discloses a kind of canine distempers, canine parvovirus detection kit, including canine parvovirus prevention monoclonal antibody CD1, CD2, anti-dog parvovirus monoclonal antibody CP1, CP2, coated slab, europium, samarium labelled reagent, chromatographic column, eluent, analysis buffer and standard items；The invention also discloses a kind of canine distemper virus, the detection method of canine parvovirus.Canine distemper of the invention, canine parvovirus detection kit use time resolution detection technique, detection sensitivity is improved, the tiny two kinds of epidemic diseases of canine distemper, dog can be detected simultaneously, provided convenience to user and reduce use cost, between that detects criticizes and batch interior difference is small, and performance is stable.
Description
Technical field
The present invention relates to technical field of virus detection, especially a kind of canine distemper, canine parvovirus detection kit.
Background technique
With the continuous improvement of people's quality of life, the number of animals raised of pet dog also increases year by year, and canine infectious disease is quick-fried
Hair causes tremendous influence to it, and the prevention and control form for these infectious diseases are also further severe.In canine epidemic disease, have
Two kinds of strong infectious diseases have tortured the life of countless dog, make the bitterly thorough heart of the owner of countless pet dogs --- and here it is dogs
Pest heat and canine parvovirus.Canine distemper is a kind of highly contagious disease as caused by canine distemper virus (CDV), and infectiousness is strong,
The death rate may be up to 80% or more.It is special that two-phase heat type, rhinitis, serious digestive disorder and respiratory inflammation etc. is presented in sick dog
Encephalitis can occur for sign, a small number of cases.The natural reservoir (of bird flu viruses) of infection is Canidae and mustelid, is mainly passed by air and droplet level
It broadcasts.In recent years, there is CDV natually morbid in Carnivora, Artiodactyla Suidae, the Macaca of Primates and clasper mesh Phocidae etc.
Report, or even also someone infects the case of CDV.Tiny dog is a kind of urgency as caused by canine parvovirus (CPV) infection puppy
Sexually transmitted disease is outwardly propagated through excrement, urine, saliva and vomitus, clinically there are two types of phenotype, hemorrhagic enteritis type with
Violent vomiting, hemorrhagic enteritis and leucocyte is significantly reduced to main feature；Myocarditis type then characterized by die by visitation of God, this
Two types disease all has the feature that disease incidence is high, the death rate is high and infectiousness is strong.Canine distemper and the tiny disease incidence of dog and
The death rate is all higher, needs quickly to detect, and accomplishes early diagnosis, early isolation, early prevents and treats the therapeutic effect that can be only achieved.
And the kit of prevalence often can only detect canine distemper on the market or dog is tiny, be detected using 2 kits
Workload is virtually increased, cost is improved.The having of kit of existing detection canine distemper and canine parvovirus in the market
Learn luminescence method, colloidal gold method and ELISA method, wherein chemoluminescence method needs the instrument of mating large size, it is difficult to realize detection by bed；
Traditional colloidal gold method, no standard measure, and detection sensitivity is low；Quantitative detection may be implemented in ELISA method, but sensitivity still can not
Meet the dynamic detection to the course of disease, treatment, prognosis.
Summary of the invention
Based on the above issues, a kind of hundstaupe is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
Heat, canine parvovirus detection kit, which can detect canine distemper, canine parvovirus simultaneously, convenient and efficient, can be to disease
Poison is quantitative, and detection sensitivity is high, save the cost.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of canine distemper, canine parvovirus detection kit, including canine parvovirus prevention monoclonal antibody CD1, CD2 resist
Canine parvovirus monoclonal antibody CP1, CP2, coated slab, europium, samarium labelled reagent, chromatographic column, eluent, analysis buffer and
Standard items.It should be noted that canine parvovirus prevention monoclonal antibody includes but is not limited to canine parvovirus prevention in this kit
The monoclonal antibody CD1 and CD2 of hemagglutinin H protein further include the other lists that can be targeted in conjunction with canine distemper virus hemagglutinin H protein
Clonal antibody, as long as the monoclonal antibody specificity is strong, affinity is high；Similarly, anti-dog parvovirus in this kit
Monoclonal antibody includes but is not limited to the monoclonal antibody CP1 and CP2 of anti-dog parvovirus VP2 albumen, further includes that can target knot
The other monoclonal antibodies for closing canine parvovirus VP2 protein albumen, as long as the monoclonal antibody specificity is strong, affinity is high.Its
The affinity of middle monoclonal antibody CD1 and CD2 can be identical, can also be variant, but poor to the affinity of hemagglutinin H protein
It is different to must not exceed 10%；Similarly, the affinity of monoclonal antibody CP1 and CP2 can be identical, can also be variant, but right
The affinity difference of VP2 albumen must not exceed 10%, in order to avoid lead to detection error.
Preferably, europium, samarium labelled reagent are Eu3+And Sm3+Labelling kit.
Preferably, the chromatographic column is Sephadex G-50 chromatographic column.
Preferably, the analysis buffer is Tris-HCl, NaCl, BSA, Tween-20 and NaN3Mixed solution.
Preferably, the analysis buffer is the Tris-HCl of 50mmol/L, is also contained in every liter of analysis buffer
The NaCl of 0.09% (W/V), 0.02% (W/V) BSA, the NaN of 0.05% (W/V) Tween-20 and 0.05% (W/V)3。
Preferably, the pH value of the analysis buffer is 7.8.
Preferably, the standard items the preparation method comprises the following steps: the standard items with the Tris-HCl (pH 7.8) of 50mmol/L are slow
Fliud flushing is dense at 0,5,25,50,100,200ng/mL 6 by canine distemper virus antigen, Canine Parvovirus antigen accurate dilutions respectively
Degree to get, wherein the standard items buffer contains 0.2% (W/V) BSA and 0.1% (W/V) NaN3。
As another aspect of the present invention, the present invention also provides a kind of canine distemper virus, the detection of canine parvovirus
Method includes the following steps:
(1) respectively to the enzyme of coating canine parvovirus prevention monoclonal antibody CD1, anti-dog parvovirus monoclonal antibody CP1
CDV/CPV standard items, sample to be tested are added on yoke plate；
(2) analysis buffer is added to step (1) gained elisa plate, concussion incubates, then cleaning solution washing is added to divide
Analyse the diluted labelled antibody Eu of buffer3+- CD2 monoclonal antibody and Sm3+The mixture of-CP2 monoclonal antibody, concussion incubate,
Cleaning solution washing；
(3) it after being eventually adding enhancement solution concussion, is detected on time-resolved fluorescence detector.
Preferably, the detection method includes the following steps:
(1) 25 μ L CDV/CPV standard items, sample to be tested are added on the elisa plate of coating CD1, CP1 antibody respectively；
(2) 200 μ L analysis buffers are added to elisa plate obtained by step (1), shake and incubates 1h, cleaning solution washing 4 times, so
After be added with the diluted labelled antibody Eu in analysis buffer 1:50~100 times3+- CD2 monoclonal antibody and Sm3+- CP2 monoclonal
200 hole μ L/ of mixture of antibody, concussion incubate 1h, and cleaning solution washs 6 times；
(3) it after being eventually adding 200 hole μ L/ of enhancement solution concussion 5min, is detected on time-resolved fluorescence detector.
In conclusion the invention has the benefit that
Canine distemper of the invention, canine parvovirus detection kit use time resolution detection technique, improve detection spirit
Sensitivity can detect the tiny two kinds of epidemic diseases of canine distemper, dog simultaneously, use cost is provided convenience and reduced to user, detection
Batch between and batch in difference it is small, performance stablize；With the antibody such as dog is coronal, dog influenza, mad dog without obvious cross reaction.
Detailed description of the invention
Fig. 1 is the standard curve of canine distemper virus antigen；
Fig. 2 is the standard curve of Canine Parvovirus antigen.
Specific embodiment
Canine distemper of the invention, canine parvovirus detection kit, as marker, are adopted using time-resolved fluorescence microballoon
With double antibody sandwich method, sensitivity and the anti-interference ability of detection are improved.Using time-resolved fluorescence microballoon as marker,
The influence that the stray lights such as background fluorescence and the exciting light in sample can effectively be avoided, improve detection sensitivity and
Anti-interference ability；And test speed is fast.Two kinds of detection methods (time resolution and double antibody sandwich method) is integrated into one by the present invention
A kit, provides convenience to user and reduces use cost.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.Unless otherwise instructed, the reagent in the application or kit can be from markets or other public
Channel obtains；Experimental method in the present invention is conventional method.
Embodiment 1
A kind of embodiment of canine distemper, canine parvovirus detection kit of the invention, including canine parvovirus prevention Dan Ke
Grand antibody CD1, CD2, anti-dog parvovirus monoclonal antibody CP1, CP2, coated slab, Eu3+And Sm3+Labelling kit,
Sephadex G-50 chromatographic column, eluent, analysis buffer and standard items；Analysis buffer be Tris-HCl, NaCl, BSA,
Tween-20 and NaN3Mixed solution.
Wherein, following method preparation is respectively adopted in each ingredient:
1, the preparation of anti-canine distemper, canine parvovirus antibody
It is immune that BALB/c mouse is carried out respectively with canine distemper virus (CDV) and canine parvovirus (CPV).Pass through cell fusion
Technology carries out monoclonal antibody preparation, and the monoclonal for filtering out the high anti-canine distemper hemagglutinin H protein of high specificity, affinity is anti-
The monoclonal antibody CP1 and CP2 of body CD1 and CD2 and anti-dog parvovirus VP2 albumen.
2, coating plate preparation
Anti- CDV, CPV monoclonal antibody CD1, CP1 of purifying Tris-HCl buffer is diluted, 3 μ g/mL are diluted to,
100 holes μ L/ are added in each hole of coated slab, and anti-CDV, CPV monoclonal antibody coating is obtained after absorption, washing, closing, drying
Lath.
3、Eu3+And Sm3+The preparation of labelled antibody
By anti-CDV, CPV monoclonal antibody CD2, CP2 to be marked be separately added into Millipore company with filter membrane from
It is centrifuged in heart pipe, for several times with label buffer repeated washing.Again by the anti-canine distemper of 200 μ L, canine parvovirus labelled antibody (CD2,
CP2 it) is mixed well respectively with europium, samarium labelled reagent, room temperature concussion is overnight.Sephadex G-50 chromatographic column is used after the completion of label
It isolates and purifies, elution is collected simultaneously efflux, measures absorbance by pipe, merges peak pipe, measures protein content, then
Respectively according to Eu3+And Sm3+Formula provided by labelling kit specification measures mark rate and protein recovery.
4, dilution is analyzed
Analysis buffer are as follows: 50mmol/L pH 7.8Tris-HCl, every liter contains 0.09% (W/V) NaCl, 0.02% (W/
V) BSA, 0.05% (W/V) Tween-20,0.05% (W/V) NaN3。
5, prepared by standard items
With containing 0.2% (W/V) BSA, 0.1% (W/V) NaN3, the standard items of the Tris-HCl (pH 7.8) of 50mmol/L are slow
Fliud flushing is by canine distemper virus antigen diluent at 0,5,25,50,100,200ng/mL series standard solution；By Canine Parvovirus antigen
It is diluted to 0,5,25,50,100,200ng/mL series standard solution.
Embodiment 2
A kind of embodiment of the detection method of canine distemper virus, canine parvovirus of the invention, includes the following steps:
25 μ L CDV/CPV reference standard product or sample to be tested are added on the elisa plate of coating CD1, CP1 antibody, then plus
Enter 200 μ L analysis buffers, concussion incubates 1h, and cleaning solution washs 4 times, adds with the dilution of analysis buffer 1:50~100 times
Labelled antibody Eu3+- CD2 monoclonal antibody and Sm3+200 hole μ L/ of mixture of-CP2 monoclonal antibody, concussion incubate 1h, wash
It washs liquid to wash 6 times, be detected on time-resolved fluorescence detector after being eventually adding 200 hole μ L/ of enhancement solution concussion 5min.
For double labelling canine distemper/canine parvovirus-TRFIA standard curve referring to Fig. 1, canine distemper and canine parvovirus standard are bent
The related coefficient of line is respectively R2=0.9943 and R2=0.9939, show that linear dependence is good.The detection spirit of canine distemper virus
Sensitivity is 0.43ng/mL, canine parvovirus 0.81ng/mL.
The specificity of embodiment 3 detection kit of the invention and method
It chooses several common viral antigens and carries out specific analysis, canine influenza virus, canine coronavirus, rabies viruses are used
The measurement of the method for 1 kit of embodiment and embodiment 2 the results are shown in Table 1, the kit measurement high concentration dog influenza disease of embodiment 1
When poison, canine coronavirus, rabies viruses, measurement concentration is far below theoretical concentration, illustrates the specificity of this method preferably.
1 specificity experiments result (ng/mL) of table
The precision of embodiment 4 detection kit of the invention and method
(1) Precision Experiment
Using embodiment 1 kit and embodiment 2 method to high, normal, basic three standard concentrations of accurate quantification into
Row measurement, is respectively arranged 10 multiple holes.As shown in table 2, the variation within batch coefficient of the kit of embodiment 1 and interassay coefficient of variation are equal
≤ 10%, meet kit prescribed requirement.
2 precision experiment result of table
(2) recovery experiment
Conventionally carry out recovery experiment.It the results are shown in Table 3, the rate of recovery of high, normal, basic three concentration is in 99.5-
Between 100.42%.
3 recovery experiment result of table
The stability of the kit of the invention of embodiment 5
After each component part of the kit of embodiment 1 is put in 37 DEG C of 7d that accelerate the failure, then each component is detected, is shone
Value decreases, but amplitude of variation shows kit of the present invention and its detection method is stable within 10%.
The clinical application of the kit of the invention of embodiment 6
Taking 84 parts of Guangzhou Fu Mao pet clinic canine distemper to be checked, dog, tiny (enzyme-linked immunosorbent assay kit is purchased from south respectively
Jing Senbeijia Biotechnology Co., Ltd, the refined Anda Bioisystech Co., Ltd in Beijing, article No.: SBJ-Q0004,48T/96T) it doubts
Like case sample, detected using the kit of embodiment 1.
Specific detecting step: 25 μ L samples to be tested are added on coating elisa plate, add 200 μ L analysis buffers, shake
It swings and incubates 1h, cleaning solution washs 4 times, adds with diluted 200 hole μ L/ of labelled antibody in analysis buffer 1:50~100 times, shake
It swings and incubates 1h, cleaning solution washs 6 times, is eventually adding after 200 hole μ L/ of enhancement solution concussion 5min on time-resolved fluorescence detector
It is detected.Viral antigen concentration carries out quantitative calculating according to the standard curve that standard items and corresponding RLU value are established.
It is 36 parts that enzyme-linked immunosorbent assay, which detects the canine distemper positive, and feminine gender is 48 parts, is with this kit detection positive
37 parts, feminine gender is 47 parts；It is 28 parts that enzyme-linked immunosorbent assay, which detects the tiny positive of dog, and feminine gender is 56 parts, is examined with this kit
Surveying the tiny positive of dog is 30 parts, and feminine gender is 54 parts.
The result shows that: canine distemper positive rate 44% is detected with the method for the present invention, the detection of enzyme-linked immunosorbent assay is positive
The coincidence rate of rate 42.9%, this kit test result and enzyme-linked immunosorbent assay is 97.3%；
Canine parvovirus this kit Positive rate is 35.7%, the Positive rate of enzyme-linked immunosorbent assay
33.3%, the coincidence rate of this kit test result and enzyme-linked immunosorbent assay is 93.3%.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can with modification or equivalent replacement of the technical solution of the present invention are made, without departing from technical solution of the present invention essence and
Range.
Claims (9)
1. a kind of canine distemper, canine parvovirus detection kit, which is characterized in that including canine parvovirus prevention monoclonal antibody
CD1, CD2, anti-dog parvovirus monoclonal antibody CP1, CP2, coated slab, europium, samarium labelled reagent, chromatographic column, eluent, point
Analyse buffer and standard items.
2. detection kit according to claim 1, which is characterized in that europium, samarium labelled reagent are Eu3+And Sm3+Label examination
Agent box.
3. detection kit according to claim 1, which is characterized in that the chromatographic column is Sephadex G-50 chromatography
Column.
4. detection kit according to claim 1, which is characterized in that the analysis buffer be Tris-HCl, NaCl,
BSA, Tween-20 and NaN3Mixed solution.
5. detection kit according to claim 4, which is characterized in that the analysis buffer is 50mmol/L's
Tris-HCl also contains the NaCl, 0.02% (W/V) BSA, 0.05% (W/V) of 0.09% (W/V) in every liter of analysis buffer
The NaN of Tween-20 and 0.05% (W/V)3。
6. detection kit according to claim 5, which is characterized in that the pH value of the analysis buffer is 7.8.
7. detection kit according to claim 1, which is characterized in that the standard items the preparation method comprises the following steps: with
The standard items buffer of the Tris-HCl (pH 7.8) of 50mmol/L is quasi- by canine distemper virus antigen, Canine Parvovirus antigen respectively
Really be diluted to 0,5,25,50,100,200ng/mL 6 concentration to get, wherein the standard items buffer contains 0.2% (W/
V) BSA and 0.1% (W/V) NaN3。
8. a kind of canine distemper virus, the detection method of canine parvovirus, which comprises the steps of:
(1) respectively to the elisa plate of coating canine parvovirus prevention monoclonal antibody CD1, anti-dog parvovirus monoclonal antibody CP1
Upper addition CDV/CPV standard items, sample to be tested；
(2) analysis buffer is added to elisa plate obtained by step (1), concussion incubates, cleaning solution washing, is then added slow to analyze
The diluted labelled antibody Eu of fliud flushing3+- CD2 monoclonal antibody and Sm3+The mixture of-CP2 monoclonal antibody, concussion incubate, washing
Liquid washing；
(3) it after being eventually adding enhancement solution concussion, is detected on time-resolved fluorescence detector.
9. detection method according to claim 8, which comprises the steps of:
(1) 25 μ L CDV/CPV standard items, sample to be tested are added on the elisa plate of coating CD1, CP1 antibody respectively；
(2) 200 μ L analysis buffers are added to elisa plate obtained by step (1), concussion incubates 1h, and cleaning solution washs 4 times, then plus
Enter with the diluted labelled antibody Eu in analysis buffer 1:50~100 times3+- CD2 monoclonal antibody and Sm3+- CP2 monoclonal antibody
200 hole μ L/ of mixture, concussion incubate 1h, cleaning solution wash 6 times；
(3) it after being eventually adding 200 hole μ L/ of enhancement solution concussion 5min, is detected on time-resolved fluorescence detector.
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| CN201811450672.7A CN109342725A (en) | 2018-11-29 | 2018-11-29 | A kind of canine distemper, canine parvovirus detection kit |
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