CN109655623A - A kind of the visualization quick detection kit and its application of A type antibodies against foot-and-mouth disease virus - Google Patents
A kind of the visualization quick detection kit and its application of A type antibodies against foot-and-mouth disease virus Download PDFInfo
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- CN109655623A CN109655623A CN201910096472.4A CN201910096472A CN109655623A CN 109655623 A CN109655623 A CN 109655623A CN 201910096472 A CN201910096472 A CN 201910096472A CN 109655623 A CN109655623 A CN 109655623A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- 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
- 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
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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Abstract
The present invention provides a kind of visualization quick detection kits of A type antibodies against foot-and-mouth disease virus, belong to technical field of biological.Kit provided by the invention includes coated elisa plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, standard positive serum and standard female serum, for the coated elisa plate using A type foot and mouth disease virus as envelope antigen, the peridium concentration of the envelope antigen is 1.5~3 μ g/mL.Kit provided by the invention can be used to detect A type antibodies against foot-and-mouth disease virus, determined by color change as a result, have the advantages that efficiently, it is high sensitivity, high specificity, reproducible.Kit provided by the invention is easy to operate, quick, low in cost, can under room temperature (20~22 DEG C) Visual retrieval, be suitable for being promoted in clinical application, for A type antibodies against foot-and-mouth disease virus it is quick detection reliable technological means is provided.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of visualization of A type antibodies against foot-and-mouth disease virus is quick
Detection kit and its application.
Background technique
Aftosa is a kind of acute, hot, highly contagious disease caused by foot and mouth disease virus (FMDV), main
Artiodactyl beast is encroached on, is often propagated in the animals such as sheep, ox, pig.There is blister in the lip of susceptible animal and hoof when morbidity, simultaneously
With symptoms such as fever, loss of appetite, infected animal weight and the output of milk decline to a great extent.Foot and mouth disease virus is easily passed by air
It broadcasts, infectiousness is strong, and popular quick, susceptible animal also results in death in the case where resistance is weaker, causes to raiser huge
Huge economic loss seriously hinders the development of aquaculture.
The evaluation method of the diagnostic method for A type antibodies against foot-and-mouth disease virus and immune effect of vaccine can only test at present
Room is completed, and base's detection is not suitable for, and needs to establish more sensitive, quick and easy visual detection method.
Summary of the invention
In view of technical problem present in background technique, the purpose of the present invention is to provide a kind of A type foot and mouth disease viruses
The visualization quick detection kit of antibody and its application.
The present invention provides a kind of visualization quick detection kits of A type antibodies against foot-and-mouth disease virus, including coating enzyme mark
Plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, the coated elisa plate is with A type foot and mouth disease virus
As envelope antigen, the peridium concentration of the envelope antigen is 1.5~3 μ g/mL.
Preferably, the coated elisa plate is made by confining liquid closing envelope antigen, the confining liquid be include 40~
The PBST buffer of 60g/L skimmed milk power, the closed time are 15~25min；The coated elisa plate is placed in 2~6 DEG C
It saves.
Preferably, the cleaning solution be include 0.04%~0.06% volumetric concentration Tween-20 phosphate buffer,
PH value is 7~7.4.
Preferably, the sample diluting liquid is the phosphate buffer for including 40~60g/L skimmed milk power.
Preferably, the substrate developing solution is TMB solution.
Preferably, the extension rate of the ELIAS secondary antibody is 1:(15000~25000).
Preferably, the terminate liquid is the SDS solution of 0.8~1.2% volumetric concentration.
The present invention provides above-mentioned visualization quick detection kits to carry out vaccine inoculation to A type antibodies against foot-and-mouth disease virus
The application in detection afterwards.
Preferably, the application includes the following steps:
(1) vitro samples to be detected are taken, vitro samples are diluted with the sample diluting liquid, described diluted times
Rate is 1:(30~50), obtain dilute sample；
(2) dilute sample is added in coated elisa plate, 20~22 DEG C of 30~40min of incubation, abandons reaction solution, washes
Liquid board-washing is washed, it is dry；
(3) ELIAS secondary antibody is added in the ELISA Plate after step (2) described drying, 20~22 DEG C of incubations 30~
40min, abandons reaction solution, and cleaning solution board-washing is dry；
(4) the substrate developing solution is added in the ELISA Plate after step (3) described drying, 20~22 DEG C of chromogenic reactions
10~20min judges testing result.
The utility model has the advantages that the present invention provides a kind of visualization quick detection kits of A type antibodies against foot-and-mouth disease virus, including
Coated elisa plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, the coated elisa plate is with A type mouth
Aphtovirus is 1.5~3 μ g/mL as envelope antigen, the peridium concentration of the envelope antigen.Kit energy provided by the invention
For detecting A type antibodies against foot-and-mouth disease virus, determined by color change as a result, have efficiently, it is high specificity, reproducible
Advantage.Kit provided by the invention is easy to operate, quick, low in cost, can Visual retrieval at room temperature, be suitable for facing
It is promoted in bed application, provides reliable technological means for the quick detection of A type antibodies against foot-and-mouth disease virus.
Detailed description of the invention
Fig. 1 is specific detection experimental result picture described in the embodiment of the present invention 2.
Specific embodiment
The present invention provides a kind of visualization quick detection kits of A type antibodies against foot-and-mouth disease virus, including coating enzyme mark
Plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, the coated elisa plate is with A type foot and mouth disease virus
As envelope antigen, the peridium concentration of the envelope antigen is 1.5~3 μ g/mL.
Kit provided by the invention includes coated elisa plate.In the present invention, the coated elisa plate is with A type aftosa
Virus is envelope antigen, can be captured the A type antibodies against foot-and-mouth disease virus specificity in sample to be tested when detecting.In the present invention
In, the envelope antigen is A type foot and mouth disease virus, and peridium concentration is 1.5~3 μ g/mL.The present invention is to the A type hoof-and-mouth disease
The source of poison is not particularly limited, this field conventional commercial product.
The present invention is closed envelope antigen into coated elisa plate using confining liquid.In the present invention, the confining liquid is excellent
It is selected as the PBST buffer containing skimmed milk power.Concentration of the skimmed milk power in PBST buffer is preferably 40~60g/L,
More preferably 50g/L.The closed time is preferably 15~25min, more preferably 20min.The present invention is slow to the PBST
The source of fliud flushing and the skimmed milk power is not particularly limited, this field conventional commercial product.In the embodiment of the present invention
In, the PBST, skimmed milk power are purchased from sigma.After envelope antigen closing to ELISA Plate, the present invention is preferably to packet
It is saved by ELISA Plate.The temperature of the preservation is preferably 2~6 DEG C, and more preferably 4 DEG C；It is described preservation preferably in hermetic bag into
Row.
Kit provided by the invention includes ELIAS secondary antibody.In the present invention, it is caught on the ELIAS secondary antibody energy and ELISA Plate
The combination of specificity occurs for the A type antibodies against foot-and-mouth disease virus obtained, and chromogenic reaction can occur with developing solution.In the present invention,
The ELIAS secondary antibody is purchased from sigma.The present invention is preferably when detecting diluted ELIAS secondary antibody.In the present invention, the enzyme
The extension rate for marking secondary antibody is preferably 1:(15000~25000), more preferably 1:20000.
Kit provided by the invention further includes cleaning solution, sample diluting liquid, substrate developing solution and terminate liquid.In the present invention
In, the cleaning solution is preferably the phosphate buffer containing Tween-20.Body of the Tween-20 in phosphate buffer
Product concentration is preferably 0.04%~0.06%, and more preferably 0.05%.The pH value of the phosphate buffer is preferably 7~7.4,
More preferably 7.2.In the present invention, the sample diluting liquid is preferably the phosphate buffer containing skimmed milk power.It is described de-
Concentration of the rouge milk powder in phosphate buffer is preferably 40~60g/L, more preferably 50g/L.In the present invention, the substrate
Developing solution is preferably TMB solution (3,3', 5,5'- tetramethyl biphenyl amine aqueous solution).In the present invention, the terminate liquid is preferably
SDS or sulfuric acid solution.The percentage by volume of the SDS is preferably 0.8%~1.2%, and more preferably 1%.The sulfuric acid is molten
The concentration of liquid is preferably 1~3mol/L, more preferably 2mol/L.The present invention is molten to the phosphate buffer, Tween-20, TMB
Liquid, SDS solution source be not particularly limited, this field conventional commercial product.In an embodiment of the present invention, the phosphorus
Phthalate buffer, Tween-20, TMB solution and SDS are purchased from sigma.
Kit provided by the invention further preferably includes standard positive serum and standard female serum.The standard positive blood
It is clearly A type foot and mouth disease virus positive Swine serum；The standard female serum is healthy Swine serum；The standard positive serum and mark
Quasi- negative serum can be used for the sample to be tested of check experiment in the detection process.In an embodiment of the present invention, the A type mouth
Aphtovirus positive Swine serum and healthy Swine serum are provided by Lanzhou veterinary institute.
Kit provided by the invention can be used to detect A type antibodies against foot-and-mouth disease virus, determined by color change as a result,
Has the advantages that efficient, high specificity, reproducible.
The preparation method of the kit is not particularly limited in the present invention.Each component is prepared using raw material of the present invention
Obtain the solution of respective concentration to obtain the final product.
The present invention also provides above-mentioned visualization quick detection kits to carry out inoculation epidemic disease to A type antibodies against foot-and-mouth disease virus
The application in detection after seedling.The application preferably includes following steps:
(1) vitro samples to be detected are taken, vitro samples are diluted with the sample diluting liquid, described diluted times
Rate is 1:(30~50), obtain dilute sample；
(2) dilute sample is added in coated elisa plate, 20~22 DEG C of 30~40min of incubation, abandons reaction solution, washes
Liquid board-washing is washed, it is dry；
(3) ELIAS secondary antibody is added in the ELISA Plate after step (2) described drying, 20~22 DEG C of incubations 30~
40min, abandons reaction solution, and cleaning solution board-washing is dry；
(4) the substrate developing solution is added in the ELISA Plate after step (3) described drying, 20~22 DEG C of chromogenic reactions
10~20min judges testing result.
(1) step of the invention first takes vitro samples to be detected, is diluted with the sample diluting liquid to vitro samples.
In the present invention, the diluted multiplying power is preferably 1:(30~50), dilute sample is obtained after more preferably 1:40. dilution.
The dilute sample is added in coated elisa plate by (2) step of the invention to be incubated for.In the present invention, the incubation
Temperature be preferably 20~22 DEG C.The time of the incubation is preferably 30~40min, more preferably 35min.Reaction is abandoned after incubation
Liquid, cleaning solution board-washing, the number of the board-washing are preferably 2~3 times.Dry after board-washing, the mode of the drying preferably absorbs water
Paper pats dry.
The ELIAS secondary antibody is added in the ELISA Plate after step (2) described drying by (3) step of the invention further incubates
It educates.In the present invention, the temperature being further incubated for is preferably 20-22 DEG C, and more preferably 22 DEG C.It is described to be further incubated for
Time is preferably 30~40min, more preferably 40min.Abandon reaction solution after being further incubated for, cleaning solution board-washing, the board-washing
Number is preferably 2~3 times.Dry after board-washing, the mode of the drying is preferably that blotting paper pats dry.
The substrate developing solution is added in the ELISA Plate after step (3) described drying by (4) step of the invention to develop the color.?
In the present invention, the temperature of the colour developing is preferably 20-22 DEG C, and more preferably 22 DEG C.The time of the chromogenic reaction is preferably 10
~20min, more preferably 20min.Testing result is judged after chromogenic reaction.In the present invention, the testing result is sentenced
SDS terminate liquid is added in disconnected preferably pass through, and visually observes and is judged: blue is the positive, and colourless is negative.
Kit provided by the invention is easy to operate, quick, low in cost, can Visual retrieval at room temperature, be suitable for
It is promoted in clinical application, provides reliable technological means for the quick detection of A type antibodies against foot-and-mouth disease virus.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
(1) production of coated elisa plate and the preparation of sample diluting liquid, cleaning solution, terminate liquid
0.05mol/L carbonate buffer solution is prepared as coating buffer: Na3CO31.59g NaHCO32.93g adds distilled water fixed
Hold to 1000mL, pH 9.6.A type foot and mouth disease virus is diluted using coating buffer, dilution is 2.0 μ g/mL.By the A type after dilution
Foot-and-mouth disease virus antigen is added in ELISA Plate, and every hole adds 100 μ L volumes.Then it is closed with 50g/L skimmed milk power PBST
20min obtains coated elisa plate.It is packed into hermetic bag, 4 DEG C save backup.
The phosphate buffer containing 50g/L skimmed milk power is prepared, as sample diluting liquid；
The phosphate buffer that the 0.01mol/LpH containing 0.05%Tween-20 is 7.2 is prepared as cleaning solution: NaCl
8.5g, NaH2PO4·2H2O 0.356g, NaH2PO4·12H2O 2.772g, after being mixed, then with distilled water dissolution and constant volume
To 1000mL, then addition 0.5mL Tween-20 thereto；
The sulfuric acid solution of 1% SDS or 2mol/L is prepared as terminate liquid.
2, sample detection
(1) blood serum sample to be detected is taken, using sample diluting liquid, with 1:40 times of dilute serum；
(2) the elisa plate item (coated elisa plate) for being coated with and having closed is taken out, the diluted blood of 100 μ L is added in every hole
Clearly；The control group of addition standard positive serum and standard female serum (healthy Swine serum) is set simultaneously, in room temperature (20-22 DEG C)
It is incubated for 1h, discards liquid in hole, every hole is washed 3 times with cleaning solution and patted, and liquid in hole to the greatest extent is abandoned；
(3) ELIAS secondary antibody (purchase sigma) is taken, using sample diluting liquid, dilutes ELIAS secondary antibody with 1:20000 times.Then
ELIAS secondary antibody after dilution is added in ELISA Plate, 100 μ L are added in every hole, in (20-22 DEG C) incubation 40min of room temperature, is abandoned
Liquid in dereaction hole, every hole are washed 3 times with cleaning solution and are patted, and liquid in hole to the greatest extent is abandoned；
(4) 100 μ L substrate TMB developing solutions are added into the every hole of ELISA Plate, (20-22 DEG C) is protected from light chromogenic reaction at room temperature
100 μ LSDS terminate liquids are added in 20min；Visual color, blue are the positive, colourless for feminine gender.
Embodiment 2
The specific test of kit: known swine fever virus, encephalitis B virus, pig are detected respectively as described in Example 1
Circovirus, pig parvoviral, Pseudorabies virus, reproductive and respiratory syndrome virus, influenza virus and A type foot and mouth disease virus positive blood final proof
Product, feminine gender be it is colourless, the positive for blue.
Testing result is as shown in Figure 1, Fig. 1 is from left to right followed successively by swine fever virus, A type foot and mouth disease virus, encephalitis B virus, pig
Circovirus, pig parvoviral, Pseudorabies virus, reproductive and respiratory syndrome virus and influenza virus.Fig. 1 the result shows that: the embodiment of the present invention 1
The kit of offer to swine fever virus, encephalitis B virus, pig circular ring virus, pig parvoviral, Pseudorabies virus, reproductive and respiratory syndrome virus with
And the positive serum samples testing result of influenza virus is in colourless, to the testing result of A type foot and mouth disease virus positive serum samples
It is blue.Illustrate kit provided by the invention for detecting A type antibodies against foot-and-mouth disease virus, have efficient, sensitive specificity and
The good advantage of repeatability.And it is easy to operate, quick, low in cost, can Visual retrieval at room temperature, be suitable for facing
It is promoted in bed application, provides reliable technological means for the quick detection of A type antibodies against foot-and-mouth disease virus.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of visualization quick detection kit of A type antibodies against foot-and-mouth disease virus, including coated elisa plate, cleaning solution, sample
Dilution, substrate developing solution, ELIAS secondary antibody, terminate liquid, which is characterized in that the coated elisa plate is with A type foot and mouth disease virus work
For envelope antigen, the peridium concentration of the envelope antigen is 1.5~3 μ g/mL.
2. visualization quick detection kit according to claim 1, which is characterized in that the coated elisa plate is by closing
Fluid-tight is closed envelope antigen and is made, and the confining liquid is the PBST buffer for including 40~60g/L skimmed milk power, when described closed
Between be 15~25min；The coated elisa plate is placed in 2~6 DEG C of preservations.
3. visualization quick detection kit according to claim 1, which is characterized in that the cleaning solution is to include
The phosphate buffer of the Tween-20 of 0.04%~0.06% volumetric concentration, pH value are 7~7.4.
4. visualization quick detection kit according to claim 1, which is characterized in that the sample diluting liquid is to include
The phosphate buffer of 40~60g/L skimmed milk power.
5. visualization quick detection kit according to claim 1, which is characterized in that the substrate developing solution is TMB
Solution.
6. visualization quick detection kit according to claim 1, which is characterized in that the dilution of the ELIAS secondary antibody times
Number is 1:(15000~25000).
7. visualization quick detection kit according to claim 1, which is characterized in that the terminate liquid be 0.8~
The SDS solution of 1.2% volumetric concentration.
8. visualization quick detection kit described in claim 1~7 any one is carried out to A type antibodies against foot-and-mouth disease virus
The application in detection after vaccine inoculation.
9. application according to claim 8, which comprises the steps of:
(1) vitro samples to be detected are taken, vitro samples are diluted with the sample diluting liquid, the diluted multiplying power is
1:(30~50), obtain dilute sample；
(2) dilute sample is added in coated elisa plate, 20~22 DEG C of 30~40min of incubation, abandons reaction solution, cleaning solution
Board-washing, it is dry；
(3) ELIAS secondary antibody is added in the ELISA Plate after step (2) described drying, 20~22 DEG C of 30~40min of incubation,
Reaction solution is abandoned, cleaning solution board-washing is dry；
(4) the substrate developing solution is added in the ELISA Plate after step (3) described drying, 20~22 DEG C of chromogenic reactions 10~
20min judges testing result.
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| 杨玉平 主编。: "《动物微生物》", 31 July 2012 * |
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Application publication date: 20190419 |