CN109324193A - A kind of the visualization quick detection kit and its application of antibody against swine fever virus - Google Patents
A kind of the visualization quick detection kit and its application of antibody against swine fever virus Download PDFInfo
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- CN109324193A CN109324193A CN201811494762.6A CN201811494762A CN109324193A CN 109324193 A CN109324193 A CN 109324193A CN 201811494762 A CN201811494762 A CN 201811494762A CN 109324193 A CN109324193 A CN 109324193A
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- G—PHYSICS
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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Abstract
The present invention provides a kind of visualization quick detection kits of antibody against swine fever 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 swine fever virus E2 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 the antibody of swine fever 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 (20~25 DEG C) Visual retrieval, be suitable for being promoted in clinical application, for antibody against swine fever 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 antibody against swine fever virus quickly detects examination
Agent box and its application.
Background technique
Swine fever is a kind of with epidemic disease highly infectious, is one of the Infectious Diseases for threatening pig breeding industry, it is characterized in that
It is acute, change in septic, organa parenchymatosum's bleeding, necrosis and infarct, there is generation in most of China province.Swine fever is at all seasons
It may all occur, be more with spring and summer rainy season.It is strong with infectiousness, popular quick feature.Susceptible animal is in resistance
It is easy death in the case where weaker, and then huge economic loss can be caused to raiser, the serious development for hindering aquaculture.
The cause of disease of swine fever is Alphaherpesvirinae, the swine fever virus in pestivirus (Pestivirus).It is main to pass through directly
Contact, or due to contact stain medium and fall ill.Alimentary canal, nasal membrane and the skin of rupture are routes of infection.Mesh
Preceding is mostly completed in laboratory for the diagnostic method of antibody against swine fever virus and the evaluation method of immune effect of vaccine, is not suitable for
Base's detection.It thus needs to establish more sensitive, quick and easy visual detection method.
Summary of the invention
The problem of in view of background technique, the purpose of the present invention is to provide a kind of the visual of antibody against swine fever virus
Change quick detection kit, including coated elisa plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid,
Standard positive serum and standard female serum, the coated elisa plate is using swine fever virus albumen E2 as envelope antigen, the coating
The peridium concentration of 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 sulfuric acid solution of the SDS or 1~3mol/L of 0.8~1.2% volumetric concentration.
The present invention also provides above-mentioned visualization quick detection kits after carrying out vaccine inoculation to antibody against swine fever virus
Detection in application.
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, 20~25 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, 20~25 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, 20~25 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 kit of antibody against swine fever virus, including coating
ELISA Plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, standard positive serum and standard female blood
Clearly, for the coated elisa plate using swine fever virus albumen E2 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 the antibody of swine fever virus, be determined by color change as a result, having height
Effect, high specificity, reproducible advantage.Kit provided by the invention is easy to operate, quick, low in cost, can be in room temperature
Lower Visual retrieval is suitable for being promoted in clinical application, provides reliable skill for the quick detection of antibody against swine fever virus
Art means.
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 kit of antibody against swine fever virus, including coated elisa plate, wash
Wash liquid, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, standard positive serum and standard female serum, the packet
By ELISA Plate using swine fever virus albumen E2 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 swine fever virus
For envelope antigen, the antibody against swine fever virus specificity in sample to be tested can be captured when detecting.In the present invention, the packet
It is swine fever virus albumen E2 by antigen, peridium concentration is 1.5~3 μ g/mL.Source of the present invention to the swine fever virus albumen E2
It 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 antibody against swine fever virus obtained, and chromogenic reaction can occur with developing solution.In the present invention, the enzyme
Mark secondary antibody is purchased from sigma.The present invention is preferably when detecting diluted ELIAS secondary antibody.In the present invention, the ELIAS secondary antibody
Extension rate be 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 that TMB solution (3,3', 5,5'- tetramethyl biphenyl amine aqueous solution) is purchased from sigma.In the present invention, the termination
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%.Institute
The concentration for stating sulfuric acid solution is preferably 1~3mol/L, more preferably 2mol/L.The present invention is to the phosphate buffer, tween-
20, TMB solution, SDS solution source be not particularly limited, this field conventional commercial product.In the embodiment of the present invention
In, the phosphate 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 swine fever virus positive Swine serum；The standard female serum is swine fever virus feminine gender Swine serum；The standard positive serum
It can be used for the sample to be tested of check experiment in the detection process with standard female serum.In an embodiment of the present invention, described
Swine fever virus positive Swine serum and swine fever virus feminine gender Swine serum are provided by Lanzhou veterinary institute.
Kit provided by the invention can be used to detect the antibody of swine fever virus, be determined by color change as a result, having
Have the advantages that efficiently, it is 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 after carrying out vaccine inoculation to antibody against swine fever virus
Detection in application.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, 20~25 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, 20~25 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, 20~25 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 20~25 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 20-25 DEG C, and more preferably 25 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 20-25 DEG C, and more preferably 25 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 antibody against swine fever virus.
Below with reference to embodiment to a kind of visualization quick detection kit of antibody against swine fever virus provided by the invention and
Its application is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1, the 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: Na3ABO31.59g NaHCO32.93g adds distilled water fixed
Hold to 1000mL, pH 9.6.CSFV antigen E2 is diluted using coating buffer, dilution is 2.0 μ g/mL.By the pig after dilution
Pestivirus antigen is added in ELISA Plate, and every hole adds 100 μ L volumes.Then 20min is closed with 50g/L skimmed milk power PBST, obtained
To 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 (A type foot and mouth disease virus) is set simultaneously, in room temperature (20-25
DEG C) it is incubated for 1h, liquid in hole is discarded, 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 (20-25 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, (20-25 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.Sulfuric acid can also be added to terminate
Liquid reads each hole OD value in 450nm wavelength with microplate reader, determines result.
Embodiment 2
The specific test of kit: detect respectively as described in Example 1 known foot and mouth disease virus, encephalitis B virus,
Pig circular ring virus, pig parvoviral, Pseudorabies virus, reproductive and respiratory syndrome virus and swine fever virus positive serum samples, feminine gender be it is colourless,
The positive is blue.
Testing result is as shown in Figure 1.Fig. 1 is followed successively by foot and mouth disease virus (No. 1), swine fever virus positive blood final proof from left to right
Product (No. 2), encephalitis B virus (No. 3), pig circular ring virus (No. 4), pig parvoviral (No. 5), Pseudorabies virus (No. 6), hog cholera
Malicious positive serum samples (No. 7) and reproductive and respiratory syndrome virus (No. 8).Fig. 1 the result shows that: the embodiment of the present invention 1 provide kit pair
Porcine epidemic diarrhea virus, foot and mouth disease virus, encephalitis B virus, pig circular ring virus, pig parvoviral, Pseudorabies virus and blue otopathy
Virus-positive blood serum sample testing result is blue to swine fever virus positive serum samples testing result in colourless.Illustrate this hair
Bright kit is used to detect the antibody of swine fever virus, has the advantages that efficient, sensitive specificity and repeatability are good.And it grasps
Make it is easy, quick, low in cost, can Visual retrieval at room temperature, be suitable for being promoted in clinical application, for swine fever
The quick detection of antiviral antibody provides reliable technological means.
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 antibody against swine fever virus, including the dilution of coated elisa plate, cleaning solution, sample
Liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, which is characterized in that the coated elisa plate is coating with swine fever virus albumen E2
Antigen, the peridium concentration of the envelope antigen are 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 sulfuric acid solution of the SDS or 1~3mol/L of 1.2% volumetric concentration.
8. visualization quick detection kit described in claim 1~7 any one is inoculated with to antibody against swine fever virus
The application in detection after vaccine.
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, 20~25 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, 20~25 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, 20~25 DEG C of chromogenic reactions 10~
20min judges testing result.
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