CN109765366A - A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody - Google Patents
A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody Download PDFInfo
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- CN109765366A CN109765366A CN201910096803.4A CN201910096803A CN109765366A CN 109765366 A CN109765366 A CN 109765366A CN 201910096803 A CN201910096803 A CN 201910096803A CN 109765366 A CN109765366 A CN 109765366A
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Abstract
The present invention provides a kind of kits and its detection method for detecting foot and mouth disease virus 3AB antibody, are related to Enzyme-multiplied immune technique field, the kit includes ELISA Plate and ELIAS secondary antibody；The ELISA Plate is coated with through foot and mouth disease virus 3AB albumen.The antibody of kit detection foot and mouth disease virus 3AB provided by the invention, have the advantages that efficient, sensitive specificity and repeatability are good, this method is easy to operate, quick, low in cost, it can Visual retrieval at room temperature, it is suitable for being promoted in clinical application, provides reliable technological means for the quick detection of foot and mouth disease virus 3AB antibody.
Description
Technical field
The present invention relates to Enzyme-multiplied immune technique fields, and in particular to a kind of kit for detecting foot and mouth disease virus 3AB antibody
And its detection method.
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 foot and mouth disease virus 3AB antibody 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
The purpose of the present invention is to provide a kind of kits and its detection method for detecting foot and mouth disease virus 3AB antibody.This
Invent the kit that provides can easy, quick and accurate detection foot and mouth disease virus 3AB antibody, without the instrument in laboratory
And equipment, be more suitable base detection.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of kits for detecting foot and mouth disease virus 3AB antibody, including ELISA Plate and ELIAS secondary antibody；Institute
ELISA Plate is stated to be coated with through foot and mouth disease virus 3AB albumen.
Preferably, the peridium concentration of the foot and mouth disease virus 3AB albumen is 1~2 μ g/mL.
Preferably, the kit further includes cleaning solution, serum dilution, substrate developing solution, terminate liquid, standard positive blood
Cleer and peaceful standard female serum.
Preferably, the cleaning solution includes phosphate buffer, is containing mass fraction in the phosphate buffer
0.03~0.08% Tween-20, the pH value of the phosphate buffer are 7.2.
Preferably, the serum dilution includes phosphate buffer, and skimmed milk power is contained in the buffer, described de-
The concentration of rouge milk powder is 45~55g/L.
Preferably, the substrate developing solution includes TMB solution.
Preferably, the terminate liquid includes SDS solution or sulfuric acid solution.
Preferably, the ELIAS secondary antibody includes the ELIAS secondary antibody of pig or the ELIAS secondary antibody of ox.
The present invention also provides the non-diagnostic destination detection foot and mouth disease virus 3AB of kit described in above-mentioned technical proposal is anti-
The method of body, comprising the following steps:
1) serum dilution is obtained into dilute serum with volume ratio 1:40 dilute serum sample；
2) the obtained dilute serum of step 1) is added in the hole of Xiang Suoshu ELISA Plate, it is incubated for 0.5 at 15~30 DEG C~
1.5h discards the liquid in hole, and cleaning solution washing pats dry, and obtains first and pats dry ELISA Plate；
3) it is patted dry to first that step 2) obtains and the ELIAS secondary antibody after dilution is added in the hole of ELISA Plate, at 15~30 DEG C
It is incubated for 0.5~1.5h, discards liquid in hole, cleaning solution washing pats dry, and obtains second and pats dry ELISA Plate；
4) addition substrate developing solution in the hole of ELISA Plate is patted dry to second that step 3) obtains, is protected from light at 15~30 DEG C aobvious
15~25min of color detects foot and mouth disease virus 3AB antibody when the color in hole is blue, when the color in hole is colourless
When, foot and mouth disease virus 3AB antibody is not detected.
Preferably, the ELIAS secondary antibody is after phosphate buffer dilutes 20000 times, in adding hole.
The present invention provides a kind of kits for detecting foot and mouth disease virus 3AB antibody, including ELISA Plate and ELIAS secondary antibody；Institute
ELISA Plate is stated to be coated with through foot and mouth disease virus 3AB albumen.In the present invention, after zoogenetic infection foot and mouth disease virus, it is anti-to generate 3AB
Body reaches testing goal by detection 3AB antibody.
The embodiment of the present invention is as the result is shown: the antibody of kit detection foot and mouth disease virus 3AB provided by the invention has
Efficiently, sensitive specificity and repeated good advantage, this method is easy to operate, quick, low in cost, can be at room temperature
Visual retrieval is suitable for being promoted in clinical application, provides reliably for the quick detection of foot and mouth disease virus 3AB antibody
Technological means.
Detailed description of the invention
Fig. 1 is kit test result provided by the invention, wherein from left to right, 1, hog cholera serum；2, encephalitis B virus
Serum；3, pig circular ring virus serum；4, pig parvoviral serum；5, foot and mouth disease virus serum；6, Pseudorabies virus serum；7, it is blue
Otopathy serum virus；8, swine flu serum.
Specific embodiment
The present invention provides a kind of kits for detecting foot and mouth disease virus 3AB antibody, including ELISA Plate and ELIAS secondary antibody；Institute
ELISA Plate is stated to be coated with through foot and mouth disease virus 3AB albumen.
In the present invention, the ELISA Plate is coated with through foot and mouth disease virus 3AB albumen, and the coated concentration is preferably 1~2
μg/mL.The present invention is not particularly limited the adopted method of packet, is coated with using conventional.
In the present invention, the ELISA Plate is preferably closed through the skimmed milk power solution that concentration is 50g/L.
In the present invention, the ELIAS secondary antibody preferably includes the ELIAS secondary antibody of pig or the ELIAS secondary antibody of ox, the enzyme of the pig
The ELIAS secondary antibody for marking secondary antibody or ox is preferably the product being commercialized.
In the present invention, the kit further preferably includes cleaning solution, serum dilution, substrate developing solution, terminate liquid, sun
Property serum and negative serum.
In the present invention, the cleaning solution preferably includes phosphate buffer, contains quality in the phosphate buffer
Score is preferably 0.03~0.08% Tween-20, more preferably 0.05%；The pH value of the phosphate buffer is 7.2.
In the present invention, the serum dilution preferably includes phosphate buffer, contains defatted milk in the buffer
Powder, the concentration of the skimmed milk power are preferably 45~55g/L, more preferably 50g/L.
In the present invention, the substrate developing solution preferably includes TMB solution, and the present invention does not have the concentration of the TMB solution
There is particular determination, when being developed the color using conventional substrate, required TMB concentration.
In the present invention, the terminate liquid preferably includes SDS solution or sulfuric acid solution.The present invention to the SDS solution or
The concentration of sulfuric acid solution is not particularly limited, and terminates concentration needed for enzyme linked immunoassay using conventional.
In the present invention, the standard positive serum is preferably the serum for infecting aftosa, and the standard female serum is excellent
It is selected as the serum of healthy animal.In embodiments of the present invention, the standard female serum is preferably A type foot and mouth disease virus feminine gender blood
Clearly；Standard positive serum is preferably A type foot and mouth disease virus positive serum.
The present invention also provides the non-diagnostic destination detection foot and mouth disease virus 3AB of kit described in above-mentioned technical proposal is anti-
The method of body, comprising the following steps:
1) serum dilution is obtained into dilute serum with volume ratio 1:40 dilute serum sample；
2) the obtained dilute serum of step 1) is added in the hole of Xiang Suoshu ELISA Plate, it is incubated for 0.5 at 15~30 DEG C~
1.5h discards the liquid in hole, and cleaning solution washing pats dry, and obtains first and pats dry ELISA Plate；
3) it is patted dry to first that step 2) obtains and the ELIAS secondary antibody after dilution is added in the hole of ELISA Plate, at 15~30 DEG C
It is incubated for 0.5~1.5h, discards liquid in hole, cleaning solution washing pats dry, and obtains second and pats dry ELISA Plate；
4) addition substrate developing solution in the hole of ELISA Plate is patted dry to second that step 3) obtains, is protected from light at 15~30 DEG C aobvious
15~25min of color detects foot and mouth disease virus 3AB antibody when the color in hole is blue, when the color in hole is colourless
When, foot and mouth disease virus 3AB antibody is not detected.
The serum dilution with volume ratio 1:40 dilute serum sample, is obtained dilute serum by the present invention.
The dilute serum that the present invention is added into the hole of the ELISA Plate is incubated for 0.5~1.5h at 15~30 DEG C,
The liquid in hole is discarded, cleaning solution washing pats dry, and obtains first and pats dry ELISA Plate.
In the present invention, the amount for dilute serum being added in the hole of the ELISA Plate is preferably 100 μ L.
In the present invention, it is incubated for after dilute serum being added in the hole of the ELISA Plate, the temperature of the incubation is 15
~30 DEG C, preferably 18~28 DEG C, more preferably 20~25 DEG C；The time of the incubation be 0.5~1.5h, preferably 1.8~
1.2h, most preferably 1h.
In the present invention, the number of the washing is preferably 3 times.
The present invention pats dry the ELIAS secondary antibody being added after dilution in the hole of ELISA Plate to first obtained, incubates at 15~30 DEG C
0.5~1.5h is educated, liquid in hole is discarded, cleaning solution washing pats dry, and obtains second and pats dry ELISA Plate.
In the present invention, in the hole of first ELISA Plate, the amount of the ELIAS secondary antibody after dilution is added is preferably 100 μ L.
In the present invention, the ELIAS secondary antibody is preferably diluted with phosphate buffer, and extension rate is preferably 20000 times.
In the present invention, the described first ELIAS secondary antibody for patting dry after dilution is added in the hole of ELISA Plate is incubated for, described
The temperature of incubation is 15~30 DEG C, preferably 18~28 DEG C, more preferably 20~25 DEG C；The time of the incubation be 0.5~
1.5h, preferably 0.8~1.2h, most preferably 1h.
In the present invention, the number of the washing is preferably 3 times.
The present invention is patted dry to second obtained is added substrate developing solution in the hole of ELISA Plate, colour developing is protected from light at 15~30 DEG C
15~25min detects foot and mouth disease virus 3AB antibody when the color in hole is blue, when the color in hole is colourless,
Foot and mouth disease virus 3AB antibody is not detected.
In the present invention, the additional amount of the substrate developing solution is preferably 100 holes μ L/.In the present invention, the colour developing
Temperature is preferably 15~30 DEG C, more preferably 18~28 DEG C, most preferably 20~25 DEG C；The time of the colour developing is preferably 18~
23min, more preferably 20min.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
1, the preparation of sample diluting liquid, cleaning solution, terminate liquid
Coating buffer is 0.05M carbonate buffer solution: Na2CO31.59g NaHCO32.93g adds distilled water to be settled to
1000mL, pH 9.6；
Sample diluting liquid is the cleaning solution of the skimmed milk power containing 50g/L；
Cleaning solution is the phosphate buffer that the 0.01MpH containing 0.05%Tween-20 is 7.2, formula are as follows:
NaCl:8.5g, NaH2PO4·2H2O:0.356g, NaH2PO4·12H2O:2.772g, after being mixed, then with steam
Distilled water dissolves and is settled to 1000mL, then 0.5mL Tween-20 is added thereto and obtains above-mentioned cleaning solution；
Terminate liquid are as follows: configuration 1%SDS solution.
2, the determination of the optimal dilution of the most suitable peridium concentration and serum of antigen
As the result is shown when the dilution of antigen is the 2.0 every holes μ g/mL, serum is diluted with 1:40 times, and ELIAS secondary antibody is with 1:
20000 times of dilutions.
3, the optimization of action time
The best off-period of antigen is 20min, and the seroreaction time is 1h, and the best incubation time of ELIAS secondary antibody is
40min, best developing time are 20min.
4. the determination of operation sequence
According to optimum operation condition determined by the above items, determining operation sequence are as follows: taking-up has been coated with and has closed
Elisa plate item, serum to be checked is 1:40 times with serum dilution and is diluted, be added each hole of ELISA Plate, every 100 μ L of hole, respectively plus
One hole, A type foot and mouth disease virus is negative, positive serum respectively adds two holes (positive serum and negative serum are respectively to infect aftosa
Serum and healthy animal serum dilute 100 times with PBS respectively), room temperature acts on 1h；Liquid in hole is discarded, every hole is washed with cleaning solution
It 3 times and pats, abandons liquid in hole to the greatest extent；100 μ L ELIAS secondary antibodies are added in every hole, and room temperature acts on 1h；Discard liquid in hole, every hole is with washing
It washs liquid to wash 3 times and pat, abandons liquid in hole to the greatest extent；It is aobvious that 100 μ LTMB (3,3', 5,5'- tetramethyl benzidine) substrate is added in every hole
After color liquid, room temperature is protected from light colour developing 20min, and 100 μ LSDS terminate liquids are added；Visual color, blue are the positive, colourless for yin
Property.
Embodiment 2
The specific test of kit:
Known swine influenza virus, swine fever virus, encephalitis B virus, pig annulus are detected respectively by the method that embodiment 1 is established
Virus, pig parvoviral, Pseudorabies virus, reproductive and respiratory syndrome virus and foot and mouth disease virus 3AB positive serum samples, feminine gender be it is colourless,
The positive is blue.The result is shown in Figure 1.
As can be seen from FIG. 1, aftosa can accurately be detected using kit provided by the invention, with other viruses without intersecting
Reaction has specificity.
By above embodiments, it can be concluded that, the antibody of kit detection foot and mouth disease virus 3AB provided by the invention has height
Effect, sensitive specificity and repeated good advantage, this method is easy to operate, quick, low in cost, can at room temperature may be used
It is detected depending on changing, is suitable for being promoted in clinical application, provide reliable skill for the quick detection of foot and mouth disease virus 3AB antibody
Art 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 (10)
1. a kind of kit for detecting foot and mouth disease virus 3AB antibody, which is characterized in that including ELISA Plate and ELIAS secondary antibody；
The ELISA Plate is coated with through foot and mouth disease virus 3AB albumen.
2. kit according to claim 1, which is characterized in that the peridium concentration of the foot and mouth disease virus 3AB albumen is 1
~2 μ g/mL.
3. kit according to claim 1 or 2, which is characterized in that the kit further includes cleaning solution, serum dilution
Liquid, substrate developing solution, terminate liquid, standard positive serum and standard female serum.
4. kit according to claim 3, which is characterized in that the cleaning solution includes phosphate buffer, the phosphorus
The Tween-20 for being 0.03~0.08% containing mass fraction in phthalate buffer, the pH value of the phosphate buffer are 7.2.
5. kit according to claim 3, which is characterized in that the serum dilution includes phosphate buffer, institute
It states and contains skimmed milk power in buffer, the concentration of the skimmed milk power is 45~55g/L.
6. kit according to claim 3, which is characterized in that the substrate developing solution includes TMB solution.
7. kit according to claim 3, which is characterized in that the terminate liquid includes that SDS solution or sulfuric acid are molten
Liquid.
8. kit according to claim 1, which is characterized in that the ELIAS secondary antibody includes ELIAS secondary antibody or the ox of pig
ELIAS secondary antibody.
9. utilizing the side of according to any one of claims 1 to 88 described in any item non-diagnostic destination detection foot and mouth disease virus 3AB antibody of kit
Method, which comprises the following steps:
1) serum dilution is obtained into dilute serum with volume ratio 1:40 dilute serum sample；
2) dilute serum that step 1) obtains is added in the hole of Xiang Suoshu ELISA Plate, 0.5~1.5h is incubated at 15~30 DEG C, is abandoned
The liquid in hole is removed, cleaning solution washing pats dry, and obtains first and pats dry ELISA Plate；
3) it is patted dry to first that step 2) obtains and the ELIAS secondary antibody after dilution is added in the hole of ELISA Plate, be incubated at 15~30 DEG C
0.5~1.5h discards liquid in hole, and cleaning solution washing pats dry, and obtains second and pats dry ELISA Plate；
4) addition substrate developing solution in the hole of ELISA Plate is patted dry to second that step 3) obtains, and colour developing 15 is protected from light at 15~30 DEG C
~25min detects foot and mouth disease virus 3AB antibody when the color in hole is blue, when the color in hole is colourless, not
Detect foot and mouth disease virus 3AB antibody.
10. detection method according to claim 9, which is characterized in that the ELIAS secondary antibody is diluted through phosphate buffer
After 20000 times, in adding hole.
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Application publication date: 20190517 |