CN110346566A - PPR virus antibody assay kit - Google Patents
PPR virus antibody assay kit Download PDFInfo
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- CN110346566A CN110346566A CN201811121776.3A CN201811121776A CN110346566A CN 110346566 A CN110346566 A CN 110346566A CN 201811121776 A CN201811121776 A CN 201811121776A CN 110346566 A CN110346566 A CN 110346566A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to PPR virus antibody assay kits.Detection kit of the invention includes one or more solid carriers, and particular polypeptide or the particular polypeptide combination being independently connected on one or more of solid carriers.
Description
The application is that (applying date is 04 month 2018 application No. is 201810310284.2 Chinese invention patent application
0130 day, entitled PPR virus antibody assay kit) divisional application.
Technical field
The invention mainly relates to diagnostic kit for animals and diagnostic methods.Specifically, the present invention relates to detections pair
As biological source biological sample in the presence or absence of anti-PPR virus antibody kit.
Background technique
Peste des petits ruminants (peste des petits ruminants, PPR) is commonly called as sheep pest also known as small ruminates the false ox of beast
Pest (pseudorinderpest), pneumoenteritis (pneumoenteritis), the compound disease (stomatitis- of stomatitis pneumoenteritis
Pneumoenteritis complex), it is a kind of acute viral infectious disease as caused by PPR virus, it is main to feel
Small ruminant is contaminated, characterized by fever, stomatitis, diarrhea, pneumonia.
Nineteen forty-two, this disease occurred in the Ivory Coast for the first time, and thereafter, African Senegal, Ghana, Togo, Benin etc. have this
Disease is reported, this disease also has occurred in the sheep and goat of Nigeria, and cause heavy losses.Some countries in Asia also report
This disease of road, according to World Organization for Animal Health (OIE) 1993 " world animal health " reports, the goat of Bangladesh had this
The disease of similar rinderpest has occurred in disease generation, India De Labang and some areas sheep of horse Harrar Shi Telabang, it is last true
It examines as peste des petits ruminants, hereafter, safe Mir's ladd nation is also infected report.1993, Israel's first time report had small
Epizootic disease is ruminated, source of infection is unknown, and to prevent this disease from propagating, Israel is vaccinated with the sheep and goat of its northern territory
Rinderpest vaccines.1992, this disease-specific antibody was had found in the sheep and goat of Jordan, 1993, there are 11 farms to occur
Clinical case, more than 100 sheep and goat death.1993, Saudi Arabia found 133 cases for the first time.In July, 2007,
The epidemic disease is passed to China Tibet region for the first time.
PPR virus category paramyxovirus section Morbillivirus.There is similar physical chemistry with rinderpest virus and is immunized
Learn characteristic.Virus is in pleomorphism, usually coarse spherical shape.Virion is big compared with rinderpest virus, and nucleocapsid is spiral hollow stem
Shape simultaneously has characteristic subunit, there is cyst membrane.Virus can be in tire sheep renal, testicular cell, the Vero cell of tire sheep and newborn sheep
Upper proliferation, and cytopathy (CPE) is generated, form plasomidum.
The small ruminants such as this disease main infection goat, sheep, U.S. white-tailed deer are popular in African western, middle part and Asia
The some areas in continent.In epidemic-stricken area, this disease is that fragmentary generation can happening and prevelence when susceptible animal increases.This disease mainly passes through
Immediate contagion, the secretion and excreta of ill domestic animal are the infections sources, and the sick sheep for facing the type of examining in Asia is especially dangerous.It is small to ruminate beast
Epidemic disease incubation period is 4-5 days, longest 21 days.Natural occurrence is detected in goat and sheep.Goat morbidity is serious, and sheep also occasionally has seriously
Case occurs.The lip of some rehabilitation goats forms aphtha sample lesion.Infection animal clinical symptom is similar to rinderpest infected cattle.It is acute
Type body temperature can rise to 41 DEG C, and continue 3~5 days.Infection animal is had the fidgets, and dorsal body setae is unglazed, and mouth and nose are dry, appetite stimulator.
Mucus purulent rhinorrhea is flowed, foul gas is breathed out.At first 4 days of fever, mucous membrane of mouth was congested, cheek mucous membrane progressive popularity damage
Evil leads to polysialia, gangrenosum acne lesion then occurs, starts mucous membrane of mouth and small coarse red superficial necrotic lesion occurs, with
After become pink, infection site includes lower lip, lower gums etc..The visible necrotic lesion of several cases involves tooth pad, palate, cheek
And its nipple, tongue etc..There is band blood watery diarrhea in later period, and serious dehydration is thin, therewith temperature decline.There is cough, exhale
It inhales abnormal.Disease incidence is up to 100%, and when seriously breaking out, the death rate 100%, in slight occur, the death rate is no more than
50%.Growing animal morbidity severe morbidity and dead all very high, a kind of disease delimited for China.
Because this virus has special affinity to gastrointestinal lymphoid cell and epithelial cell, therefore feature venereal disease can be caused
Become.Generally occur acidophilia cytoplasmic inclusions and multinucleate giant cell in infection cell.In lymphoid tissue, peste des petits ruminants disease
Poison can cause lymphocyte downright bad.Spleen, tonsillotome, lymph node cells are destroyed.The multicore of the cytoplasmic inclusions containing acidophilia is huge
Cell occurs, rarely intranuclear inclusion.In digestive system, it is bad that virus causes the epithelial cell in Ma Erjishi layer depth portion to occur
Extremely, infection cell generates karyopycnosis and karyorrhexis, and it is big and small to form the multicore containing acidophilia cytoplasmic inclusions in stratum germinativum epidermidis
Born of the same parents.
Currently, effective ways there is no to treat peste des petits ruminants, vaccine inoculation, epidemic situation can only be taken to slaughter and periodically after occurring
The method of sero monitoring is controlled.Therefore, the serodiagnosis of the disease and the assessment of immune effect of vaccine seem with monitoring
It is particularly important.
World Organization for Animal Health recommends the PPR virus antibody detection method used mainly to have virus to neutralize examination
Test (VNT) and enzyme-linked immunosorbent assay (ELISA).Wherein, the testing result of virus neutralization tests method is accurate, and being that detection is small ruminates
The goldstandard of epizootic disease virus, but this method detection time is long and is unsuitable for detecting great amount of samples.In contrast, enzyme-linked immunosorbent assay
Specificity and sensibility is higher, detection time is shorter than virus neutralization tests and therefore the suitable a large amount of samples of detection are answered extensively
Detection for PPR virus antibody.
Enzyme-linked immunosorbent assay method for PPR virus antibody test mainly includes competitive enzyme-linked immune measurement
(c-ELISA), enzyme-linked immunosorbent assay (b-ELISA) and indirect enzyme-linked immunosorbent measurement (indirect ELISA) are blocked.C-ELISA and b-
The specificity and sensibility of ELISA is relatively high, is by clinical generally accepted PPR virus antibody detection method, example
C-ELISA method is used if the peste des petits ruminants diagnostic kit in the French laboratory BIRAD general in the world.But
C-ELISA and b-ELISA is both needed to using monoclonal antibody, this causes testing cost to greatly improve；Moreover, c-ELISA and b-
The cumbersome of ELISA, detection time long (although having been shortened relative to VNT), criterion are complicated.On the other hand, traditional
Indirect ELISA use from PPR virus intact proteins (such as H protein, N protein, F protein etc.) or recombination egg
White as envelope antigen to detect the antibody in serum, cost is lower than c-ELISA and b-ELISA；But since this method makes
It uses intact proteins as antigen, is easy to happen the wrong identification and non-specific identification of antibody, therefore, specificity and sensibility
All not as good as c-ELISA and b-ELISA.
Therefore, it is necessary to develop, testing cost is low, detection time is short, easy to operate, specific and high sensibility small anti-
Hay epizootic disease virus antibody assay kit.
Summary of the invention
In view of above-mentioned problems of the prior art, low, detection that the purpose of the present invention is to provide a kind of testing costs
Time short, easy to operate, specific and high sensibility PPR virus antibody assay kit, and can be used in making
The polypeptide or polypeptides in combination of the standby kit.
Based on the indirect ELISA method of antigen epitope polypeptide due to use antigen epitope polypeptide (20 amino acid or so
Single polypeptide usually only includes an epitope) it is used as envelope antigen.Inventor's discovery: the sensibility of this method is often inclined
Low, the sensibility of single polypeptide does not exceed 50% generally；And the polypeptide that sensitivity is high, false positive rate are also high, i.e., it is specific low.
If all high antigen epitope polypeptide of sensibility and specificity can be found, so that it may the shortcomings that overcoming traditional indirect ELISA,
Developing specificity and sensibility can match in excellence or beauty c-ELISA and b-ELISA, even higher PPR virus antibody test
Kit.
Inventor has made intensive studies to solve above-mentioned technical problem, as a result, it has been found that more shown in SEQ ID NO:1
Peptide with the sensibility that the polypeptide individually detects PPR virus antibody is 70.2%, specificity 99.4%.In particular,
When the polypeptide shown in SEQ ID NO:1 is with the detection PPR virus antibody of polypeptides in combination shown in SEQ ID NO:2,
Sensibility is 88.7%, specificity 94.9%；And work as shown in the polypeptide shown in SEQ ID NO:1 and SEQ ID NO:3
When polypeptides in combination detects PPR virus antibody, sensibility 87.2%, specificity 91.3%；It completely can be with c-
ELISA method and b-ELISA method match in excellence or beauty.
Inventor also found, when the polypeptide shown in SEQ ID NO:1 and polypeptides in combination shown in SEQ ID NO:4 detect
When PPR virus antibody, sensibility 86.5%, specificity 95.2%；And when more shown in SEQ ID NO:1
When peptide is with the detection PPR virus antibody of polypeptides in combination shown in SEQ ID NO:5, sensibility 85.7%, specificity are
94.4%；It can match in excellence or beauty completely with c-ELISA method and b-ELISA method.
Inventor also found, when the polypeptide shown in SEQ ID NO:1 and polypeptides in combination shown in SEQ ID NO:6 detect
When PPR virus antibody, sensibility 85%, specificity 92.7%.Better effect is, when with SEQ ID NO:1
Shown in polypeptide, polypeptides in combination detects PPR virus shown in polypeptide and SEQ ID NO:7 shown in SEQ ID NO:6
When antibody, sensibility 89%, specificity 89.9%；It can match in excellence or beauty completely with c-ELISA method and b-ELISA method.
Therefore, the present invention includes:
Polypeptide shown in 1.SEQ ID NO:1.
Polypeptide shown in 2.SEQ ID NO:2.
Polypeptide shown in 3.SEQ ID NO:3.
The combination of polypeptide shown in 4.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:2.
The combination of polypeptide shown in 5.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:3
6. aforementioned polypeptides or polypeptides in combination are in the biological sample that preparation is used for test object biological source with the presence or absence of anti-
Purposes in the kit of the antibody (IgG) of PPR virus.
7. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, and
The following polypeptides in combination 1 being independently connected on one or more of solid carriers；
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
8. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, and
The following polypeptides in combination 2 being independently connected on one or more of solid carriers；
Polypeptides in combination 2
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:3.
9. PPR virus antibody (IgG) detection kit according to claim 7 or 8 does not include it
Its probe molecule (such as polypeptide, albumen or nucleic acid).
10. following polypeptides in combination 1 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
11. following polypeptides in combination 2 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 2
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:3.
The invention also includes:
Polypeptide shown in 12.SEQ ID NO:4.
Polypeptide shown in 13.SEQ ID NO:5.
The combination of polypeptide shown in 14.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:4.
The combination of polypeptide shown in 15.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:5.
16. aforementioned polypeptides or polypeptides in combination are in the biological sample that preparation is used for test object biological source with the presence or absence of anti-
Purposes in the kit of the antibody (IgG) of PPR virus.
17. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, with
And it is independently connected to following polypeptides in combination 1 on one or more of solid carriers；
Polypeptides in combination 3
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:4.
18. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, with
And it is independently connected to following polypeptides in combination 2 on one or more of solid carriers；
Polypeptides in combination 4
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:5.
19. PPR virus antibody (IgG) detection kit described in 7 or 18 according to claim 1, does not include
Other probe molecules (such as polypeptide, albumen or nucleic acid).
20. following polypeptides in combination 3 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 3
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:4.
21. following polypeptides in combination 4 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 4
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:5.
In addition, the invention also includes:
Polypeptide shown in 22.SEQ ID NO:6.
Polypeptide shown in 23.SEQ ID NO:7.
The combination of polypeptide shown in 24.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:6.
Polypeptide shown in 25.SEQ ID NO:1, polypeptide shown in polypeptide and SEQ ID NO:7 shown in SEQ ID NO:6
Combination.
26. aforementioned polypeptides or polypeptides in combination are in the biological sample that preparation is used for test object biological source with the presence or absence of anti-
Purposes in the kit of the antibody (IgG) of PPR virus.
27. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, with
And it is independently connected to following polypeptides in combination 1 on one or more of solid carriers；
Polypeptides in combination 5
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:6.
28. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, with
And it is independently connected to following polypeptides in combination 6 on one or more of solid carriers；
Polypeptides in combination 6
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:6, and
Polypeptide shown in SEQ ID NO:7.
29. PPR virus antibody (IgG) detection kit, does not include according to claim 27 or 28
Other probe molecules (such as polypeptide, albumen or nucleic acid).
30. following polypeptides in combination 5 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 5
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:6.
31. following polypeptides in combination 6 are small anti-with the presence or absence of resisting in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of hay epizootic disease virus；
Polypeptides in combination 6
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:6, and
Polypeptide shown in SEQ ID NO:7.
In the present specification, the object organisms are preferably small ruminant, more preferably goat or sheep.
In the present specification, the biological sample can be whole blood, blood plasma or serum.
Aforementioned polypeptides and kit, which can be used for whether there is in the biological sample of test object biological source, resists small ruminate
The antibody (IgG) of epizootic disease virus.In general, the PPR virus antibody in biological sample is to be ruminated due to object organisms by small
Epizootic disease virus infection is generated by peste des petits ruminants vaccine immunity, and therefore, aforementioned polypeptides and kit can be used for diagnosing
Whether object organisms are by PPR virus infection or peste des petits ruminants vaccine immunity.
The specific embodiment of invention
Firstly, the present invention provides polypeptide shown in NO:1~7 SEQ ID, these polypeptides are as test object biological source
Biological sample in the presence or absence of the detection instrument of antibody (IgG) of anti-PPR virus be useful.Moreover, inventor
It was found that polypeptide shown in SEQ ID NO:1 individually detects, the sensibility of PPR virus antibody is 70.2%, specificity is
99.4%.In the indirect ELISA method based on antigen epitope polypeptide, the sensibility phase for being usually no more than 50% of single polypeptide
Be than, the sensibility of polypeptide shown in SEQ ID NO:1 it is surprising, therefore, achieve unexpected technical effect.
In this specification, sensibility refers to: in the positive sample with the confirmation of " goldstandard " method, being measured as by other methods
The ratio of positive sample.Specificity refers to: in the negative sample with the confirmation of " goldstandard " method, being measured as feminine gender by other methods
The ratio of sample.For the detection of PPR virus antibody, " goldstandard " of the art is that virus neutralizes examination
Test (VNT).
Polypeptide shown in the SEQ ID NO:1 can be used as the life that detection probe is used to prepare test object biological source
With the presence or absence of the kit of the antibody of anti-PPR virus in object sample.
In addition, the present invention also provides polypeptides shown in NO:2~7 SEQ ID.
In addition, the present invention also provides following polypeptides in combination 1~6.
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
Polypeptides in combination 2
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:3.
Polypeptides in combination 2-1
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:2, and
Polypeptide shown in SEQ ID NO:3.
Polypeptides in combination 3
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:4.
Polypeptides in combination 4
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:5.
Polypeptides in combination 4-1
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
Polypeptides in combination 5
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:6.
Polypeptides in combination 6
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:6, and
Polypeptide shown in SEQ ID NO:7.
Polypeptides in combination 6-1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:7.
Aforementioned polypeptides are used in the biological sample of test object biological source with the presence or absence of anti-PPR virus
Antibody, sensibility and specificity (and are using VNT method validation, are tested using contrast agents cassette method 85% or more
Card), it is sufficient to it matches in excellence or beauty using c-ELISA method or b-ELISA method.
Therefore, aforementioned polypeptides combination can be used as detection probe and be used to prepare in the biological sample of test object biological source
With the presence or absence of the kit of the antibody of anti-PPR virus.
Therefore, the present invention also provides a kind of PPR virus antibody assay kits comprising one or more is solid
One or more of body carrier, and the aforementioned polypeptides combination being independently connected on one or more of solid carriers.
In the present specification, solid carrier can be one, be also possible to it is multiple, but preferably one, i.e., whole polypeptides
It is independently connected on same solid carrier.In the present invention, solid carrier is not particularly limited, as long as solid or
The carrier of insoluble material.The connection of polypeptide and solid carrier can be using well known to a person skilled in the art polypeptide and admittedly
The connection method of body carrier carries out.
In the present specification, the object organisms are preferably small ruminant, more preferably goat or sheep.
In the present specification, the biological sample can be whole blood, blood plasma or serum.
It is stated in the biological sample of kit test object biological source in use with the presence or absence of anti-PPR virus
Antibody in the case where, when any one or more polypeptide in the polypeptides in combination has the biological sample in object organisms source
When response, it is determined as in the biological sample in the object organisms source that there are the antibody of anti-PPR virus (i.e. positive)；Instead
It is determined as this when not having any polypeptide to have response to the biological sample in object organisms source in the polypeptides in combination
There is no the antibody of anti-PPR virus (i.e. negative) in the biological sample in object organisms source.
In the present specification, " response " refers to: signal-to-noise ratio (SNR) is greater than or equal to 2, wherein signal-to-noise ratio=(polypeptide point letter
Number value-negative control point signal value)/negative control point signal value.
Embodiment
1. the preparation and confirmation of polypeptide
The polypeptide of NO:1~7 SEQ ID is synthesized by gill biochemistry (Shanghai) Co., Ltd., and is carried out by mass spectrum to it
Confirmation.Wherein,
SEQ ID NO:1:SAEALFRLQAMAKILEDQEE.
SEQ ID NO:2:TKRTRSGKPRGETPGPLLPE.
SEQ ID NO:3:GETPGPLLPEIMQEDELSRE.
SEQ ID NO:4:TKNVRPIQTLTPGRRTRRFV.
SEQ ID NO:5:SSQNPREAQRSAEALFRLQA.
SEQ ID NO:6:NQDPDKLLTVIASDKCPVVE.
SEQ ID NO:7:TPGRRTRRFVGAVLAGVALG.
2. the preparation of kit (detection chip) 1~3
Kit 1
Using conventional method on conventional ELISA solid phase carrier polypeptide shown in the above-mentioned SEQ ID NO:1 of point sample
Solution, in addition one goat IgG of point sample is prepared for detection core as positive quality control point and a PB point as negative Quality Control point
Piece.
Kit 2
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 2 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 2.Kit 3
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 3 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 3.Kit 4
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 4 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 4.Kit 5
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 5 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 5.Kit 6
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 6 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 6.Kit 7
In addition on solid phase carrier distinguish above-mentioned SEQ ID NO:1,6 and 7 of point sample shown in polypeptide solution other than, according to
The identical preparation method of mentioned reagent box 1 is prepared for detection chip, as kit 7.
3. being detected with kit
Checking procedure
(1), with purified water by 20 × concentrated cleaning solutions (TBST:0.4M Tris-HCl, 2.74M NaCl, 2%
Tween20, pH7.2 ± 0.2) it is diluted by 1:20, obtain cleaning solution.To make detection chip surface complete wetting, liquid relief is used
About 200 μ L cleaning solutions are added to detection chip surface by rifle, and impregnate detection chip certain time.
It (2), will with Sample dilution (0.05MPBS, 1%BSA, 0.2%PVP, 0.5%Tween20, pH7.2 ± 0.2)
Test serum sample is diluted according to 1:50.
(3), for having discarded the detection chip of cleaning solution, in the state that surface is completely wet, after drawing 200 μ L dilution
Serum sample be added in detection chip.
(4), it will test chip in constant-temperature table with 150 revs/min to be incubated for 30 minutes, temperature is room temperature.
(5), serum sample is discarded, the surface of detection chip is cleaned with cleaning solution.
(6), clean after, be added in detection chip 200 μ L enzyme labelled antibody solution (rabbit-anti goat IgG-HRP, Sigma,
A5420), will test chip in constant-temperature table with 150 revs/min to be incubated for 30 minutes, temperature is room temperature.
(7), enzyme labelled antibody solution is discarded, chip surface is will test with cleaning solution and cleans up.
(8), after the completion of cleaning, luminous substrate liquid (Thermo, the Prod# of 20 μ L are uniformly sprawled on detection chip surface
37074)。
(9), chemiluminescence imaging will be carried out to detection chip with gel imager, and determines result.
(10), result judgement: for every a serum, whether each polypeptide counted in the kit respectively has response
(that is, signal-to-noise ratio (SNR) is more than or equal to 2), and determined.That is,
For mentioned reagent box 1, when detecting that polypeptide shown in SEQ ID NO:1 has response, it is judged to small ruminating beast
Epidemic disease virus antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 2, when detecting that polypeptide shown in SEQ ID NO:1 and/or 2 has response, it is determined as small
Ruminate epizootic disease virus antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 3, when detecting that polypeptide shown in SEQ ID NO:1 and/or 3 has response, it is determined as small
Ruminate epizootic disease virus antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 4, when detecting that polypeptide shown in SEQ ID NO:1 and/or 4 has response, it is determined as small
Ruminate epizootic disease virus antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 5, when detecting that polypeptide shown in SEQ ID NO:1 and/or 5 has response, it is determined as small
Ruminate epizootic disease virus antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 6, when detecting that polypeptide shown in SEQ ID NO:1 and/or 6 has response, it is determined as small
Ruminate epizootic disease virus antibody positive；Conversely, being determined as feminine gender.
Mentioned reagent box 7 is sentenced when detecting that polypeptide shown in SEQ ID NO:1 and/or 6 and/or 7 has response
It is set to PPR virus antibody positive；Conversely, being determined as feminine gender.
Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point
Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the feminine gender that software is read
The chemiluminescence intensity value of control point.
For 755 parts of goats and sheep serum sample from China regions, it is respectively adopted and is prepared in above-mentioned steps 2
Kit (the step of pressing above-mentioned 3) and virus neutralization tests (VNT, by OIE (2010) " PPR virus neutralization test "
The method) it is detected, testing result is as follows.
Table 1
Sensibility=280/399*100%=70.2%
Specificity=354/356=99.4%
Table 2
Sensibility=354/399*100%=88.7%
Specificity=338/356=94.9%
Table 3
Sensibility=348/399*100%=87.2%
Specificity=325/356=91.3%
Table 4
Sensibility=345/399*100%=86.5%
Specificity=339/356=95.2%
Table 5
Sensibility=342/399*100%=85.7%
Specificity=336/356=94.4%
Table 6
Sensibility=339/399*100%=85.0%
Specificity=330/356=92.7%
Table 7
Sensibility=35539/399*100%=89.0%
Specificity=320/356=89.9%
As known from the above, the sensibility and specificity of kit 2~7 (and is tested using VNT method 85% or more
Card is verified using contrast agents cassette method), it is sufficient to the kit to match in excellence or beauty using c-ELISA method or b-ELISA method.
Sequence table
<110>Suzhou Ai Bituo Bioisystech Co., Ltd
<120>PPR virus antibody assay kit
<130> PB00133D2
<160> 3
<170> PatentIn version 3.2
<210> 1
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 1
SAEALFRLQA MAKILEDQEE 20
<210> 2
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 2
TKRTRSGKPR GETPGPLLPE 20
<210> 3
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 3
GETPGPLLPE IMQEDELSRE 20
<210> 4
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 4
TKNVRPIQTL TPGRRTRRFV 20
<210> 5
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 5
SSQNPREAQR SAEALFRLQA 20
<210> 6
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 6
NQDPDKLLTV IASDKCPVVE 20
<210> 7
<211> 20
<212>polypeptide
<213>artificial sequence
<400> 7
TPGRRTRRFV GAVLAGVALG 20
Claims (9)
1. a kind of PPR virus antibody (IgG) detection kit comprising one or more solid carriers, and it is independent
Ground is connected to following polypeptides in combination 5 or polypeptides in combination 6 on one or more of solid carriers；
Polypeptides in combination 5
Polypeptide shown in polypeptide shown in SEQ ID NO:1 and SEQ ID NO:6；
Polypeptides in combination 6
Polypeptide shown in SEQ ID NO:1, polypeptide shown in polypeptide and SEQ ID NO:7 shown in SEQ ID NO:6.
2. whether kit according to claim 1 is used in the biological sample by test object biological source deposit
In the antibody (IgG) of anti-PPR virus, diagnose whether object organisms by PPR virus infection or small ruminate beast
Epidemic disease vaccine immunity.
3. kit according to claim 2, wherein the object organisms small ruminant.
4. kit according to claim 2, wherein the object organisms are goat or sheep.
5. kit according to claim 2, wherein the biological sample is whole blood, blood plasma or serum.
6. following polypeptides in combination 5 or polypeptides in combination 6 whether there is in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgG) of anti-PPR virus；
Polypeptides in combination 5
Polypeptide shown in polypeptide shown in SEQ ID NO:1 and SEQ ID NO:6；
Polypeptides in combination 6
Polypeptide shown in SEQ ID NO:1, polypeptide shown in polypeptide and SEQ ID NO:7 shown in SEQ ID NO:6.
7. purposes according to claim 6, wherein the object organisms small ruminant.
8. purposes according to claim 6, wherein the object organisms are goat or sheep.
9. purposes according to claim 6, wherein the biological sample is whole blood, blood plasma or serum.
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| CN110346567A (en) | 2019-10-18 |
| CN110346567B (en) | 2022-08-12 |
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| CN110346565B (en) | 2022-05-24 |
| CN110346566B (en) | 2022-04-12 |
| CN111537713B (en) | 2023-03-24 |
| CN111537713A (en) | 2020-08-14 |
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