CN105116144A - Antibody detection kit of porcine epidemic diarrhea virus IgG - Google Patents
Antibody detection kit of porcine epidemic diarrhea virus IgG Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and concretely relates to an antibody detection kit of porcine epidemic diarrhea virus IgG. The kit prepared by the invention has a simple operation flow for clinic sample detection, the detection is completed during 3.5 hours, only a common inverted microscope is needed for determination of result, special apparatuses are not needed, specific dyeing is visible to naked eye, non-specific dyeing is easy to be distinguished, so the kit prepared by the invention is convenient for laboratory and clinic on-site detection. A secondary antibody used in the kit prepared by the invention is an enzyme-labeling SPA which can be combined with the mammal IgG with good specificity; the kit provided by the invention can be used for detecting pigs which are infected by the porcine epidemic diarrhea virus or specific IgG antibody in the vaccine inoculated pig, simultaneously is used for evaluating level of the porcine epidemic diarrhea virus antibody IgG during the porcine epidemic diarrhea virus immunization research using mice or rabbit as experiment animal.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Porcine epidemic diarrhea virus IgG antibody detection kit.
Background technology
Porcine epidemic diarrhea virus disease is one of Major Epidemic type infectious disease of harm China pig industry, is a kind of deadly infectious disease with high degree in contact of pig, has fall ill anxious and mortality ratio high.The popular new strain of current China all has different cultivars and the pig at age and infects new, and in 1 week age of newborn piglet, infection rate is up to 100%, and mortality ratio reaches 50% ~ 100%.Old strain in the past only causes endemic conditions, and since 2011, China starts the new variant this virus being detected, brings about great losses.Have report to show, the U.S., between year May in March, 2013 to 2014, is infected by this new circulating virus strains and causes 5,000,000 piglets that are born dead, lose startling.This disease is popular more than 3 years on a large scale in China, and the economic loss caused also does not have concrete data at present.From the reaction of a clinical line, new epidemic diarrhea virus strain is incited somebody to action the negative effect that pig industry produces also can continuing fermentation.Therefore, while exploitation effective vaccine, the handy root canal preparation being equipped with vaccine effect evaluation and clinical antibody test seems very necessary.
Detection method for new epidemic diarrhea virus strain specific antibody mainly contains neutralization test, enzyme linked immunosorbent assay (ELISA), the methods such as colloidal gold method.Wherein neutralization test, need virus, the cell of living, material source not in time, need technology and equipment condition higher, be difficult to high flux detect, the impact of operation human factor is large, and be only limitted to the use of Individual testwas chamber interior, cannot produce by bulk industrial, the use of a more impossible line detection in plant of basic unit.Collaurum is convenient to the clinical characterizing antibody positive and negative problem, and cannot accomplish accurate quantification.ELISA be one domestic and international general, at present in a kind of method that each detection field technology is the most ripe, commercial kit is also very handy.But technical force domestic at present does not reach corresponding world level, slowly there is no the Porcine epidemic diarrhea virus ELISA antibody assay kit listing of China.And import Porcine epidemic diarrhea virus ELISA antibody assay kit is expensive, clinical application cost is high, considerably increases the burden of raiser.
In view of the foregoing, develop a kind of Porcine epidemic diarrhea virus antibody assay kit that can be applicable to industrialized mass production, quality controllable, stable performance, high flux, with low cost, specificity and susceptibility good very necessary, particularly importantly, the cost of this kit can not be too high, otherwise, be difficult to apply.Based on above all backgrounds, establish the present invention.
Summary of the invention
The object of this invention is to provide a kind of Porcine epidemic diarrhea virus IgG antibody detection kit, can detect fast and accurately.
The present invention for the taked technical scheme that solves the problem is: a kind of Porcine epidemic diarrhea virus IgG antibody detection kit, be provided with in this kit bag by 96 hole immune detection reaction plates 5 pieces of Porcine epidemic diarrhea virus antigen, 120mL10 times concentrated cleaning solution 1 bottle, 120mL sample diluting liquid 1 bottle, 50 μ L positive serums 1 are managed, 50 μ L negative serums 1 are managed, the SPA enzyme labelled antibody 1 of 20 μ L horseradish peroxidases marks is managed, the substrate nitrite ion B liquid 2 of the every pipe of 60mL substrate nitrite ion A liquid 1 bottle, 1.5mL is managed.
Described bag is as follows by the preparation method of Porcine epidemic diarrhea virus antigen 96 hole immune detection reaction plate:
(1), by the nutrient culture media of individual layer monkey-kidney cells in culture flask outwell, then in culture flask, add PBS cleansing solution, every cm
2pBS cleansing solution corresponding to individual layer monkey-kidney cells is 0.027mL, shakes culture flask afterwards, makes cleansing solution cover individual layer monkey-kidney cells face, to remove nutrient culture media residual in culture flask, for subsequent use after outwelling PBS cleansing solution;
(2) pancreas enzyme-EDTA-Na that mass concentration is 0.25% is added in the culture flask, processed to step (1)
2solution, every cm
2pancreas enzyme-EDTA-the Na that individual layer monkey-kidney cells is corresponding
2solution is 0.013mL, and shake culture flask, until pancreas enzyme-EDTA-Na
2solution covers individual layer monkey-kidney cells, after left at room temperature 2min, outwells the pancreas enzyme-EDTA-Na in bottle
2solution, at room temperature leaves standstill 5min, and vibration culture flask is until individual layer monkey-kidney cells all comes off, for subsequent use;
(3) add MEM nutrient culture media in the culture flask, to step (2) processed, and in MEM nutrient culture media, the mass concentration of hyclone is 5%, dispels monkey-kidney cells with liquid-transfering gun, until monkey-kidney cells is single distribution, namely obtained cell suspension, for subsequent use;
(4), by cell suspension and hyclone mass concentration be 5% MEM nutrient culture media mix by the volume ratio of 1:5 and stir, obtained nutrient solution,
(5), with nutrient solution prepared by volley of rifle fire aspiration step (4), and join in 96 hole immune detection reaction plates with every hole 120 μ L, vibration immune detection reaction plate, nutrient solution is uniformly distributed in hole, and afterwards immune detection reaction plate being put into temperature is 37 DEG C, CO
2volumetric concentration is cultivate 24h in the incubator of 5%, outwells the nutrient solution in immune detection reaction plate, and with PBS wash solution cleaning 1-2 time, for subsequent use;
(6) add the diluted Porcine epidemic diarrhea virus of serum free medium in the 96 hole immune detection reaction plates processed to step (5), make every hole virus infections plural number be 5TCID
50, backward immune detection reaction plate in add the trypsase that concentration is 3 μ g/mL, putting into temperature after mixing is 37 DEG C, CO
2volumetric concentration is cultivate 48h in the incubator of 5%, is outwelled by the liquid of 96 hole immune detection reaction plates, and cleans 3 times with PBS wash solution, is the vacuum drying instrument inner drying of 30 DEG C in temperature, until white circle appears in bore edges place;
(7) immobile liquid is added in the 96 hole immune detection reaction plates, processed to step (6), immobile liquid is discarded after leaving standstill 30min, put into the vacuum drying instrument inner drying that temperature is 30 DEG C, until white circle appears in bore edges, described immobile liquid acetone and PBS wash solution is mixed by the volume ratio of 33:67 and obtains after stirring;
(8), by the 96 hole immune detection reaction plates that step (7) is obtained vacuumize to be placed at temperature is-20 DEG C and preserve, namely obtained bag is by 96 hole immune detection reaction plates of Porcine epidemic diarrhea virus antigen.
The preparation method of often liter of 10 times of concentrated cleaning solutions is by the Na of KCl, 14.2g of NaCl, 2g of 80g
2hPO
4, 2.7g KH
2pO
4be added in 900mL deionized water, and be settled to 1000mL, at temperature is 120 DEG C, sterilizing is after 30 minutes, and proceeding to temperature is airtight at 4 DEG C preservation.
The preparation method of described substrate nitrite ion A liquid be by deionization sterilized water, concentration of volume percent be 30% hydrogen peroxide and pH be 5.0, volumetric molar concentration is that the acetate buffer of 0.1moL/L mixes by the volume ratio of 5:0.026:5 and obtained after stirring.
The preparation method of described substrate nitrite ion B liquid adds 4mg3-amino-9-ethyl carbazole in every milliliter of dimethyl formamide, mixes and after stirring.
Substrate nitrite ion A liquid and substrate nitrite ion B liquid mix according to the volume ratio of 20:1 and obtained after stirring by the preparation method of described substrate nitrite ion.
beneficial effect
1, the kit prepared by the present invention, when clinical sample detects, operating process is simple, detection can be completed within 3.5 hours, result judges only to need the inverted microscope that a Daepori is logical, do not need special instrument, the visible specific stain of naked eyes, unspecific staining is very easily distinguished, and facilitates the use of laboratory and clinical sites detection.
2, kit of the present invention adopt two anti-be can with the enzymic change foci of mammal IgG combination, its specificity is fine, kit of the present invention can not only be used for detecting the specific IgG antibodies in Porcine epidemic diarrhea virus infected pigs or its vaccine immunity pig body, also may be used for when adopting mouse or rabbit to carry out Porcine epidemic diarrhea virus immune Research as animal used as test simultaneously, evaluating Porcine epidemic diarrhea virus IgG antibody level.That is, if research object---Porcine epidemic diarrhea virus does not change, when changing other mammals into as research model, do not need to change two in kit and resist, this has the incomparable advantage of general kit.
3, the present invention adopts totivirus antigen preparation feedback plate, the antibody of all structural proteins of encoding viral can be detected, and therefore, its detection is very responsive.
4, it is stable, single, controlled that the kit prepared by the present invention has material source; Vero cell of the present invention is continuous cell line, international material, and background is clear, can preserve for a long time, without the need to cell chulture during Site Detection after antigen-reactive plate prepared by the present invention; In security, this kit wrap quilt for totivirus inactivation antigen, in bio-safety, have good guarantee, effectively can avoid virus diffusion.
5, relative to high cost, hi-tech preparation technology and the working condition required for the preparation of existing commercial Porcine epidemic diarrhea virus ELISA antibody assay kit, kit prepared by the present invention, lower cost for material, preparation technology are simple, and its stability, sensitivity and high-throughout characteristic can match in excellence or beauty with commercial Porcine epidemic diarrhea virus ELISA antibody assay kit.
Embodiment
A kind of Porcine epidemic diarrhea virus IgG antibody detection kit, be provided with in this kit bag by 96 hole immune detection reaction plates 5 pieces of Porcine epidemic diarrhea virus antigen, 120mL10 times concentrated cleaning solution 1 bottle, 120mL sample diluting liquid 1 bottle, 50 μ L positive serums 1 are managed, 50 μ L negative serums 1 are managed, the SPA enzyme labelled antibody 1 of 20 μ L horseradish peroxidases marks is managed, the substrate nitrite ion B liquid 2 of the every pipe of 60mL substrate nitrite ion A liquid 1 bottle, 1.5mL is managed.
Described bag is as follows by the preparation method of Porcine epidemic diarrhea virus antigen 96 hole immune detection reaction plate:
(1), by the nutrient culture media of individual layer monkey-kidney cells in culture flask outwell, then in culture flask, add PBS cleansing solution, every cm
2pBS cleansing solution corresponding to individual layer monkey-kidney cells is 0.027mL, shakes culture flask afterwards, makes cleansing solution cover individual layer monkey-kidney cells face, to remove nutrient culture media residual in culture flask, for subsequent use after outwelling PBS cleansing solution;
(2) pancreas enzyme-EDTA-Na that mass concentration is 0.25% is added in the culture flask, processed to step (1)
2solution, every cm
2pancreas enzyme-EDTA-the Na that individual layer monkey-kidney cells is corresponding
2solution is 0.013mL, and shake culture flask, until pancreas enzyme-EDTA-Na
2solution covers individual layer monkey-kidney cells, after left at room temperature 2min, outwells the pancreas enzyme-EDTA-Na in bottle
2solution, at room temperature leaves standstill 5min, and vibration culture flask is until individual layer monkey-kidney cells all comes off, for subsequent use;
(3) add MEM nutrient culture media in the culture flask, to step (2) processed, and in MEM nutrient culture media, the mass concentration of hyclone is 5%, dispels monkey-kidney cells with liquid-transfering gun, until monkey-kidney cells is single distribution, namely obtained cell suspension, for subsequent use;
(4), by cell suspension and hyclone mass concentration be 5% MEM nutrient culture media mix by the volume ratio of 1:5 and stir, obtained nutrient solution,
(5), with nutrient solution prepared by volley of rifle fire aspiration step (4), and join in 96 hole immune detection reaction plates with every hole 120 μ L, vibration immune detection reaction plate, nutrient solution is uniformly distributed in hole, and afterwards immune detection reaction plate being put into temperature is 37 DEG C, CO
2volumetric concentration is cultivate 24h in the incubator of 5%, outwells the nutrient solution in immune detection reaction plate, and with PBS wash solution cleaning 1-2 time, for subsequent use;
(6) add the diluted Porcine epidemic diarrhea virus of serum free medium in the 96 hole immune detection reaction plates processed to step (5), make every hole virus infections plural number be 5TCID
50, backward immune detection reaction plate in add the trypsase that concentration is 3 μ g/mL, putting into temperature after mixing is 37 DEG C, CO
2volumetric concentration is cultivate 48h in the incubator of 5%, is outwelled by the liquid of 96 hole immune detection reaction plates, and cleans 3 times with PBS wash solution, is the vacuum drying instrument inner drying of 30 DEG C in temperature, until white circle appears in bore edges place;
(7) immobile liquid is added in the 96 hole immune detection reaction plates, processed to step (6), immobile liquid is discarded after leaving standstill 30min, put into the vacuum drying instrument inner drying that temperature is 30 DEG C, until white circle appears in bore edges, described immobile liquid acetone and PBS wash solution is mixed by the volume ratio of 33:67 and obtains after stirring;
(8), by the 96 hole immune detection reaction plates that step (7) is obtained vacuumize to be placed at temperature is-20 DEG C and preserve, namely obtained bag is by 96 hole immune detection reaction plates of Porcine epidemic diarrhea virus antigen.
The preparation method of often liter of 10 times of concentrated cleaning solutions is by the Na of KCl, 14.2g of NaCl, 2g of 80g
2hPO
4, 2.7g KH
2pO
4be added in 900mL deionized water, and be settled to 1000mL, at temperature is 120 DEG C, sterilizing is after 30 minutes, and proceeding to temperature is airtight at 4 DEG C preservation.
The preparation method of described substrate nitrite ion A liquid be by deionization sterilized water, concentration of volume percent be 30% hydrogen peroxide and pH be 5.0, volumetric molar concentration is that the acetate buffer of 0.1moL/L mixes by the volume ratio of 5:0.026:5 and obtained after stirring.
The preparation method of described substrate nitrite ion B liquid adds 4mg3-amino-9-ethyl carbazole in every milliliter of dimethyl formamide, mixes and after stirring.
Substrate nitrite ion A liquid and substrate nitrite ion B liquid mix according to the volume ratio of 20:1 and obtained after stirring by the preparation method of described substrate nitrite ion.
A kind of Porcine epidemic diarrhea virus IgG antibody detection kit, its concrete using method comprises the following steps:
One, reagent prepares:
1. blood serum sample prepare: fresh, refrigeration (4 DEG C) and freezing pig anteserum sample all can be used for detection.Freezing serum thaws at 4 DEG C, then the centrifugal 10min of 3000r/min, gets supernatant for detecting.
2. cleansing solution: 10 times of concentrated cleansing solutions should return to room temperature, and mix fully, guarantees that the salt of crystallization dissolves; 1:10 dilution is done before use with distilled water or deionized water.The cleansing solution prepared under aseptic condition can be preserved 1 week at 4 DEG C.Such as, 200mL cleansing solution need add 180mL distilled water with 20mL concentrated cleaning solution and fully mixes.
3. enzyme labelled antibody working fluid: before use, does 1:4000 dilution with sample diluting liquid by enzyme labelled antibody, mixing, notes matching while using.Such as, the enzyme labelled antibody working fluid of 10mL, the enzyme labelled antibody getting 3 μ L joins in the sample diluting liquid of 9mL and mixes, and notes matching while using.
4. substrate nitrite ion: before colour developing, mixes substrate A liquid and substrate B liquid according to the ratio of 20:1, notes matching while using.
Two, operation steps
1. pre-temperature: before use, according to detection serum sample quantity, gets immune detection reaction plate (quantity per sample will not closed with hole plastic foil), cleansing solution and dilution and returns to room temperature, gently evenly.
2. soak plate: the cleansing solution of 200 μ L is injected in every hole, puts room temperature and embathes once, throw away washing lotion gently, tapping on thieving paper.
3. Sample Dilution: with sample diluting liquid, measuring samples, negative serum control product and positive serum controls product are made 1:100 and doubly dilute.Such as: respectively to be checked and control serum samples 3 μ L are added 300 μ L sample diluting liquids, mixing.If want the titre of IgG in working sample can to dilute according to the gradient dilution method of 10 times.
4. react with blood serum sample: the blood serum sample getting above-mentioned dilution joins corresponding aperture respectively, every hole 100 μ L, inject reaction plate hole successively, record each sample position, reaction plate is inserted in wet box, in 37 DEG C of incubators, hatch 1h.
5. wash plate: 200 μ L cleansing solutions are injected in every hole, get rid of washing lotion immediately, tapping on thieving paper, repeat 3 times.
6. react with enzyme labelled antibody: the antibody of the horseradish peroxidase-labeled that 100 μ L dilute is injected in every hole, is inserted by reaction plate in wet box, in 37 DEG C of incubators, hatches 1h.
7. wash plate: undertaken by step 5.
8. substrate colour developing: 100 μ L substrates nitrite ion (matching while using) are injected in every hole, and lucifuge, reacts 15min in 37 DEG C of incubators.
9. cessation reaction: throw away reactant liquor, every hole is injected 200 μ L and is distilled washing once, and every hole adds 100 μ LddH
2o observations.
10. examine under a microscope each hole colour developing situation, carry out sample result judgement.
Three, result judges
In brownish red in immune detection reaction plate inner cell matter, nucleus non-coloring, is judged to the positive; Non-coloring reaction is judged to be feminine gender in tenuigenin or around nucleus.During to detect seropositivity terminal, the inverse of most high dilution represents antibody titer.
Kit quality standard of the present invention and calibrating:
Specificity: 60 parts of negative serums, 60 parts of positive serums, adopt import ELISA kit and this kit to detect respectively, coincidence rate is not less than 98%.
Sensitivity: the 6400 times of dilutions of Porcine epidemic diarrhea virus positive serum can detect, and (namely 1 μ L serum is added in 6400 μ L sample diluting liquids, gets wherein 100 μ L and adds sample detection hole) can detect.
Stability: kit is placed in 37 DEG C and is no less than 2 days with 4 DEG C of kits deposited and synchronously detects 10 increment product, its coincidence rate is 100%.
Test 1
Newborn piglet Porcine epidemic diarrhea virus IgG antibody level is detected: from Hunan A with kit of the present invention, the pig farm that B two is different, 3 nests are all chosen on each pig farm, 10, every nest, the sodium selenite (10 age in days) (note: the sow of its correspondence had Porcine epidemic diarrhea virus inactivated vaccine to inoculate history) of totally 30 non-Pigs Inoculated epidemic diarrhea virus vaccines, two pig farms gather serum 60 parts altogether, epidemic diarrhea virus IgG antibody is detected by Porcine epidemic diarrhea virus IgG antibody detection kit, detect according to Porcine epidemic diarrhea virus IgG antibody detection kit trace routine.
Test findings
The health pig IgG antibody testing result of non-Pigs Inoculated epidemic diarrhea virus vaccine: the health pig serum sample gathering 60 parts of non-Pigs Inoculated epidemic diarrhea virus vaccines from Hunan A, B two pig farms altogether carries out the detection of Porcine epidemic diarrhea virus IgG antibody, result shows (table 1): 22 parts, A pig farm is for positive, and 8 parts is negative; 6 parts, B pig farm is positive, and 24 parts is negative.Wherein in the positive serum of A pig farm, the highest tiring is 800 times (800 ×), and minimum tiring is 10 times (10 ×), and in the positive serum of B pig farm, the highest tiring is 40 times (40 ×), and minimum tiring is 10 times of (10 ×) (data are in table 1 and tables 2).All former times of all negative serum samples are detected as negative findings.These results show, in the nest piglet of A pig farm 3, Porcine epidemic diarrhea virus IgG antibody positive rate is 73.3%, B pig farm positive rate is 20%.These data illustrate, A pig farm sow immune swine epidemic diarrhea virus vaccine effect is better, and piglet obtains good maternal antibody after lactation, has certain resistibility to disease; And B pig farm positive rate is very low, and antibody specific antibody horizontal is very low, and its sow vaccine inoculation poor effect is described, piglet faces the risk of infected pigs's epidemic diarrhea virus.
Contrast test: it is that Porcine epidemic diarrhea virus ELISA antibody kit synchronously detects that 60 parts of blood serum samples adopt commercial abroad simultaneously, in result display A field, 8# sample is the weak positive, and No. 1-7 is feminine gender, and other are strong positive.In B field, 7#, 8#, 9# and 10# are the weak positive, and other are negative findings.These data show, kit of the present invention and commercial Porcine epidemic diarrhea virus ELISA antibody kit have good accordance.
Table 1 Porcine epidemic diarrhea virus IgG antibody detection kit detects A pig farm 10, Hunan age in days piglet serum antibody
Table 2 Porcine epidemic diarrhea virus IgG antibody detection kit detects B pig farm 10, Hunan age in days piglet serum antibody
Test 2
IgG dynamic change in Porcine epidemic diarrhea virus inactivated vaccine immune swine body is detected: choose 2, certain pig farm Porcine epidemic diarrhea virus negative antibody (adopting commercial ELISA kit to detect), 60 age in days child care pigs with kit of the present invention, commercial inactivated vaccine is adopted to inoculate, immunity 2 times, 21 days, interval, after first 3 days of first immunisation and the 2nd immunity, the 7th, 14,21 gather serum sample, adopt this kit program to carry out IgG antibody detection.
Test findings
IgG dynamic change in Porcine epidemic diarrhea virus inactivated vaccine immune swine body is detected: to 5 Porcine epidemic diarrhea virus inactivated vaccine inoculation test pigs in this research with kit of the present invention, 7th, 14,21 gather serum samples afterwards in first 3 days of first immunisation and the 2nd immunity (twice immunization interval 2 weeks), adopt this kit program to carry out IgG antibody detection.Result shows, in preimmune serum, Porcine epidemic diarrhea virus antibody is feminine gender (detection of former times of serum), and in the serum that after 2 immunity, the 7th, 14,21 day gathers, IgG antibody is obvious ascendant trend.After time immunity of 1# pig, the 7th, 14,21 day IgG titre reaches 800 times, 3200 times, 6400 times respectively.After the immunity of 2# pig second time, the 7th, 14,21 day IgG titre reaches 800 times, 3200 times, 12800 times respectively.
Test 3
Porcine epidemic diarrhea virus N protein specific IgG dynamic change in the rabbit body of Porcine epidemic diarrhea virus N protein immunity is detected with kit of the present invention:
Prepare immunogen immune new zealand white rabbit after adopting Porcine epidemic diarrhea virus N protein and Freund's adjuvant emulsification, carry out 2 immunity inoculations, 21 days, interval, each inoculation protein content is 1mg, and dosage is 1mL.Blood sampling: blood sampling once before immunity, after first immunisation, blood sampling in 21 days 1 time, every blood sampling in 7 days 1 time after secondary immunity, continues blood sampling 3 times, collects blood serum sample 5 parts altogether.Kit of the present invention is adopted to detect IgG antibody.
Test findings
Porcine epidemic diarrhea virus N protein specific IgG dynamic change in the rabbit body of Porcine epidemic diarrhea virus N protein immunity is detected with kit of the present invention:
Prepare immunogen immune 2 new zealand white rabbits after adopting Porcine epidemic diarrhea virus N protein and adjuvant emulsion, collect the blood serum sample 5 parts that different time points gathers altogether, adopt kit of the present invention to detect IgG antibody.Result shows, before immunity, in the serum that in 2 rabbit bodies, Porcine epidemic diarrhea virus IgG antibody gathers after being feminine gender (detection of former times of serum) 2 immunity on the the 7th, 14,21 day, IgG antibody is obvious ascendant trend.After 1 pig time immunity, the 7th, 14,21 day IgG titre reaches 50 times, 200 times, 400 times respectively.After the immunity of 2# pig second time, the 7th, 14,21 day IgG titre reaches 200 times, 400 times, 400 times respectively.Result shows, Porcine epidemic diarrhea virus N protein has certain immunogenicity, and body can be stimulated to produce corresponding antibodies, but in relative specific embodiment 3, inactivated vaccine is to the immunogenicity of pig, and the immunogenicity of N protein is not very desirable.This embodiment can illustrate, kit of the present invention can detect the specific antibody of the N protein in infected pigs or vaccine immunity pig body.This kit, it is the enzymic change foci that can combine with mammal IgG that two of employing resists, and its specificity is fine.Therefore, this kit also may be used for carrying out the use of Porcine epidemic diarrhea virus immune Research as animal used as test, to evaluate antibody horizontal employing mouse or rabbit.
Test 1, test the quality arbitration of 2 and test 3 reagent:
Specificity: Porcine epidemic diarrhea virus IgG antibody detection kit and ELISA detect 20 parts of negative serums, 10 positive quality control serum respectively, and coincidence rate is 98.5%;
Sensitivity: it is the Porcine epidemic diarrhea virus positive serum prepared with Porcine epidemic diarrhea virus inactivated vaccine immunity test pig that quality controlled serum used is examined and determine in the sensitivity of two kinds of reagent.Verification result shows: the sensitivity of Porcine epidemic diarrhea virus IgG antibody detection kit can reach 1:6400.
In specific embodiment 2 and 3, when measuring pig epidemic diarrhea antibody titer in serum, serum is done doubling dilution, and by specification operating process detects, and most heel specifically implements the same result of determination of kind decision method in 1, calculates IgG and tires.
Claims (6)
1. a Porcine epidemic diarrhea virus IgG antibody detection kit, is characterized in that: be provided with in this kit bag by 96 hole immune detection reaction plates 5 pieces of Porcine epidemic diarrhea virus antigen, 120mL10 times concentrated cleaning solution 1 bottle, 120mL sample diluting liquid 1 bottle, 50 μ L positive serums 1 are managed, 50 μ L negative serums 1 are managed, the SPA enzyme labelled antibody 1 of 20 μ L horseradish peroxidases marks is managed, the substrate nitrite ion B liquid 2 of the every pipe of 60mL substrate nitrite ion A liquid 1 bottle, 1.5mL is managed.
2. a kind of Porcine epidemic diarrhea virus IgG antibody detection kit according to claim 1, is characterized in that: described bag is as follows by the preparation method of Porcine epidemic diarrhea virus antigen 96 hole immune detection reaction plate:
(1), by the nutrient culture media of individual layer monkey-kidney cells in culture flask outwell, then in culture flask, add PBS cleansing solution, every cm
2pBS cleansing solution corresponding to individual layer monkey-kidney cells is 0.027mL, shakes culture flask afterwards, makes cleansing solution cover individual layer monkey-kidney cells face, to remove nutrient culture media residual in culture flask, for subsequent use after outwelling PBS cleansing solution;
(2) pancreas enzyme-EDTA-Na that mass concentration is 0.25% is added in the culture flask, processed to step (1)
2solution, every cm
2pancreas enzyme-EDTA-the Na that individual layer monkey-kidney cells is corresponding
2solution is 0.013mL, and shake culture flask, until pancreas enzyme-EDTA-Na
2solution covers individual layer monkey-kidney cells, after left at room temperature 2min, outwells the pancreas enzyme-EDTA-Na in bottle
2solution, at room temperature leaves standstill 5min, and vibration culture flask is until individual layer monkey-kidney cells all comes off, for subsequent use;
(3) add MEM nutrient culture media in the culture flask, to step (2) processed, and in MEM nutrient culture media, the mass concentration of hyclone is 5%, dispels monkey-kidney cells with liquid-transfering gun, until monkey-kidney cells is single distribution, namely obtained cell suspension, for subsequent use;
(4), by cell suspension and hyclone mass concentration be 5% MEM nutrient culture media mix by the volume ratio of 1:5 and stir, obtained nutrient solution,
(5), with nutrient solution prepared by volley of rifle fire aspiration step (4), and join in 96 hole immune detection reaction plates with every hole 120 μ L, vibration immune detection reaction plate, nutrient solution is uniformly distributed in hole, and afterwards immune detection reaction plate being put into temperature is 37 DEG C, CO
2volumetric concentration is cultivate 24h in the incubator of 5%, outwells the nutrient solution in immune detection reaction plate, and with PBS wash solution cleaning 1-2 time, for subsequent use;
(6) add the diluted Porcine epidemic diarrhea virus of serum free medium in the 96 hole immune detection reaction plates processed to step (5), make every hole virus infections plural number be 5TCID
50, backward immune detection reaction plate in add the trypsase that concentration is 3 μ g/mL, putting into temperature after mixing is 37 DEG C, CO
2volumetric concentration is cultivate 48h in the incubator of 5%, is outwelled by the liquid of 96 hole immune detection reaction plates, and cleans 3 times with PBS wash solution, is the vacuum drying instrument inner drying of 30 DEG C in temperature, until white circle appears in bore edges place;
(7) immobile liquid is added in the 96 hole immune detection reaction plates, processed to step (6), immobile liquid is discarded after leaving standstill 30min, put into the vacuum drying instrument inner drying that temperature is 30 DEG C, until white circle appears in bore edges, described immobile liquid acetone and PBS wash solution is mixed by the volume ratio of 33:67 and obtains after stirring;
(8), by the 96 hole immune detection reaction plates that step (7) is obtained vacuumize to be placed at temperature is-20 DEG C and preserve, namely obtained bag is by 96 hole immune detection reaction plates of Porcine epidemic diarrhea virus antigen.
3. a kind of Porcine epidemic diarrhea virus IgG antibody detection kit according to claim 1, is characterized in that: the preparation method of often liter of 10 times of concentrated cleaning solutions is by the Na of KCl, 14.2g of NaCl, 2g of 80g
2hPO
4, 2.7g KH
2pO
4be added in 900mL deionized water, and be settled to 1000mL, at temperature is 120 DEG C, sterilizing is after 30 minutes, and proceeding to temperature is airtight at 4 DEG C preservation.
4. a kind of Porcine epidemic diarrhea virus IgG antibody detection kit according to claim 1, is characterized in that: the preparation method of described substrate nitrite ion A liquid be by deionization sterilized water, concentration of volume percent be 30% hydrogen peroxide and pH be 5.0, volumetric molar concentration is that the acetate buffer of 0.1moL/L mixes by the volume ratio of 5:0.026:5 and obtained after stirring.
5. a kind of Porcine epidemic diarrhea virus IgG antibody detection kit according to claim 1, it is characterized in that: the preparation method of described substrate nitrite ion B liquid adds 4mg3-amino-9-ethyl carbazole in every milliliter of dimethyl formamide, to mix and after stirring.
6. a kind of Porcine epidemic diarrhea virus IgG antibody detection kit according to claim 1, is characterized in that: substrate nitrite ion A liquid and substrate nitrite ion B liquid mix according to the volume ratio of 20:1 and obtained after stirring by the preparation method of described substrate nitrite ion.
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| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
Citations (2)
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| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
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