CN108303541A - A kind of porcine circovirus type 2 antibody testing kit and its detection method - Google Patents
A kind of porcine circovirus type 2 antibody testing kit and its detection method Download PDFInfo
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Abstract
The invention discloses a kind of porcine circovirus type 2 antibody testing kit and its detection method, kit of the invention using the coated antigen of magnetic particle, luminescent label albumen and sample to be tested in antibody specific reaction occur and carry out the measurement of antibody content in sample to be tested.The kit of the present invention realizes the quantitative detection of porcine circovirus 2 type antibody, and high sensitivity, detection range is wide, quickly, reproducible, the effect of full automatic working and high-throughput detection.
Description
Technical field
The present invention relates to porcine circovirus 2 type antibody detection technique field more particularly to a kind of porcine circovirus 2 type antibodies
Detection kit and the detection method being detected using the kit.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) is the zoosis of presently found minimum
Poison, virion diameter about 17nm are covalence closed, and cyclic annular, Single-stranded DNA virus, there are two types of genotype for PCV tools：PCV1 and
PCV2.PCV1 no pathogenicities, but be widely present in pig body and pig source continuous cell line, PCV2 have pig pathogenic, mainly draw
Send out pmws (Post-weaning multisystemic wasting syndrome, PMWS).
Other than causing PMWS, PCV2 also with piglet congenital tremors, pigskin inflammation nephritic syndrome, porcine respiratory syndrome (PDNS),
The diseases such as sow breeding difficulty are related.Since 1991 since Canada reports for the first time, many countries and regions all report this
The generation of disease.In recent years, the generation of the disease is in rising trend, and sizable economic loss is caused to the pig breeding industry in the world.
In China, immunity inoculation is to prevent the main policies of pig annulus.Pig circular ring virus Cap protein plays important work
With and necessary structural proteins, can generate protectiveness neutralizing antibody after immune.Rear pig circular ring virus antibody titer is immunized and attacks
Poison protection has apparent correlation, so it is to improve to formulate reasonable, effective immune programme by the monitoring of antibody level
The guarantee of herd immunity level.
Carrying Cap gene of porcine circovirus type 2 is the main neutrality antigen protein of pig annulus, and antibody is resisted after vaccine immunity
The main target that antibody level detects after the horizontal monitoring of body and virus infection, antibody level height and its immune or infection state
It is directly related, so the current demand for thering is porcine circovirus 2 type antibody quantitatively to detect.There are veterinary station, epidemic disease in existing testing agency
There is a large amount of samples for Miao companies, farm etc., also to the porcine circovirus type 2 antibody testing kit and instrument of big flux
There is demand.The method of existing ELISA, colloidal gold etc. is difficult to meet.
It establishes at present and the porcine circovirus 2 type antibody detection method applied has ELISA method, virus neutralization tests, exempts from
The methods of epidemic disease fluorescence method, colloidal gold strip, wherein ELISA method are currently the most common method of porcine circovirus 2 type antibody,
The range of the surveyed OD values of ELISA defines that measured antibody range is relatively narrow, can only accomplish sxemiquantitative in 0.1-3.5, reaction
Time, the time was longer usually at 2 hours or so；Virus neutralization tests be by porcine circovirus 2 type and antibody on cell in
Antigen-antibody reaction is carried out to judge antibody titer, can reflect that the height of neutralizing antibody, overall process are titration of virus, resist more comprehensively
The processes such as body neutralization, result judgement need 5-7 days time, overall process that can only manually operate, judge, poor repeatability；Immunofluorescence
The shortcomings of method is generally also to be operated on cell, and it is not high that there are sensitivity, poor repeatability；Colloidal gold method can quickly obtain knot
Fruit, but can only be qualitative, it is difficult to it is quantitative, limit application range.
Patent CN104237513A (date of publication 2014.12.24) discloses a kind of thyroid peroxidase antibody magnetic
Particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, including TPO-Ab calibration objects, TPO-Ab dilutions, coupling have Streptavidin
Magnetic particle suspension, the TPO-Ab antigens of biotin labeling, the anti-human enzyme mark conjugate of mouse, TPO-Ab quality-control products, chemistry hair
Light liquid A and B, 20 times of concentration washing lotions and reaction tube, the kit carry out quantitative survey to the content of thyroid peroxidase antibody
It is fixed, realize high sensitivity, specificity, accuracy, the effect having good stability.The kit is widely used in human antibody, but
It is not had been reported that also on animal's antibody.
Pig annulus Cap antibody tests at present mainly can not meet quantitative demand using the ELISA method of sxemiquantitative,
This point is mainly due to lacking pig annulus Cap antibody standard substances and quantitative criterion, without the detection method of accurate repeatability
Caused by.It is different from animal doctor, in people doctor field, the marker of each disease, thyroid peroxidase antibody as escribed above,
It has all been well studied, has established general national standard, international standard or professional standard, it is quantitative simple and practicable, therefore
The product of different manufacturers production has obtained specification, and testing result consistency is preferable.And in veterinary applications, current pig annulus Cap
Antibody test market is largely occupied by the ELISA qualitative detection reagents of import, and the product testing result difference of each producer
It is very big, it is unfavorable for livestock breeding industry development, there is an urgent need for establish quantitative criterion and accurate and reliable repeated good detection method.
Invention content
In order to solve present in existing animal doctor's detection technique detection time is long, be difficult to quantitative, poor repeatability, be difficult to it is automatic
The problem of change etc., the object of the present invention is to provide a kind of porcine circovirus type 2 antibody testing kits, it can quantify detection pig
The content of circovirus 2 type antibody.For this purpose, the present invention also provides a kind of method being detected using the kit, the present invention
Detection method high sensitivity, quantitative detection and detection range is wide, detection time is short, reproducible.
To achieve the goals above, the technical solution adopted by the present invention：
The first aspect of the present invention provides a kind of porcine circovirus type 2 antibody testing kit, including 2 porcine circovirus
Type Cap protein is coupled or the solution of indirect conjugation magnetic particle, the protein solution of luminescent label, luminous substrate.
Wherein, the coupling includes being condensed to form amide, magnetic particle ammonia by magnetic particle carboxyl and histone amino
Base forms five carbon bridges with histone amino by glutaraldehyde cross-linking, tosyl magnetic particle is connected with histone amino covalent coupling
It connects.
Wherein, the indirect conjugation includes the coupling mediated in the following manner：What Streptavidin-biotin mediated
Coupling, anti-FITC antibody-FITC couplings.
Wherein, the preparation method of the solution of the carrying Cap gene of porcine circovirus type 2 conjugated magnetic particle includes following step
Suddenly：
S1 takes the solution containing magnetic particle, using buffer solution for cleaning magnetic particle, and is suspended in buffer solution；
The carrying Cap gene of porcine circovirus type 2 of purifying is added in S2；
Crosslinking agent or catalyst, oscillating reactions is added in S3；
S4, magnet adsorption magnetic particle, washing are suspended in the solution containing sealer.
Wherein, the ratio of the solution of the magnetic particle and carrying Cap gene of porcine circovirus type 2 is 10mg:1-5nmol.
Wherein, the preparation method of the solution of the carrying Cap gene of porcine circovirus type 2 indirect conjugation magnetic particle includes as follows
Step：
1) magnetic particle combination Avidin
S1 takes the solution containing magnetic particle, using buffer solution for cleaning magnetic particle, and is suspended in buffer solution；
Avidin is added in S2；
Crosslinking agent or catalyst, oscillating reactions is added in S3；
S4, magnet adsorption magnetic particle, washing are suspended in the solution containing sealer, constitute magnetic particle-Avidin
Compound；
2) carrying Cap gene of porcine circovirus type 2 combination biotin
S1 takes carrying Cap gene of porcine circovirus type 2, dialysis purification；
S2 sequentially adds biotin and sealer, reaction；
S3, dialysis remove unbonded biotin, obtain carrying Cap gene of porcine circovirus type 2-biotin.
3) magnetic particle-Avidin is mixed with carrying Cap gene of porcine circovirus type 2-biotin composite, by Avidin with
The binding force connection magnetic particle and carrying Cap gene of porcine circovirus type 2 of biotin.
Wherein, the preparation method of the protein solution of the luminescent label includes the following steps：
S1 takes the albumen specifically bound with carrying Cap gene of porcine circovirus type 2 or with porcine circovirus 2 type antibody, dialysis
Purifying；
Luminous marker, reaction is added in S2；
Sealer, reaction is added in S3；
S4, the unbonded luminous marker of dialysis separation.
Wherein, the carrying Cap gene of porcine circovirus type 2 is selected from carrying Cap gene of porcine circovirus type 2 overall length, natural Cap protein
Segment, the carrying Cap gene of porcine circovirus type 2 overall length of recombinant expression, the Cap protein segment of recombinant expression, Cap protein polypeptide, Cap
One or more combinations in protein chemistry synthetic, the magnetic particle are with Fe3O4For core, surface is covered with polymerization
Object coating, and import the particle of hydroxyl, carboxyl, sulfonyl or amino active group.
Wherein, a diameter of 0.1-5 μm of the magnetic particle.
Preferably, a diameter of 1-3 μm of the magnetic particle, the magnetic particle diameter CV ＜ 3%.
Wherein, any one of the luminous marker in acridinium ester, alkaline phosphatase, peroxidase.
Wherein, the albumen of the luminescent label is selected from porcine circovirus 2 type antigen, monoclonal antibody, polyclonal
One or more combinations in antibody, genetic engineering antibody, anti-pig IgG antibody, anti-pig IgM antibody.
Wherein, the luminous substrate is corresponded with luminous marker.
Preferably, when the luminous marker is acridinium ester, luminous substrate is by the first luminous substrate and the second luminous bottom
Object form, the first luminous substrate be the solution containing 0.1mol/L nitric acid, 0.1% hydrogen peroxide, the second luminous substrate be containing
The solution of 2%Tween-20,0.25mol/L NaOH；When the luminous marker is alkaline phosphatase, luminous substrate is with gold
Substrate solution based on rigid alkane；When the luminous marker is peroxidase, luminous substrate is by the first luminous substrate and second
Luminous substrate forms, and the first luminous substrate is the solution containing 0.5g/L luminols, 0.1g/L p-iodophenols, and the second luminous substrate is
0.625g/L urea peroxide solution.
Wherein, in the kit further include dilution, quality-control product, calibration object and cleaning solution, dilution is buffer solution, ox
Haemocyanin, blocking agent, monoclonal antibody, one or more combinations in polyclonal antibody.
To establish reliable quantitative criterion, present invention employs calibration object calibrations, it is ensured that the repeatability of experiment.Calibration object
Trace to the source to classical virus neutralization tests, the serum antibody titer obtained according to virus neutralization tests, it is determined that calibration object it is dense
Degree.Wherein, calibration object is divided into 8 concentration gradients, is followed successively by 0,3.13,6.25,12.5,25,50,100,200U/mL.
Wherein, the quality-control product is porcine circovirus 2 type antibody low value quality-control product and high level quality-control product, the low value Quality Control
The Quality Control of product ranging from 22-36U/mL, the Quality Control ranging from 92-142U/mL of high level quality-control product；The cleaning solution 0.05mol/L
Phosphate (PBS) buffer solution that trishydroxymethylaminomethane (Tris) buffer solution or 0.01mol/L pH that pH is 8.0 are 7.0,
Respectively containing 0.1% Tween-20 in the Tris buffer solutions and PBS buffer solution.
The second aspect of the present invention provides a kind of detection method of porcine circovirus 2 type antibody, is tried using above-mentioned detection
Agent box, includes the following steps：
S1 sequentially adds 10-100 μ L samples to be tested or calibration object, carrying Cap gene of porcine circovirus type 2 into reaction vessel
The solution of coupling or indirect conjugation magnetic particle；
S2 reacts 10-20 minutes at 35-39 DEG C；
S3 is adsorbed with magnet, sucks supernatant, and the washing of 200-500 μ L cleaning solutions is added, discards cleaning solution；
The protein solution of 100-200 μ L luminescent labels is added into S3 by S4；
The step of S5, repetition S2 and S3；
S6, into S5, addition luminous substrate is reacted 0.5-10 minutes at 35-39 DEG C；
S7, detects luminous value with Chemiluminescence Apparatus, draws calibration curve, and antibody in test serum is calculated according to calibration curve
Concentration.
The sample to be tested includes blood sample, humoral sample and tissue sample.
Compared with prior art, the advantageous effect that the present invention realizes：
1. the kit of the present invention uses the magnetic particle of 0.1-5 μm of diameter to be coated with carrier, magnetic particle is sphere, is had
Magnetism has the characteristics that surface area is big, can be coated with more carrying Cap gene of porcine circovirus type 2；Magnetic particle can be suspended in liquid
In, it is possible to fully, in all directions contacts and react with reactant, and existing porcine circovirus 2 type antibody ELISA is examined
The coating carrier of the methods of survey method, virus neutralization tests, immunofluorescence is ELISA Plate surface or cell plate surface, is coated with or holds
The albumen received is limited.Therefore the detection method of the present invention is compared with the methods of ELISA, virus neutralization tests, immunofluorescence：
1) coated albumen is more, and detection range is wide；
2) detection method of the invention is the liquid phase reactor that can be come into full contact with, and the reaction time is short, usual 5~10 minutes.
2. the detection method of the present invention is corresponded with developing solution and luminous marker.The hair that detection method uses
Signal object can be following one kind：Acridinium ester and its derivative, alkaline phosphatase (AP), peroxidase (HRP).Acridinium ester
And its derivative is that chemiluminescent agent is marked directly on albumen, is shone by starting luminescence reagent (NaOH-H2O2) and acting on；
Protein labeling alkaline phosphatase, peroxidase are that enzymatic shines, and the substrate of alkaline phosphatase is that adamantane and its derivative are matched
The substrate of the solution set, peroxidase is luminol and its solution of derivative configuration.And current porcine circovirus 2 type antibody
The ELISA chromogenic substrates of detection method are typically TMB, and developing sensitivity, intensity are far below chemiluminescence.
3. detection method is because magnetic particle is very easily cleaned and detached under magnet effect, easy to implement to add
Sample, reaction, cleaning, separation, detection full automatic working, it is easy to accomplish quickly, high throughput, automatically detect, whole process is by examining
It surveys instrument to accurately control, testing result favorable repeatability；And it is existing detection method such as ELISA, virus neutralization tests, immune glimmering
The detection methods such as light, colloidal gold are difficult to realize full automatic working, poor repeatability.
4. the present invention uses highly homogeneous liquid phase reactor, and coordinates the accuracy of calibration object, quality-control product Control experiment, make
Repeatability has obtained very big promotion, improves the comparativity of different batches test bay testing result, be more suitable for vaccine immunity it
Monitoring afterwards.And pig annulus antibody detection method is limited by its principle, operation at present, different batches testing inspection result is difficult to weight
It is multiple.
Description of the drawings
Below in conjunction with the drawings and specific embodiments, present invention be described in more detail：
Fig. 1 is the most suitable carrying Cap gene of porcine circovirus type 2 dosage curve of carboxyl magnetic particle of the present invention；
Fig. 2 is the most suitable carrying Cap gene of porcine circovirus type 2 dosage curve of tosyl magnetic particle of the present invention；
Fig. 3 is the calibration curve in the embodiment of the present invention one；
Fig. 4 is the calibration curve in the embodiment of the present invention two；
Fig. 5 is the calibration curve in the embodiment of the present invention three；
Fig. 6 is the calibration curve in the embodiment of the present invention four.
Specific implementation mode
Carrying Cap gene of porcine circovirus type 2 for use in the present invention or its segment are not particularly limited, and may include the day
The overall length or its segment of right or recombination Cap protein.Preferably, it may include carrying Cap gene of porcine circovirus type 2 full length sequence is such as
SEQ ID NO.:Shown in 1, contain 233 amino acid, molecular weight 29KD；Carrying Cap gene of porcine circovirus type 2 fragment sequence
Such as SEQ ID NO.:Shown in 2, contain 102 amino acid, molecular weight 12.5KD；Carrying Cap gene of porcine circovirus type 2 polypeptide
Sequence such as SEQ ID NO.:Shown in 3, contain 43 amino acid, molecular weight 5KD.
Those skilled in the art can purify the polypeptide with the purified technology of protein of standard.Substantially pure polypeptide exists
Single master tape can be generated in non-reducing polyacrylamide gel.The purity of the polypeptide can also use amino acid sequence into traveling
One step is analyzed.The albumen or its segment of the present invention can be recombination, natural, synthesis albumen or its segment.This hair
The bright albumen or its segment can be native purified product or chemically synthesized product, or use recombinant technique from original
It is generated in core or eucaryon host (for example, bacterium, yeast, plant).
Magnetic particle refers to inside and is magnetic core, the particle of external coated polymer.Clad contains active group, can be with
The couplings such as albumen, polypeptide have no effect on the immunocompetence of albumen, polypeptide；Magnetic core makes particle can be directed under external magnetic fields
Mobile aggregation, leaving magnetic field later can be evenly dispersed in the solution, and the liquid phase reactor and antigen to take into account antigen-antibody are anti-
The separation of nanocrystal composition and unreacting substance.
Magnetic particle for use in the present invention is not particularly limited, and can any have magnetic core, surface with poly-
Close the magnetic-particle of object.The core that can be used for magnetic particle of the present invention is iron oxide (Fe3O4)；It can be used for magnetic-particle of the present invention
The polymer on surface includes polystyrene, acrylic resin, polymethyl methacrylate etc..The size of magnetic particle of the present invention is excellent
It is selected as 0.1-5 μm, more preferably 1-3 μm.Magnetic particle for use in the present invention exists usually in the form of Particle Swarm solution, leads to
Often, in the Particle Swarm solution, particle size shape height is uniform, grain size CV<3%.
Magnetic particle for use in the present invention can also contain multiple active groups, thus will by way of chemical crosslinking
Albumen, polypeptide are incorporated into magnetic particle surface.Preferably, the active group includes that hydroxyl, carboxyl, sulfonyl or amino are lived
Property group.Magnetic particle containing active group can be prepared by this field routine techniques or directly it is commercially available.Example
Such as it is purchased from Japanese JSR companies, article No.：The magnetic particle containing carboxyl of MagnosphereTM MS300/Caboxyl；Or it is purchased from
Japanese JSR companies, article No.：MagnosphereTM MS300/Tosyl contain the magnetic particle of tosyl.
It can be by being directly or indirectly coupled between carrying Cap gene of porcine circovirus type 2 and magnetic particle in the present invention.For example,
The direct coupling includes being condensed to form amide, magnetic particle amino and albumen ammonia by magnetic particle carboxyl and histone amino
Base forms five carbon bridges by glutaraldehyde cross-linking, tosyl magnetic particle is connected with histone amino covalent coupling.Described
Indirect conjugation includes the coupling mediated in the following manner：Streptavidin-the coupling of biotin mediation, anti-FITC antibody-
FITC is coupled.Preferably mode is：Streptavidin is coated on magnetic particle, and biotin is coupled at pig breeding and is integrated with breathing
In sign virus, bonding porcine reproductive and breath syndrome virus and magnetic particle are acted on by Streptavidin-biotin；Anti-FITC
Antibody is coated on magnetic particle, and FITC is crosslinked on porcine reproductive and respiratory syndrome virus, passes through anti-FITC antibody-FITC phases
Interaction bonding porcine reproductive and breath syndrome virus and magnetic particle.
A kind of porcine circovirus type 2 antibody testing kit, including carrying Cap gene of porcine circovirus type 2 coupling or indirectly even
Join the solution of magnetic particle, the protein solution of luminescent label, luminous substrate.
A kind of detection method of porcine circovirus 2 type antibody is included the following steps using above-mentioned detection kit：
S1 sequentially adds 10-100 μ L test serums or calibration object, carrying Cap gene of porcine circovirus type 2 into reaction vessel
The solution of coupling or indirect conjugation magnetic particle；
S2 reacts 10-20 minutes at 35-39 DEG C；
S3 is adsorbed with magnet, sucks supernatant, and the washing of 200-500 μ L cleaning solutions is added, discards cleaning solution；
The protein solution of 100-200 μ L luminescent labels is added into S3 by S4；
The step of S5, repetition S2 and S3；
S6, into S5, addition luminous substrate is reacted 0.5-10 minutes at 35-39 DEG C；
S7, detects luminous value with Chemiluminescence Apparatus, draws calibration curve, and antibody in test serum is calculated according to calibration curve
Concentration.
By taking carboxyl magnetic particle and tosyl magnetic particle as an example, magnetic particle, porcine circovirus 2 type Cap are carried out
Albumen optimum dose is tested.
One, carboxyl magnetic particle, the experiment of pig circular ring virus Cap protein optimum dose
Using 3 kinds of pig circular ring virus Cap proteins, wherein the addition of pig circular ring virus Cap protein overall length be 15.7,
31.3,62.5,125,250,500 μ g, the amount of tie substance is 0.54,1.08,2.16,4.31,8.62,17.24nmol；Pig is justified
The addition of circovirus virus Cap protein segment is 6.3,12.5,25,50,100,200 μ g, the amount of tie substance is 0.5,1,2,4,
8、16nmol；The addition of pig circular ring virus Cap protein polypeptide is 3.2,6.3,12.5,25,50,100 μ g, the amount of tie substance
For 0.64,1.26,2.5,5,10,20nmol, the addition of carboxyl magnetic particle is 10mg.
Above-mentioned pig circular ring virus Cap virus proteins and carboxyl magnetic particle are respectively used to prepare coating pig circular ring virus
Kit comprising the magnetic suspension liquid is respectively used to detection pig circular ring virus Cap antibody sun by the magnetic suspension liquid of Cap protein
Property serum, the results are shown in Table 1, Fig. 1 be coating protein dosage curve corresponding with its luminous value.
The result shows that increasing with albumen dosage, the luminous value rate of climb gradually slows down, and illustrates that magnetic particle binding protein connects
Nearly saturation；And albumen dosage continues growing, luminous value decreases instead, illustrates albumen excess, more albumen occurs certainly
Body is crosslinked.
Therefore, for carboxyl magnetic particle, pig circular ring virus Cap protein overall length, segment, polypeptide optimum dose be 1.0-
5nmol/10mg magnetic particles.Although three kinds of albumen amino acid quantity, molecular weight differences are very big, calculated by the amount of substance, most
Good coating protein dosage is not much different.
Table 1
Two, tosyl magnetic particle, the experiment of pig circular ring virus Cap protein optimum dose
Using 3 kinds of pig circular ring virus Cap proteins, wherein the addition of pig circular ring virus Cap protein overall length be 15.7,
31.3,62.5,125,250,500 μ g, the amount of tie substance is 0.54,1.08,2.16,4.31,8.62,17.24nmol；Pig is justified
The addition of circovirus virus Cap protein segment is 6.3,12.5,25,50,100,200 μ g, the amount of tie substance is 0.5,1,2,4,
8、16nmol；The addition of pig circular ring virus Cap protein polypeptide is 3.2,6.3,12.5,25,50,100 μ g, the amount of tie substance
For 0.64,1.26,2.5,5,10,20nmol.
Above-mentioned pig circular ring virus Cap virus proteins and tosyl magnetic particle are respectively used to prepare coating pig annulus
Kit comprising the magnetic suspension liquid is respectively used to detection pig circular ring virus Cap and resisted by the magnetic suspension liquid of viral Cap protein
Body positive serum, the results are shown in Table 2, and Fig. 2 is coating protein dosage curve corresponding with its luminous value.
The result shows that increasing with albumen dosage, the luminous value rate of climb gradually slows down, and illustrates that magnetic particle binding protein connects
Nearly saturation.Therefore, for tosyl magnetic particle, pig circular ring virus Cap protein overall length, segment, polypeptide optimum dose
More than 1.0nmol/10mg magnetic particles.Although three kinds of albumen amino acid quantity, molecular weight differences are very big, the amount of substance is pressed
It calculates, best coating protein dosage is not much different, and considers cost factor, albumen dosage should not be excessive, in conjunction with carboxyl magnetic particle
As a result, determine three kinds of albumen optimum amount ranging from 1.0-5nmol/10mg magnetic particles.
Table 2
Embodiment one
A kind of kit of porcine circovirus 2 type antibody detection, includes the magnetism of coating carrying Cap gene of porcine circovirus type 2
Suspension, the porcine circovirus 2 type antibody solution of alkali phosphatase enzyme mark, dilution, calibration object, quality-control product, cleaning solution shine
Substrate.
It is coated with the preparation of the magnetic suspension liquid of carrying Cap gene of porcine circovirus type 2：
(1) 1mL is taken (to be purchased from Japanese JSR companies, article No. containing magnetic particle：MagnosphereTMMS300/Caboxyl)
Solution, a concentration of 10mg/mL, 2-morpholine ethane sulfonic acid (MES) buffer solution for cleaning 2 times for being 5.0 with 0.1mol/L pH, most rear overhang
Float in the MES buffer solutions that 1mL 0.1mol/L pH are 5.0；
(2) carrying Cap gene of porcine circovirus type 2 (being purchased from YEBIO Bioengineering Co., Ltd of Qingdao) 70 μ of purifying are added
g；
(3) 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) are weighed, the MES for being 5.0 with 0.1mol/L pH
Buffer solution makes a concentration of 10mg/mL of EDC；
(4) the 100 μ L of EDC solution in (3) are taken to be added in (2), oscillating reactions 2 hours at 37 DEG C；
(5) magnet adsorption removes supernatant, the PBS solution (Tween-20 for containing 0.1%) for being 7.4 with 0.01mol/L pH
Cleaning 3 times is finally suspended in the PBS solution (BSA for containing 1%) that 0.01mol/L pH are 7.4, and is added 0.1%
ProClinTM300 (are purchased from Sigma companies, article No.：48914-U).
The magnetic particle is the magnetic particle containing carboxylic group.
The preparation of the porcine circovirus 2 type antibody solution of alkali phosphatase enzyme mark：
(1) 1mg alkaline phosphatases are taken, are diluted to the 0.05mol/L pH carbonate buffer solutions (CB buffer solutions) for being 9.5
10mg/mL；
(2) sodium metaperiodate (NaIO is weighed4) and be 9.5 CB buffer solutions with 0.05mol/L pH, make NaIO4It is dense
Degree is 12.5mg/mL；
(3) NaIO in (2) is taken4100 μ L of solution are added in (1), vibrate mixing, are protected from light at 2 DEG C 1 hour；
(4) 10 μ L ethylene glycol are taken, the CB buffer solutions that 1mL 0.05mol/L pH are 9.5 are added, obtain ethylene glycol solution；
(5) it takes 100 μ L of ethylene glycol solution in (4) to be added in (3), is protected from light in 6 DEG C 1 hour；
(6) porcine circovirus 2 type monoclonal antibody 0.5mg is taken to be added in (5), it is 9.5 that 0.05mol/L pH are used after mixing
CB buffer solutions be protected from light at 2 DEG C dialysis 20 hours；
(7) sodium borohydride (NaBH is weighed4) be dissolved in pure water, prepare the NaBH of 2mg/mL4Solution；
(8) NaBH in (7) is taken410 μ L of solution are added in (6), are protected from light in 2 DEG C 2 hours；
(9) the unbonded alkaline phosphatase of over-molecular sieve purifies and separates and porcine circovirus 2 type monoclonal antibody；
(10) by the 3- N-morpholinyls that the pH containing 1%BSA is 7.0 0.05M of the antibody-solutions in (9) after purification
(MOPS) buffer solution dilution is spare.
Diluted concentration is 0.1-0.5 μ g/mL.
Calibration object is the buffer solution of the proven porcine circovirus 2 type antibody containing known concentration.
The preparation of calibration object：
(1) by the porcine circovirus 2 type antibody strong positive serum obtained after pig annulus vaccine reinforced immunological in 60 DEG C of heat
Inactivation 1 hour；
(2) the strong positive serum after inactivation in (1) is added 0.1% through 0.2 μm of micro-filtrate membrane filtration
ProClinTM300；
(3) serum in (2) is demarcated, is diluted by a certain concentration, obtains 0,3.13,6.25,12.5,25,50,
100,200U/mL series of calibration product.
Quality-control product is proven porcine circovirus 2 type antibody positive Swine serum.It is divided into low value quality-control product and high level Quality Control
Product, the wherein Quality Control of low value quality-control product ranging from 22-36U/mL, the Quality Control ranging from 92-142U/mL of high level quality-control product.Quality Control
Product are used for the validity of Control experiment, and periodic detection quality-control product must be re-scaled if exceeding Quality Control range using calibration object.
The preparation of quality-control product：
10 parts of selection or more porcine circovirus 2 type antibody positive serum, 60 DEG C of heat inactivate 1 hour, through 0.2 μm after mixing
0.1% ProClin is added in micro-filtrate membrane filtrationTM300。
Dilution is containing 1%BSA, pH 7.4, the PBS buffer solution of a concentration of 0.01mol/L；Cleaning solution is to contain 0.1%
The Tris buffer solutions that the 0.05mol/L pH of Tween-20 are 8.0；Luminous substrate is based on adamantane and its derivative
Solution, the luminous substrate in this is Lumi-Phos 530, is purchased from Lumigen companies, article No.：P-5000.
A kind of detection method of porcine circovirus 2 type antibody, includes the following steps：
S1, several reaction tubes are taken, sequentially adds 20 μ L test serums or calibration object, 100 μ L dilutions and 25 μ L coating pigs
The magnetic suspension liquid of circovurus type 2 Cap protein；
S2, it reacts 10 minutes at 37 DEG C；
S3, magnet adsorption suck supernatant, and 300 μ L cleaning solutions are added in each reaction tube, and repeated washing 3 times discards cleaning
Liquid；
S4, the porcine circovirus 2 type antibody solution that 100 μ L alkali phosphatase enzyme marks are added into S3；
S5, it reacts 10 minutes at 37 DEG C；
Cleaning step in S6, repetition step S3；
100 μ L luminous substrates are added in S7, S6；
S8, it reacts 5 minutes at 37 DEG C；
S9, luminous value is detected with Chemiluminescence Apparatus, draws calibration curve, antibody in test serum is calculated according to calibration curve
Concentration.
It takes logarithm as X-axis using concentration value, takes Logit as Y-axis using luminous value, carry out linear fit, draw calibration curve.
Sn：Calibration object (in addition to calibration object zero) or sample luminous value；
S0：The luminous value of calibration object zero.
Table 3 is the luminous value corresponding to the calibration object of various concentration, and Fig. 3 is the calibration curve drawn.
Table 3
One, sensitivity experiment
With the porcine circovirus type 2 antibody testing kit of kit and the production of famous foreign producer in the present embodiment
(enzyme-linked immunosorbent assay, hereinafter referred to as ELISA kit) while the porcine circovirus 2 type for detecting different extension rates are anti-
Body positive serum, wherein the kit of the present embodiment is done 10 repetitions to every part of blood sample and detected, and the coefficient of variation (CV% is calculated
Standard deviation/arithmetic mean of instantaneous value of=10 test results).The results show that the kit quantification in the present embodiment is accurate.With
CV%<20% minimum concentration is as sensitivity, the present embodiment medium sensitivity<3.05U/mL is better than ELISA kit.
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
Table 4 be embodiment one kit and ELISA kit remolding sensitivity compared with
Two, repeated experiment
3 porcine circovirus 2 type antibody positive serums are taken, are detected with this kit, per 2 batches of detections of natural gift, 3 parts of blood of every batch of
Clear respectively to do 2 tests, two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 detection datas, meter
The coefficient of variation of its concentration is calculated, as a result such as table 5.The results show that 3 parts of Virus monitory result repeatability are good.
Table 5
Three, coincidence rate is tested
This kit detects more parts of Swine serums, testing result such as table 6 simultaneously with ELISA kit.The results show that this reagent
Box and ELISA kit positive coincidence rate 93.5%, negative match-rate 94.6%, overall coincidence rate 93.7%.
Table 6
Four, the kit of the present embodiment is carried out to the immunologic surveillance of pig annulus vaccine
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
After with pig annulus vaccine immunity, 3 are randomly selected, extracts blood examination within 1,7,14,21 day after immune
Porcine circovirus 2 type antibody, and as a contrast with non-immune swine, testing result such as table 7.The results show that 14 days after immune, 21
It, pig annulus antibody turns sun by the moon, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；Except 3# is immune
14 days ELISA results are feminine gender and the present embodiment result is outside the positive, and two kinds of reagent testing results are consistent.
Table 7
Embodiment two
A kind of kit of porcine circovirus 2 type antibody detection, includes the magnetism of coating carrying Cap gene of porcine circovirus type 2
Suspension, the anti-pig IgG antibody of goat (being purchased from Beijing Suo Laibao Science and Technology Ltd) solution of acridinium ester label, dilution, school
Quasi- product, quality-control product, cleaning solution, the first luminous substrate and the second luminous substrate.
It is coated with the preparation of the magnetic suspension liquid of carrying Cap gene of porcine circovirus type 2：
(1) solution of the 1mL containing magnetic particle, a concentration of 10mg/mL, the borate buffer for being 9.5 with 0.1mol/L pH are taken
Cleaning 2 times is finally suspended in the borate buffer that 1mL 0.1mol/L pH are 9.5；
(2) the 80 μ g of porcine circovirus type 2 Cap antigen of purifying, vortex mixing is added；
(3) borate buffer (containing 3mol/L ammonium sulfate) 0.5mL that 0.1mol/L pH are 9.5 is added, is vibrated at 37 DEG C
Reaction 20 hours；
(4) the BSA aqueous solutions of 0.5mL 10%, vortex mixing, 37 DEG C of oscillating reactions 12 hours is added；
(5) magnet adsorption removes supernatant, the PBS solution (Tween-20 for containing 0.1%) for being 7.4 with 0.01mol/L pH
Cleaning 3 times is finally suspended in the PBS solution (containing 1%BSA) of 0.01mol/L pH7.4, and is added 0.1%
ProClinTM300。
The magnetic particle is the magnetic particle containing toluenesulphonyl group.
The preparation of the anti-pig IgG antibody-solutions of acridinium ester label goat：
(1) the anti-pig IgG antibody of 1mg goats is taken, the CB buffer solutions for being 9.5 with 0.05mol/L pH dialysed overnight at 8 DEG C；
(2) the acridine ester solution containing 0.2mg acridinium esters is taken to be added in (1), reacting at normal temperature without light 2 hours；
(3) 100 μ L 0.1g/mL lysine solutions, reacting at normal temperature without light 2 hours is added；
(4) dialysis 24 hours is protected from light at 8 DEG C with the 0.05mol/L pH CB buffer solutions for being 9.5；
(5) antibody-solutions in (4) are standby with the MOPS buffer solutions dilution that the pH containing 1%BSA is 7.0 0.05mol/L
With.
The preparation method of calibration object is identical as embodiment one.
The preparation method of quality-control product is identical as embodiment one.
Dilution is containing 1%BSA, pH 7.4, the PBS buffer solution of a concentration of 0.01mol/L；Cleaning solution is to contain 0.1%
The PBS buffer solution that the 0.01mol/L pH of Tween-20 are 7.0；First luminous substrate is to contain 0.1mol/L nitric acid, 0.1% mistake
The solution of hydrogen oxide, the second luminous substrate are the solution containing 2%Tween-20,0.25mol/L NaOH.
A kind of detection method of porcine circovirus 2 type antibody, includes the following steps：
S1, several reaction tubes are taken, sequentially adds 10 μ L test serums or calibration object, 100 μ L dilutions and 20 μ L coating pigs
The magnetic suspension liquid of circovurus type 2 Cap protein；
S2, it reacts 15 minutes at 35 DEG C；
S3, magnet adsorption suck supernatant, and 200 μ L cleaning solutions are added in each reaction tube, and repeated washing 3 times discards cleaning
Liquid；
S4, the 150 anti-pig IgG antibody-solutions of μ L acridinium ester label goats are added into S3；
S5, it reacts 15 minutes at 35 DEG C；
Cleaning step in S6, repetition step S3；
100 the first luminous substrates of μ L and 100 the second luminous substrates of μ L are sequentially added in S7, S6；
S8, luminous value is detected with Chemiluminescence Apparatus, draws calibration curve, antibody in test serum is calculated according to calibration curve
Concentration.
Table 8 is the luminous value corresponding to different calibration object concentration, and Fig. 4 is the calibration curve drawn.
One, sensitivity experiment
The porcine circovirus 2 type for detecting different extension rates simultaneously with the kit and ELISA kit of the present embodiment is anti-
Body positive serum, wherein this reagent does 10 repetitions to every part of blood sample and detects, and the coefficient of variation is calculated, as a result such as table 9.As a result
It proves, the kit quantification of the present embodiment is accurate, and sensitivity<1.60U/mL is better than ELISA kit.
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
Table 9 be the present embodiment kit and ELISA kit remolding sensitivity compared with
Two, repeated experiment
3 porcine circovirus 2 type antibody positive serums are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift,
3 parts of serum of every batch of respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 inspections
Measured data calculates the coefficient of variation of its concentration, as a result such as table 10.The results show that 3 parts of Virus monitory result repeatability are good.
Table 10
Three, coincidence rate is tested
The kit of the present embodiment detects more parts of Swine serums simultaneously with ELISA kit, as a result such as table 11.The present embodiment
Kit and ELISA kit positive coincidence rate 94.6%, negative match-rate 94.6%, overall coincidence rate 94.6%.
Table 11
Four, the kit of the present embodiment is carried out to the immunologic surveillance of pig annulus vaccine
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
After with pig annulus vaccine immunity, 3 are randomly selected, extracts blood examination within 1,7,14,21 day after immune
Porcine circovirus 2 type antibody, and as a contrast with non-immune swine, as a result such as table 12.The results show that 14 days, 21 days after immune,
Pig annulus antibody turns sun by the moon, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；Except 3# is immunized 14 days
ELISA results are feminine gender and the present embodiment result is outside the positive, and two kinds of reagent testing results are consistent.
Table 12
Embodiment three
A kind of kit of porcine circovirus 2 type antibody detection, includes the magnetism of coating carrying Cap gene of porcine circovirus type 2
Suspension, the porcine circovirus type 2 Cap antigen solution of horseradish peroxidase-labeled, dilution, calibration object, quality-control product, cleaning
Liquid, the first luminous substrate and the second luminous substrate.
It is coated with the preparation of the magnetic suspension liquid of carrying Cap gene of porcine circovirus type 2：
(1) solution of the 1mL containing magnetic particle, a concentration of 10mg/mL, the phosphate for being 7.4 with 0.01mol/L pH are taken
(PBS) buffer solution for cleaning 2 times are finally suspended in the PBS buffer solution that 1mL 0.01mol/L pH are 7.4；
(2) glutaraldehyde solution of 0.1mL 25% (v/v), 37 DEG C of oscillating reactions 2 hours is added；
(3) it is cleaned 3 times with the 1mL 0.01mol/L pH PBS buffer solution for being 7.4；
(4) the porcine circovirus 2 type Cap recombinant antigens 30 μ g of purifying are added, 37 DEG C of oscillating reactions 20 hours；
(5) 10% bovine serum albumin(BSA)s of 0.5mL (BSA) aqueous solution, vortex mixing, 37 DEG C of oscillating reactions 2 hours is added；
(6) PBS solution for being 7.4 with 0.01mol/L pH (Tween-20 for containing 0.1%) is cleaned 3 times, is finally suspended in
In the PBS solution (containing 1%BSA) of 0.01mol/L pH7.4, and the ProClinTM300 of addition 0.1%.
The magnetic particle is the magnetic particle containing amino group.
The preparation of horseradish peroxidase-labeled porcine circovirus type 2 Cap antigen：
(1) 1mg horseradish peroxidases are taken, 10mg/mL is diluted to the 0.05mol/L pH CB buffer solutions for being 9.5；
(2) NaIO is weighed4It is 9.5 CB buffer solutions that 0.05mol/L pH, which are used in combination, makes NaIO4A concentration of 12.5mg/
mL；
(3) NaIO in (2) is taken4100 μ L of solution are added in (1), vibrate mixing, are protected from light at 8 DEG C 1 hour；
(4) 10 μ L ethylene glycol are taken, the CB buffer solutions that 1mL 0.05mol/L pH are 9.5 are added, obtain ethylene glycol solution；
(5) it takes ethylene glycol solution 1mL in (4) to be added in (3), is protected from light in 2 DEG C 1 hour；
(6) porcine circovirus type 2 Cap antigen 1mg is taken to be added in (5), the CB for being 9.5 with 0.05mol/L pH after mixing is slow
Fliud flushing is protected from light dialysis 22 hours at 8 DEG C；
(7) NaBH is weighed4It is dissolved in pure water, prepares the NaBH of 2mg/mL4Solution；
(8) NaBH in (7) is taken410 μ L of solution are added in (6), are protected from light in 8 DEG C 2 hours；
(9) over-molecular sieve purifies；
(10) by the MOPS buffer solutions of the pH7.0 0.05mol/L containing 1%BSA of the antigenic solution in (9) after purification
It dilutes spare.
The preparation method of calibration object is identical as embodiment one.
The preparation method of quality-control product is identical as embodiment one.
Dilution is containing 1%BSA, pH 7.4, the PBS buffer solution of a concentration of 0.01mol/L；Cleaning solution is to contain 0.1%
The PBS buffer solution of the 0.01mol/L pH7.0 of Tween-20；First luminous substrate is 0.5g/L luminols, 0.1g/L p-iodophenols
Solution, the second luminous substrate be 0.625g/L urea peroxide solution.
A kind of detection method of porcine circovirus 2 type antibody, includes the following steps：
S1, several reaction tubes are taken, sequentially adds 50 μ L test serums or calibration object, 100 μ L dilutions and 50 μ L coating pigs
The magnetic suspension liquid of circovurus type 2 Cap protein；
S2, it reacts 20 minutes at 39 DEG C；
S3, magnet adsorption suck supernatant, and 500 μ L cleaning solutions are added in each reaction tube, and repeated washing 3 times discards cleaning
Liquid；
S4, the porcine circovirus type 2 Cap antigen solution that 200 μ L horseradish peroxidase-labeleds are added into S3；
S5, it reacts 20 minutes at 39 DEG C；
Cleaning step in S6, repetition step S3；
50 the first luminous substrates of μ L and 50 the second luminous substrates of μ L are sequentially added in S7, S6；
S8, it reacts 10 minutes at 35 DEG C；
S9, luminous value is detected with Chemiluminescence Apparatus, draws calibration curve, antibody in test serum is calculated according to calibration curve
Concentration.
Table 13 is the corresponding luminous value of calibration object various concentration, and Fig. 5 is the calibration curve drawn.
One, sensitivity experiment
The porcine circovirus 2 type for detecting different extension rates simultaneously with the kit and ELISA kit of the present embodiment is anti-
Body positive serum, wherein this reagent does 10 repetitions to every part of blood sample and detects, and the coefficient of variation is calculated, as a result such as table 14.As a result
It proves, this reagent quantitative is accurate, and sensitivity<0.82U/mL is better than ELISA kit.
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
Table 14
Two, repeated experiment
3 porcine circovirus 2 type antibody positive serums are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift,
3 parts of serum of every batch of respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 inspections
Measured data calculates the coefficient of variation of its concentration, as a result such as table 15.The results show that 3 parts of Virus monitory result repeatability are good.
Table 15
Three, coincidence rate is tested
The kit of the present embodiment detects more parts of Swine serums, testing result such as table 16 simultaneously with ELISA kit.This implementation
The kit of example and ELISA kit positive coincidence rate 92.3%, negative match-rate 94.6%, overall coincidence rate 92.7%.
Table 16
Four, the kit of the present embodiment is monitored for pig annulus vaccine immunity
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
After with pig annulus vaccine immunity, 3 are randomly selected, extracts blood examination within 1,7,14,21 day after immune
Pig annulus antibody, and as a contrast with non-immune swine.The results show that 14 days, 21 days after immune, pig annulus antibody is turned by the moon
Sun, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；It is feminine gender except 14 days ELISA results are immunized in 3#
And the present embodiment result is positive outer, two kinds of reagent testing results are consistent.
Table 17
Example IV
A kind of kit of porcine circovirus 2 type antibody detection, includes the magnetism of coating carrying Cap gene of porcine circovirus type 2
Suspension, the porcine circovirus 2 type antibody of alkali phosphatase enzyme mark, biotinylated antigen, calibration object, quality-control product, cleaning solution, hair
Light substrate and reaction tube.
It is coated with the preparation of the magnetic suspension liquid of carrying Cap gene of porcine circovirus type 2：
(1) solution of the 1mL containing magnetic particle, a concentration of 10mg/mL, the 1- morpholine second sulphurs for being 5.0 with 0.1mol/L pH are taken
Sour (MES) buffer solution for cleaning 2 times is finally suspended in the MES buffer solutions that 1mL 0.1mol/L pH are 5.0；
(2) 350 μ g of Streptavidin are added；
(3) EDC is weighed, the MES buffer solutions for being 5.0 with 0.1mol/L pH make a concentration of 10mg/mL of EDC；
(4) the 100 μ L of EDC solution in (3) are taken to be added in (2), oscillating reactions 2 hours at 37 DEG C；
(5) magnet adsorption removes supernatant, the PBS solution (Tween-20 for containing 0.1%) for being 7.4 with 0.01mol/L pH
Cleaning 3 times is finally suspended in the PBS solution (containing 1%BSA) that 0.01mol/L pH are 7.4, and is added 0.1%
ProClinTM300。
The magnetic particle is the magnetic particle containing carboxylic group.
The preparation of the porcine circovirus 2 type antibody solution of alkali phosphatase enzyme mark：
(1) 1mg alkaline phosphatases are taken, 10mg/mL is diluted to the 0.05mol/L pH CB buffer solutions for being 9.5；
(2) NaIO is weighed4It is 9.5 CB buffer solutions that 0.05mol/L pH, which are used in combination, makes NaIO4A concentration of 12.5mg/
mL；
(3) NaIO in (2) is taken4100 μ L of solution are added in (1), vibrate mixing, are protected from light at 8 DEG C 1 hour；
(4) 10 μ L ethylene glycol are taken, the CB buffer solutions that 1mL 0.05mol/L pH are 9.5 are added, obtain ethylene glycol solution；
(5) it takes ethylene glycol solution 1mL in (4) to be added in (3), is protected from light in 6 DEG C 1 hour；
(6) porcine circovirus 2 type monoclonal antibody 1mg is taken to be added in (5), it is 9.5 that 0.05mol/L pH are used after mixing
CB buffer solutions are protected from light dialysis 24 hours at 8 DEG C；
(7) NaBH is weighed4It is dissolved in pure water, prepares the NaBH of 2mg/mL4Solution；
(8) NaBH in (7) is taken410 μ L of solution are added in (6), are protected from light in 2 DEG C 2 hours；
(9) over-molecular sieve purifies；
(10) MOPS that the pH containing 1%BSA is 7.0 0.05mol/L of the antibody-solutions in (9) after purification is buffered
Liquid dilution is spare.
The preparation of biotin antigen：
(1) 2 type Cap antigens of 1mg pigs annulus are taken, were dialysed at 8 DEG C with the 0.01mol/L pH PBS buffer solution for being 7.4
Night；
(2) preactivated biotin is dissolved in pure water, prepares the biotin solution of 50mmol/L；
(3) 20 μ L of biotin solution in (2) are taken to be added in (1), normal-temperature reaction 1 hour；
(4) 100 μ L 0.1g/mL lysine solutions, normal-temperature reaction 1 hour will be added in (3)；
(5) solution in (4) is dialysed 24 hours at 5 DEG C with the 0.01mol/L pH PBS buffer solution for being 7.4.
(6) solution in (5) is spare with the MOPS buffer solutions dilution that the pH containing 1%BSA is 7.0 0.05mol/L.
The preparation method of calibration object is identical as embodiment one.
The preparation method of quality-control product is identical as embodiment one.
Cleaning solution is the Tris buffer solutions of the 0.05M pH8.0 containing 0.1%Tween-20；Luminous substrate be with adamantane and
Solution based on its derivative.Luminous substrate in this is Lumi-Phos 530, is purchased from Lumigen companies, article No.：P-
5000。
A kind of detection method of porcine circovirus 2 type antibody, includes the following steps：
S1, several reaction tubes are taken, sequentially adds 100 μ L test serums or calibration object, 100 μ L biotinylated antigens and 25 μ L
It is coated with the magnetic suspension liquid of carrying Cap gene of porcine circovirus type 2；
S2, it reacts 10 minutes at 37 DEG C；
S3, magnet adsorption suck supernatant, and 500 μ L cleaning solutions are added in each reaction tube, and repeated washing 3 times discards cleaning
Liquid；
S4, the porcine circovirus 2 type antibody solution that 200 μ L alkali phosphatase enzyme marks are added into S3；
S5, it reacts 10 minutes at 37 DEG C；
Cleaning step in S6, repetition step S3；
100 μ L luminous substrates are added in S7, S6；
S8, it reacts 0.5 minute at 39 DEG C；
S9, luminous value is detected with Chemiluminescence Apparatus, draws calibration curve, antibody in test serum is calculated according to calibration curve
Concentration.
Table 18 is the corresponding luminous value of calibration object various concentration, and Fig. 6 is the calibration curve drawn.
Table 18
One, sensitivity experiment
The porcine circovirus 2 type for detecting different extension rates simultaneously with the kit and ELISA kit of the present embodiment is anti-
Body positive serum, wherein the kit of the present embodiment is done 10 repetitions to every part of blood sample and detected, and calculates the coefficient of variation, as a result
Such as table 19.The results show that this reagent quantitative is accurate, and sensitivity<1.87U/mL is better than ELISA kit.
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
Table 19
Two, repeated experiment
3 porcine circovirus 2 type antibody positive serums are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift,
3 parts of serum of every batch of respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 inspections
Measured data calculates the coefficient of variation of its concentration, as a result such as table 20.The results show that 3 parts of Virus monitory result repeatability are good.
Table 20
Three, coincidence rate is tested
The kit of the present embodiment detects more parts of Swine serums simultaneously with ELISA kit, as a result such as table 21.The present embodiment
Kit and ELISA kit positive coincidence rate 92.3%, negative match-rate 92.7%, overall coincidence rate 92.4%.
Table 21
Four, the kit of the present embodiment is monitored for pig annulus vaccine immunity
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/P<
0.40 is determined as that feminine gender, S/P >=0.40 are determined as the positive.
After with pig annulus vaccine immunity, 3 are randomly selected, extracts blood examination within 1,7,14,21 day after immune
Pig annulus antibody, and as a contrast with non-immune swine.The results show that 14 days, 21 days after immune, pig annulus antibody is turned by the moon
Sun, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；It is feminine gender except 14 days ELISA results are immunized in 3#
And the present embodiment result is positive outer, two kinds of reagent testing results are consistent.
Table 22
The present invention is used as coating carrier using the magnetic particle of specified particle diameter, using specific porcine circovirus 2 type Cap eggs
Mixed and reacted with the ratio between magnetic particle in vain, obtain coating homogeneous, stable structure carrying Cap gene of porcine circovirus type 2
Conjugated magnetic particle also saves coating protein raw material, and coated albumen is abundant, detection range is wider, and sensitivity is higher, anti-
Between seasonable very short (only needing 5-10 minutes), and with high-throughput, automation, repeatable excellent characteristics.
The above specific embodiments are only exemplary, is to preferably make skilled artisans appreciate that originally
Patent, be not to be construed as include to this patent range limitation；As long as appointing according to made by spirit disclosed in this patent
How with change or modification, the range that this patent includes is each fallen within.
Sequence table
<110>Upper sea noise victory bio tech ltd
<120>A kind of porcine circovirus type 2 antibody testing kit and its detection method
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<212>PRT
<213>Artificial sequence
<400>3
RNVDHVGLGT AFENSKYDQD YNIRVTMYVQ FREFNLKDPP LKP 43
Claims (10)
1. a kind of porcine circovirus type 2 antibody testing kit, which is characterized in that be coupled including carrying Cap gene of porcine circovirus type 2
Or the solution of indirect conjugation magnetic particle, the protein solution and luminous substrate of luminescent label.
2. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the 2 porcine circovirus
The solution manufacturing method of type Cap protein conjugated magnetic particle includes the following steps：
S1 takes the solution containing magnetic particle, using buffer solution for cleaning magnetic particle, and is suspended in buffer solution；
The carrying Cap gene of porcine circovirus type 2 of purifying is added in S2；
Crosslinking agent or catalyst, oscillating reactions is added in S3；
S4, magnet adsorption magnetic particle, washing are suspended in the solution containing sealer.
3. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the 2 porcine circovirus
The preparation method of the solution of type Cap protein indirect conjugation magnetic particle includes the following steps：
1) magnetic particle combination Avidin
S1 takes the solution containing magnetic particle, using buffer solution for cleaning magnetic particle, and is suspended in buffer solution；
Avidin is added in S2；
Crosslinking agent or catalyst, oscillating reactions is added in S3；
S4, magnet adsorption magnetic particle, washing are suspended in the solution containing sealer, and it is compound to constitute magnetic particle-Avidin
Object；
2) carrying Cap gene of porcine circovirus type 2 combination biotin
S1 takes carrying Cap gene of porcine circovirus type 2, dialysis；
Biotin, reaction is added in S2；
Sealer, reaction is added in S3；
S3, dialysis remove unbonded biotin, obtain carrying Cap gene of porcine circovirus type 2-biotin.
3) magnetic particle-Avidin is mixed with carrying Cap gene of porcine circovirus type 2-biotin composite, passes through Avidin and biology
The binding force connection magnetic particle and carrying Cap gene of porcine circovirus type 2 of element.
4. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the luminous marker
The preparation method of the protein solution of label includes the following steps：
S1 takes the albumen with carrying Cap gene of porcine circovirus type 2 or porcine circovirus 2 type antibody specific binding, dialysis；
Luminous marker, reaction is added in S2；
Sealer, reaction is added in S3；
S4, the unbonded luminous marker of dialysis separation.
5. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the 2 porcine circovirus
Type Cap protein is selected from the porcine circovirus 2 type of carrying Cap gene of porcine circovirus type 2 overall length, natural Cap protein segment, recombinant expression
Cap protein overall length, the Cap protein segment of recombinant expression, one kind in Cap protein polypeptide, Cap protein synthetics are described
Magnetic particle be with Fe3O4For core, surface is covered with polymer coating, and imports hydroxyl, carboxyl, sulfonyl or amino activity
The particle of group.
6. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the luminous marker
The albumen of any one in acridinium ester, alkaline phosphatase, peroxidase, the luminescent label is justified selected from pig
In 2 type Cap antigens of circovirus virus, monoclonal antibody, polyclonal antibody, genetic engineering antibody, anti-pig IgG antibody, anti-pig IgM antibody
Any one.
7. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that the luminous substrate with
Luminous marker corresponds.
8. porcine circovirus type 2 antibody testing kit as described in claim 1, which is characterized in that in the kit also
Including dilution, quality-control product, calibration object and cleaning solution, dilution is selected from buffer solution, bovine serum albumin(BSA), blocking agent, monoclonal
Antibody, one or more combinations in polyclonal antibody.
9. porcine circovirus type 2 antibody testing kit as claimed in claim 8, which is characterized in that the quality-control product is pig
Circovirus 2 type antibody low value quality-control product and high level quality-control product, the Quality Control ranging from 22-36U/mL of the low value quality-control product are high
It is worth the Quality Control ranging from 92-142U/mL of quality-control product；The cleaning solution is the trihydroxy methyl amino first that 0.05mol/L pH are 8.0
Phosphate (PBS) buffer solution that alkane (Tris) buffer solution or 0.01mol/L pH are 7.0, the Tris buffer solutions and PBS bufferings
Respectively containing 0.1% Tween-20 in liquid.
10. a kind of detection method of porcine circovirus 2 type antibody, using claim 1-9 any one of them detection kits,
It is characterised in that it includes following steps：
S1 sequentially adds 10-100 μ L samples to be tested or calibration object, carrying Cap gene of porcine circovirus type 2 coupling into reaction vessel
Or the solution of indirect conjugation magnetic particle；
S2 reacts 10-20 minutes at 35-39 DEG C；
S3 is adsorbed with magnet, sucks supernatant, and the washing of 200-500 μ L cleaning solutions is added, discards cleaning solution；
The protein solution of 100-200 μ L luminescent labels is added into S3 by S4；
The step of S5, repetition S2 and S3；
S6, into S5, addition luminous substrate is reacted 0.5-10 minutes at 35-39 DEG C；
S7, with Chemiluminescence Apparatus detect luminous value, draw calibration curve, according to calibration curve calculate test serum in antibody it is dense
Degree.
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| CN201710019645.3A CN108303541B (en) | 2017-01-11 | 2017-01-11 | Porcine circovirus type 2 antibody detection kit and detection method thereof |
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| CN201710019645.3A CN108303541B (en) | 2017-01-11 | 2017-01-11 | Porcine circovirus type 2 antibody detection kit and detection method thereof |
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