CN108303530A - A kind of pseudorabies gB antibody assay kits and its detection method - Google Patents
A kind of pseudorabies gB antibody assay kits and its detection method Download PDFInfo
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- CN108303530A CN108303530A CN201710019499.4A CN201710019499A CN108303530A CN 108303530 A CN108303530 A CN 108303530A CN 201710019499 A CN201710019499 A CN 201710019499A CN 108303530 A CN108303530 A CN 108303530A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/032—Pseudorabies virus, i.e. Aujetzky virus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a kind of pseudorabies gB antibody assay kits 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 pseudorabies gB 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 pseudorabies gB antibody tests technical fields more particularly to a kind of pseudorabies gB antibody tests to try
Agent box and the detection method being detected using the kit.
Background technology
Porcine pseudorabies (Pseudorabies, PR) are caused by Pseudorabies virus (Pseudorabies Virus, PRV)
A kind of acute infectious disease.Adult Pig is often stealthy infection, and miscarriage, production stillborn foetus and the mummification of fetus occur for pregnant sow sense；It is pseudo- mad
Dog disease virus congenital infection or early infection draw the nervous symptoms and high mortality of suckling pig；For child care pig and growing and fattening pigs,
Pseudorabies virus is the important pathogen of respiratory system epidemic disease syndrome (PRDC), causes its Respiratory symptoms and growth retardation.
This disease is widely present in China, is important one of infectious disease.For animal once infected, virus can be in long-term latent sense in vivo
Dye state, the several years that can survive in vivo are even lifelong, it is difficult to remove.Under certain condition, virus can also be reactivated and
Toxin expelling is propagated.Therefore, the purification and elimination of the disease are particularly significant.
In China, immunity inoculation is to prevent the main policies of pseudoabies.Pseudorabies vaccine currently used in the market
The overwhelming majority is the attenuated vaccine of certain gE genes and TK genes.Pseudorabies virus gB and gD membrane glycoprotein plays important work
With and necessary structural proteins, can generate protectiveness neutralizing antibody after immune.Rear pseudorabies gB antibody titers are immunized and attack
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.It establishes at present and the method applied has ELISA method, virus neutralization tests, colloidal gold strip
The methods of.
Pseudorabies gB albumen is the main neutrality antigen protein of porcine pseudorabies virus, 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 there is the current demand that pseudorabies gB antibody quantitatively detects.Existing testing agency has veterinary station, vaccine public
There is a large amount of samples for department, farm etc., and also having to the pseudorabies gB antibody assay kits and instrument of big flux needs
It asks.The method of existing ELISA, colloidal gold etc. is difficult to meet.
It establishes at present and the pseudorabies gB antibody detection methods applied has ELISA method, virus neutralization tests, immune glimmering
The methods of light method, colloidal gold strip.ELISA method is currently the most common method of pseudorabies gB antibody, the surveyed OD of ELISA
The range of value defines that measured antibody range is relatively narrow, can only accomplish sxemiquantitative, the time of reaction is usually 2 in 0.1-3.5
Hour or so, the time is longer；Virus neutralization tests is that middle progress antigen is anti-on cell by pseudorabies gB albumen and antibody
Precursor reactant judges antibody titer, can reflect the height of neutralizing antibody more comprehensively, and overall process is that titration of virus, antibody neutralize, and is needed
5-7 days time, overall process can only be operated manually, be judged, poor repeatability；Immunofluorescence technique is generally also to be operated on cell,
The shortcomings of it is not high that there are sensitivity, poor repeatability；Colloidal gold method energy rapid results, 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.
Pseudorabies gB Protein Detections mainly can not meet quantitative need using the ELISA method of sxemiquantitative at present
It asks, this point is mainly due to lacking pseudorabies gB protein standard substances and quantitative criterion, without the detection of accurate repeatability
Caused by method.It is different from animal doctor, cure field, the marker of each disease, thyroid peroxidase as escribed above in people
Antibody has all been well studied, and establishes general national standard, international standard or professional standard, 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 puppet is mad
Dog gB Protein Detections market is largely occupied by the ELISA qualitative detection reagents of import, and the product testing result of each producer
It differs greatly, 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 pseudorabies gB antibody assay kits, and it is pseudo- that it can quantify detection pig
The content of mad dog gB antibody.For this purpose, the present invention also provides a kind of method being detected using the kit, detection of the invention
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 pseudorabies gB antibody assay kits, including pseudorabies gB albumen
The solution of coupling or indirect conjugation magnetic particle, the protein solution and luminous substrate of luminescent label.
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 pseudorabies gB albumen coupling magnetic particles 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 pseudorabies gB albumen 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 pseudorabies gB albumen is 10mg:0.22-4.29nmol.
Wherein, the preparation method of the solution of the pseudorabies gB albumen 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, constitute magnetic particle-Avidin
Compound；
2) pseudorabies gB protein bindings biotin
S1 takes pseudorabies gB albumen, dialysis purification；
S2 sequentially adds biotin and sealer, reaction；
S3, dialysis remove unbonded biotin, obtain pseudorabies gB avidin-biotins.
3) magnetic particle-Avidin is mixed with pseudorabies gB avidin-biotin compounds, passes through Avidin and biotin
Binding force connection magnetic particle and pseudorabies gB albumen.
Wherein, the preparation method of the protein solution of the luminescent label includes the following steps：
S1 takes the albumen combined with pseudorabies gB albumen or with pseudorabies gB antibody specificities, dialysis purification；
Luminous marker, reaction is added in S2；
Sealer, reaction is added in S3；
S4, the unbonded luminous marker of dialysis separation.
Wherein, the pseudorabies gB albumen be selected from pseudorabies gB full length proteins, natural pseudorabies gB protein fragments,
The pseudorabies gB full length proteins of recombinant expression, the pseudorabies gB protein fragments of recombinant expression, pseudorabies gB polypeptides,
One or more combinations in pseudorabies gB protein chemistry synthetics, the magnetic particle are with Fe3O4For core, table
Face is covered with polymer coating, and imports 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 pseudorabies gB antigens, monoclonal antibody, Anti-TNF-α
The combination of any one or more in body, 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
Seralbumin, 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 pseudorabies gB antibody low value quality-control products and high level quality-control product, the low value quality-control product
Quality Control ranging from 22-36U/mL, the Quality Control ranging from 88-144U/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 pseudorabies gB antibody, using above-mentioned detection reagent
Box includes the following steps：
S1 sequentially adds 10-100 μ L samples to be tested, pseudorabies gB albumen couplings or indirect conjugation into reaction vessel
The solution of 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 pseudorabies gB albumen；Magnetic particle can float on a liquid, so
Fully, in all directions it can contact and react with reactant, and the side such as existing pseudorabies gB antibody ELISA detection methods
The coating carrier of method is ELISA Plate surface, and coating or the albumen accommodated are limited.Therefore the side such as the detection method of the present invention and ELISA
Method is compared：
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, by starting luminescence reagent (NaOH-H2O2) act on and shine；
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 pseudorabies gB antibody inspection at present
The ELISA chromogenic substrates of survey 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 not homogeneous test bay testing result, is more suitable for after vaccine immunity
Monitoring.And pseudorabies gB antibody detection methods are limited by its principle, operation at present, homogeneous testing inspection result is not 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 pseudorabies gB albumen dosage curves of carboxyl magnetic particle of the present invention；
Fig. 2 is the most suitable pseudorabies gB albumen dosage curves 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
Porcine reproductive and respiratory syndrome virus antigen protein for use in the present invention or its segment are not particularly limited, can be with
Include the overall length or its segment of described natural or recombination gB albumen.Preferably, it may include pseudorabies gB full length protein sequences
Such as SEQ ID NO.:Shown in 1, contain 914 amino acid, molecular weight 114KD；Pseudorabies gB protein fragment sequences are such as
SEQ ID NO.:Shown in 2, contain 233 amino acid, molecular weight 29KD；Pseudorabies gB polypeptides 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 porcine reproductive and respiratory syndrome virus antigen and magnetic particle in the present invention.
For example, the described direct coupling include be condensed with histone amino by magnetic particle carboxyl to be formed amide, magnetic particle amino and
Histone amino forms five carbon bridges by glutaraldehyde cross-linking, tosyl magnetic particle is connected with histone amino covalent coupling.
The indirect conjugation includes the coupling mediated in the following manner：Streptavidin-the coupling of biotin mediation, anti-FITC are anti-
Body-FITC couplings.Preferably mode is：Streptavidin is coated on magnetic particle, and biotin is coupled at pig breeding and breathing
On syndrome virus, bonding porcine reproductive and breath syndrome virus and magnetic particle are acted on by Streptavidin-biotin；It is 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 interaction bonding porcine reproductives and breath syndrome virus and magnetic particle.
A kind of pseudorabies gB antibody assay kits, including pseudorabies gB albumen couplings or indirect conjugation magnetic particle
Solution, the protein solution and luminous substrate of luminescent label.
A kind of detection method of pseudorabies gB antibody is included the following steps using above-mentioned detection kit：
S1, sequentially added into reaction vessel 10-100 μ L samples to be tested or calibration object, pseudorabies gB albumen couplings 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, 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, pseudorabies gB albumen are carried out most
Dosage is tested.
One, carboxyl magnetic particle, the experiment of porcine pseudorabies virus gB antigen protein optimum doses
Using 3 kinds of porcine pseudorabies virus gB antigen proteins, the addition of wherein porcine pseudorabies virus gB full length proteins is
15.7,31.3,62.5,125,250,500 μ g, the amount of tie substance is 0.14,0.27,0.55,1.1,2.19,4.39nmol；
The addition of porcine pseudorabies virus gB protein fragments is 3.2,6.3,12.5,25,50,100 μ g, the amount of tie substance is 0.11,
0.22、0.43、0.86、1.72、3.45nmol；The addition of porcine pseudorabies virus gB polypeptides be 0.7,1.3,2.5,5,
10,20 μ g, the amount of tie substance is 0.14,0.26,0.5,1,2,4nmol.The addition of carboxyl magnetic particle is 10mg.
Above-mentioned porcine pseudorabies virus gB virus proteins and carboxyl magnetic particle are respectively used to prepare coating porcine pseudorabies
Kit comprising the magnetic suspension liquid is respectively used to detection porcine pseudorabies virus gB by the magnetic suspension liquid of malicious gB antigen proteins
Positive Sera, the results are shown in Table 1, and Fig. 1 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；And albumen dosage continues growing, luminous value decreases instead, illustrates albumen excess, more albumen occurs certainly
Body is crosslinked.
Therefore, for carboxyl magnetic particle, porcine pseudorabies virus gB full length proteins, segment, polypeptide optimum dose be
0.22-4.29nmol/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.
Table 1
Two, tosyl magnetic particle, the experiment of porcine pseudorabies virus gB antigen protein optimum doses
Using 3 kinds of porcine pseudorabies virus gB antigen proteins, the addition of wherein porcine pseudorabies virus gB full length proteins is
15.7,31.3,62.5,125,250,500 μ g, the amount of tie substance is 0.14,0.27,0.55,1.1,2.19,4.39nmol；
The addition of porcine pseudorabies virus gB protein fragments is 3.2,6.3,12.5,25,50,100 μ g, the amount of tie substance is 0.11,
0.22、0.43、0.86、1.72、3.45nmol；The addition of porcine pseudorabies virus gB polypeptides be 0.7,1.3,2.5,5,
10,20 μ g, the amount of tie substance is 0.14,0.26,0.5,1,2,4nmol.The addition of tosyl magnetic particle is
10mg。
Above-mentioned porcine pseudorabies virus gB virus proteins and tosyl magnetic particle are respectively used to prepare coating pig puppet
Kit comprising the magnetic suspension liquid is respectively used to detection pseudorabies by the magnetic suspension liquid of rabies viruses gB antigen proteins
Viral gB Positive Seras, 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, porcine pseudorabies virus gB full length proteins, segment, polypeptide optimum dose
More than 0.22nmol/10mg magnetic particles.Although three kinds of albumen amino acid quantity, molecular weight differences are very big, by substance
Amount calculates, and best coating protein dosage is not much different, and considers cost factor, albumen dosage should not be excessive, micro- in conjunction with carboxyl magnetism
Grain as a result, determine three kinds of albumen optimum amount ranging from 0.22-4.29nmol/10mg magnetic particles.
Table 2
Embodiment one
A kind of kit of pseudorabies gB antibody tests includes the magnetic suspension liquid of coating pseudorabies gB albumen, alkali
The pseudorabies gB antibody-solutions of acid phosphatase label, dilution, calibration object, quality-control product, cleaning solution, luminous substrate.
It is coated with the preparation of the magnetic suspension liquid of pseudorabies gB albumen：
(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) pseudorabies gB albumen (being purchased from hundred million minot bio tech ltd of Hangzhou) 50 μ g of purifying are added；
(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 pseudorabies gB antibody-solutions 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) pseudorabies gB monoclonal antibodies 0.5mg is taken to be added in (5), the CB for being 9.5 with 0.05mol/L pH after mixing
Buffer solution is protected from light dialysis 20 hours at 2 DEG C；
(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 pseudorabies gB monoclonal antibodies；
(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 pseudorabies gB antibody containing known concentration.
The preparation of calibration object：
(1) the pseudorabies gB antibody strong positive serum obtained after pseudorabies vaccine reinforced immunological is gone out in 60 DEG C of heat
It is 1 hour living；
(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 pseudorabies gB antibody positive Swine serums.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 88-144U/mL of high level quality-control product.Quality-control product is used
In the validity of Control experiment, 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 or more pseudorabies gB Positive Seras of selection, 60 DEG C of heat inactivations 1 hour are micro- through 0.2 μm after mixing
0.1% ProClin is added in 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 pseudorabies gB 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 25 μ L coating pigs
The magnetic suspension liquid of pseudo- mad dog gB albumen；
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 pseudorabies gB antibody-solutions 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
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；
Pseudorabies gB antibody assay kits with kit and the production of famous foreign producer in the present embodiment are (enzyme-linked
Immunosorbent adsorption test, hereinafter referred to as ELISA kit) while detecting the pseudorabies gB antibody positive blood of different extension rates
Clearly, 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%=10 survey is calculated
Standard deviation/arithmetic mean of instantaneous value of test result).The results show that the kit quantification in the present embodiment is accurate.With CV%<20%
Minimum concentration as sensitivity, the present embodiment medium sensitivity is 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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
Table 4 be embodiment one kit and ELISA kit remolding sensitivity compared with
Two, repeated experiment
3 pseudorabies gB Positive Seras are taken, are detected with this kit, per 2 batches of detections of natural gift, 3 parts of serum of every batch of
2 tests are respectively done, two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 detection datas, calculates
The coefficient of variation of its concentration, 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 95.0%, negative match-rate 92.0%, overall coincidence rate 91.8%.
Table 6
Four, the kit of the present embodiment is carried out to the immunologic surveillance of pseudorabies vaccine
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
After with pseudorabies vaccine immunity, 4 are randomly selected, extracts blood sample inspection within 1,7,14,21 day after immune
Porcine pseudorabies virus gB antibody is surveyed, and as a contrast with non-immune swine, testing result such as table 7.The results show that 14 after immune
It, 21 days, porcine pseudorabies virus gB antibody turns sun by the moon, and concentration is gradually increasing；And the antibody content of non-immune swine is without apparent
Variation；Two kinds of reagent testing results are consistent.
Table 7
Five, specificity experiments
A variety of correlated virus antibody strong positive serum are detected with the kit of the present embodiment, including swine fever virus (CSFV),
Foot and mouth disease virus O-shaped (FMDV-O), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), cattle disease
Viral diarrhea virus (BVDV).Testing result is below 10U/mL, is feminine gender, does not find cross reaction.
Table 8
| Correlated virus | CSFV | FMD-O | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 4.86 | 5.21 | 4.37 | 3.39 | 4.21 |
Embodiment two
A kind of kit of pseudorabies gB antibody tests includes the magnetic suspension liquid of coating pseudorabies gB albumen, a word used for translation
The anti-pig IgG antibody of goat (being purchased from Beijing Suo Laibao Science and Technology Ltd) solution of pyridine ester label, dilution, calibration object, 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 pseudorabies gB albumen：
(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 60 μ g of pseudorabies gB antigens 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 pseudorabies gB 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 20 μ L coating pigs
The magnetic suspension liquid of pseudo- mad dog gB albumen；
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 9 is the luminous value corresponding to different calibration object concentration, and Fig. 4 is the calibration curve drawn.
One, sensitivity experiment
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；
Detect the pseudorabies gB antibody sun of different extension rates simultaneously with the kit and ELISA kit of the present embodiment
Property serum, wherein this kit is done 10 repetitions to every part of blood sample and is detected, and the coefficient of variation is calculated, as a result such as table 10.As a result it demonstrate,proves
Bright, the kit quantification of the present embodiment is accurate, and sensitivity<1.59U/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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
Table 10 be the present embodiment kit and ELISA kit remolding sensitivity compared with
Two, repeated experiment
3 pseudorabies gB Positive Seras are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift, every batch of
3 parts of serum respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 testing numbers
According to the coefficient of variation of its concentration being calculated, as a result such as table 11.The results show that 3 parts of Virus monitory result repeatability are good.
Table 11
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 12.The present embodiment
Kit and ELISA kit positive coincidence rate 96.3%, negative match-rate 91.2%, overall coincidence rate 92.1%.
Table 12
Four, the kit of the present embodiment is carried out to the immunologic surveillance of pseudorabies vaccine
The present embodiment concentration<10U/mL is determined as that feminine gender, concentration >=10U/mL are determined as the positive；Elisa kits S/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
After with pseudorabies vaccine immunity, 4 are randomly selected, extracts blood sample inspection within 1,7,14,21 day after immune
Porcine pseudorabies virus gB antibody is surveyed, and as a contrast with non-immune swine, as a result such as table 13.The results show that 14 days after immune, 21
It, porcine pseudorabies virus gB antibody turns sun by the moon, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；
Two kinds of reagent testing results are consistent.
Table 13
Five, specificity experiments
A variety of correlated virus antibody strong positive serum are detected with the kit of the present embodiment, including swine fever virus (CSFV),
Foot and mouth disease virus O-shaped (FMDV-O), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), cattle disease
Viral diarrhea virus (BVDV).Testing result is below 10U/mL, is feminine gender, does not find cross reaction.
Table 14
| Correlated virus | CSFV | FMD-O | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 2.52 | 1.87 | 2.61 | 2.66 | 2.08 |
Embodiment three
A kind of kit of pseudorabies gB antibody tests includes the magnetic suspension liquid of coating pseudorabies gB albumen, peppery
The pseudorabies gB antigenic solutions of root peroxidase labelling, dilution, calibration object, quality-control product, cleaning solution, the first luminous substrate
With the second luminous substrate.
It is coated with the preparation of the magnetic suspension liquid of pseudorabies gB albumen：
(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 pseudorabies gB recombinant antigens 50 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 20 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 pseudorabies gB antigens：
(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) take pseudorabies gB antigen 1s mg be added (5) in, after mixing with 0.05mol/L pH be 9.5 CB buffer solutions in
Dialysis 22 hours is protected from light 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 pseudorabies gB 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 pseudo- mad dog gB albumen；
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 pseudorabies gB antigenic solutions 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 15 is the corresponding luminous value of calibration object various concentration, and Fig. 5 is the calibration curve drawn.
One, sensitivity experiment
Detect the pseudorabies gB antibody sun of different extension rates simultaneously with the kit and ELISA kit of the present embodiment
Property serum, wherein this kit is done 10 repetitions to every part of blood sample and is detected, and the coefficient of variation is calculated, as a result such as table 16.As a result it demonstrate,proves
Bright, this reagent quantitative is accurate, and sensitivity<0.58U/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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
Table 16
Two, repeated experiment
3 pseudorabies gB Positive Seras are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift, every batch of
3 parts of serum respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 testing numbers
According to the coefficient of variation of its concentration being calculated, as a result such as table 17.The results show that 3 parts of Virus monitory result repeatability are good.
Table 17
Three, coincidence rate is tested
The kit of the present embodiment detects more parts of Swine serums, testing result such as table 18 simultaneously with ELISA kit.This implementation
The kit of example and ELISA kit positive coincidence rate 91.3%, negative match-rate 94.9%, overall coincidence rate 91.1%.
Table 18
Four, the kit of the present embodiment is monitored for pseudorabies 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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
After with pseudorabies vaccine immunity, 4 are randomly selected, extracts blood sample inspection within 1,7,14,21 day after immune
Porcine pseudorabies virus gB antibody is surveyed, and as a contrast with non-immune swine.The results show that 14 days, 21 days after immune, pseudorabies
Viral gB 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# immune rear 14
Its blood sample ELISA kit testing result is feminine gender, and this reagent testing result is positive outer, two kinds of reagent detections of other blood samples
As a result consistent.
Table 19
Five, specificity experiments
A variety of correlated virus antibody strong positive serum are detected with the kit of the present embodiment, including swine fever virus (CSFV),
Foot and mouth disease virus O-shaped (FMDV-O), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), cattle disease
Viral diarrhea virus (BVDV).Testing result is below 10U/mL, is feminine gender, does not find cross reaction.
Table 20
| Correlated virus | CSFV | FMD-O | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 1.09 | 1.56 | 1.62 | 1.23 | 1.21 |
Example IV
A kind of kit of pseudorabies gB antibody tests includes the magnetic suspension liquid of coating pseudorabies gB albumen, alkali
The pseudorabies gB antibody of acid phosphatase label, biotinylated antigen, calibration object, quality-control product, cleaning solution, luminous substrate.
It is coated with the preparation of the magnetic suspension liquid of pseudorabies gB albumen：
(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 pseudorabies gB antibody-solutions 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 pseudorabies virus gB monoclonal antibodies 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) 1mg porcine pseudorabies virus gB antigens are taken, are dialysed at 8 DEG C with the 0.01mol/L pH PBS buffer solution for being 7.4
Overnight；
(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 pseudorabies gB 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 pseudorabies gB albumen；
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 pseudorabies gB antibody-solutions 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 21 is the corresponding luminous value of calibration object various concentration, and Fig. 6 is the calibration curve drawn.
Table 21
One, sensitivity experiment
Detect the pseudorabies gB antibody sun of different extension rates simultaneously with the kit and ELISA kit of the present embodiment
Property serum, wherein the kit of the present embodiment is done 10 repetitions to every part of blood sample and is detected, and the coefficient of variation is calculated, as a result such as table
22.The results show that this kit quantification is accurate, and sensitivity<1.19U/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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
Table 22
Two, repeated experiment
3 pseudorabies gB Positive Seras are taken, are detected with the kit of the present embodiment, per 2 batches of detections of natural gift, every batch of
3 parts of serum respectively do 2 tests, and two batches experiment is at least spaced 2 hours, continuous detection 5 days.Every part of serum obtains 20 testing numbers
According to the coefficient of variation of its concentration being calculated, as a result such as table 23.The results show that 3 parts of Virus monitory result repeatability are good.
Table 23
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 24.The present embodiment
Kit and ELISA kit positive coincidence rate 91.9%, negative match-rate 93.4%, overall coincidence rate 90.8%.
Table 24
Four, the kit of the present embodiment is monitored for pseudorabies 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/N≤
0.60 is determined as that the positive, 0.60-0.70 are determined as suspicious, S/N>0.70 is determined as feminine gender.
After with pseudorabies vaccine immunity, 4 are randomly selected, extracts blood sample inspection within 1,7,14,21 day after immune
Porcine pseudorabies virus gB antibody is surveyed, and as a contrast with non-immune swine.The results show that 14 days, 21 days after immune, pseudorabies
Viral gB antibody turns sun by the moon, and concentration is gradually increasing；And the antibody content of non-immune swine is without significant change；Two kinds of reagent detections
As a result consistent.
Table 25
Five, specificity experiments
A variety of correlated virus antibody strong positive serum are detected with the kit of the present embodiment, including swine fever virus (CSFV),
Foot and mouth disease virus O-shaped (FMDV-O), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), cattle disease
Viral diarrhea virus (BVDV).Testing result is below 10U/mL, is feminine gender, does not find cross reaction.
Table 26
| Correlated virus | CSFV | FMD-O | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 3.25 | 2.18 | 2.89 | 2.72 | 3.34 |
The present invention is used as coating carrier using the magnetic particle of specified particle diameter, using specific pseudorabies gB albumen and magnetic
Property the ratio between particle mixed and reacted, obtain coating homogeneous, stable structure pseudorabies gB albumen coupling magnetic particles,
Coating protein raw material is also saved, coated albumen is abundant, detection range is wider, and sensitivity is higher, the reaction time is 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 pseudorabies gB antibody assay kits and its detection method
<160> 3
<170> PatentIn version 3.5
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<213>Artificial sequence
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MPAGGGLWRG PRGHRPGHHG GAGLGRLWPA PHHAAAARGA VALALLLLAL AATPTCGAAA 60
VTRAASASPA PGTGATPDGF SAEESLEEID GAVSPGPSDA PDGEYGDLDA RTAVRAAATE 120
RDRFYVCPPP SGSTVVRLEP EQACPEYSQG RNFTEGIAVL FKENIAPHKF KAHIYYKNVI 180
VTTVWSGSTY AAITNRFTDR VPVPVQEITD VIDRRGKCVS KAEYVRNNHK VTAFDRDENP 240
VEVDLRPSRL NALGTRGWHT TNDTYTKIGA AGFYHTGTSV NCIVEEVEAR SVYPYDSFAL 300
STGDIVYMSP FYGLREGAHG EHIGYAPGRF QQVEHYYPID LDSRLRASES VTRNFLRTPH 360
FTVAWDWAPK TRRVCSLAKW REAEEMIRDE TRDGSFRFTS RALGASFVSD VTQLDLQRVH 420
LGDCVLREAS EAIDAIYRRR YNNTHVLAGD KPEVYLARGG FVVAFRPLIS NELAQLYARE 480
LERLGLAGVV GPASPAAARR ARRSPGPAGT PEPPAVNGTG HLRITTGSAE FARLQFTYDH 540
IQAHVNDMLS RIAAAWCELQ NKDRTLWGEM SRLNPSAVAT AALGQRVSAR MLGDVMAISR 600
CVEVRGGVYV QNSMRVPGER GTCYSRPLVT FEHNGTGVIE GQLGDDNELL ISRDLIEPCT 660
GNHRRYFKLG GGYVYYEDYS YVRMVEVPET ISTRVTLNLT LLEDREFLPL EVYTREELAD 720
TGLLDYSEIQ RRNQLHALKF YDIDRVVKVD HNVVLLRGIA NFFQGLGDVG AAVGKVVLGA 780
TGAVISAVGG MVSFLSNPFG ALAIGLLVLA GLVAAFLAYR HISRLRRNPM KALYPVTTKA 840
LKEDGVEEDD VDEAKLDQAR DMIRYMSIVS ALEQQEHKAR KKNSGPALLA SRVGAMATRR 900
RHYQRLESED PDAL 914
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GAAAVTRAAS ASPAPGTGAT PDGFSAEESL EEIDGAVSPG PSDAPDGEYG DLDARTAVRA 60
AATERDRFYV CPPPSGSTVV RLEPEQACPE YSQGRNFTEG IAVLFKENIA PHKFKAHIYY 120
KNVIVTTVWS GSTYAAITNR FTDRVPVPVQ EITDVIDRRG KCVSKAEYVR NNHKVTAFDR 180
DENPVEVDLR PSRLNALGTR GWHTTNDTYT KIGAAGFYHT GTSVNCIVEE VEA 233
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Claims (10)
1. a kind of pseudorabies gB antibody assay kits, which is characterized in that including pseudorabies gB albumen couplings or indirectly even
Join the solution of magnetic particle, the protein solution and luminous substrate of luminescent label.
2. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gB albumen
The solution manufacturing method of 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 pseudorabies gB albumen 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. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gB albumen
The preparation method of the solution of 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) pseudorabies gB protein bindings biotin
S1 takes pseudorabies gB albumen, dialysis；
Biotin, reaction is added in S2；
Sealer, reaction is added in S3；
S3, dialysis remove unbonded biotin, obtain pseudorabies gB avidin-biotins.
3) magnetic particle-Avidin is mixed with pseudorabies gB avidin-biotin compounds, passes through the knot of Avidin and biotin
Resultant force connection magnetic particle and pseudorabies gB albumen.
4. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the luminescent label
The preparation method of protein solution include the following steps：
S1 takes the albumen combined with pseudorabies gB albumen or pseudorabies gB antibody specificities, dialysis；
Luminous marker, reaction is added in S2；
Sealer, reaction is added in S3；
S4, the unbonded luminous marker of dialysis separation.
5. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gB albumen
Selected from pseudorabies gB full length proteins, natural pseudorabies gB protein fragments, pseudorabies gB full length proteins of recombinant expression, again
The pseudorabies gB protein fragments of group expression, one in pseudorabies gB polypeptides, pseudorabies gB protein chemistry synthetics
Kind, the magnetic particle is with Fe3O4For core, surface is covered with polymer coating, and imports hydroxyl, carboxyl, sulfonyl or ammonia
The particle of base active group.
6. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the luminous marker is selected from
The albumen of any one in acridinium ester, alkaline phosphatase, peroxidase, the luminescent label is selected from pseudorabies
It is any one in gB antigens, monoclonal antibody, polyclonal antibody, genetic engineering antibody, anti-pig IgG antibody, anti-pig IgM antibody
Kind.
7. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the luminous substrate with shine
Marker corresponds.
8. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that further include in the kit
Dilution, quality-control product, calibration object and cleaning solution, dilution are selected from buffer solution, bovine serum albumin(BSA), blocking agent, monoclonal antibody,
One or more combinations in polyclonal antibody.
9. pseudorabies gB antibody assay kits as described in claim 1, which is characterized in that the quality-control product is that pig puppet is mad
Dog gB antibody low value quality-control products and high level quality-control product, the Quality Control ranging from 22-36U/mL of the low value quality-control product, high level quality-control product
Quality Control ranging from 88-144U/mL；The cleaning solution is the trishydroxymethylaminomethane (Tris) that 0.05mol/L pH are 8.0
Phosphate (PBS) buffer solution that buffer solution or 0.01mol/L pH are 7.0, in the Tris buffer solutions and PBS buffer solution respectively
Contain 0.1% Tween-20.
10. a kind of detection method of pseudorabies gB antibody, using claim 1-9 any one of them detection kits,
It is characterized in that, includes the following steps：
S1 sequentially adds 10-100 μ L samples to be tested or calibration object, pseudorabies gB albumen couplings or indirect into reaction vessel
The solution of conjugated 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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| CN108303530B (en) | 2023-06-09 |
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