CN108303540A - A kind of pseudorabies gE antibody assay kits and its detection method - Google Patents
A kind of pseudorabies gE antibody assay kits and its detection method Download PDFInfo
- Publication number
- CN108303540A CN108303540A CN201710019644.9A CN201710019644A CN108303540A CN 108303540 A CN108303540 A CN 108303540A CN 201710019644 A CN201710019644 A CN 201710019644A CN 108303540 A CN108303540 A CN 108303540A
- Authority
- CN
- China
- Prior art keywords
- pseudorabies
- antibody
- solution
- magnetic particle
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of pseudorabies gE 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 qualitative detection of pseudorabies gE 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 gE antibody tests technical fields more particularly to a kind of pseudorabies gE 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 missing from the attenuated vaccine of gE genes and TK genes.After vaccine immunity, it cannot be detected for gE in animal body
The antibody of albumen, and wild strain contains gE genes, therefore, can detect whether contain gE antibody in blood serum sample to determine whether
By wild virus infection.
The pseudorabies gE antibody detection methods established and applied at present have ELISA method, latex agglutination experiment, colloidal gold
The methods of test strips.ELISA method is currently the most common method of pseudorabies gE antibody, and the range of the surveyed OD values of ELISA exists
0.1-3.5, sensitivity is low, and the time of reaction, the time was longer usually at 2 hours or so；Latex agglutination experiment is by pseudo- mad dog
Can antigen and antibody generate agglutination to judge, sensitivity, accuracy are not very high, and application is less；Colloidal gold method can be fast
Speed is obtained a result, but sensitivity is low, limits application range.
Invention content
In order to solve, detection time present in existing animal doctor's detection technique is long, sensitivity is low, poor repeatability, is difficult to automatically
The problem of change etc., the object of the present invention is to provide a kind of pseudorabies gE antibody assay kits, it can enzyme rapidly and sensitively be examined
Survey pseudorabies gE 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, detection time are 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 gE antibody assay kits, including pseudorabies gE 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 gE 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 gE 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 gE albumen is 10mg:0.5-5.5nmol.
Preferably, the ratio of the solution of the magnetic particle and pseudorabies gE albumen is 10mg:1-3nmol.
Wherein, the preparation method of the solution of the pseudorabies gE 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 gE protein bindings biotin
S1 takes pseudorabies gE albumen, dialysis purification；
S2 sequentially adds biotin and sealer, reaction；
S3, dialysis remove unbonded biotin, obtain pseudorabies gE avidin-biotins.
3) magnetic particle-Avidin is mixed with pseudorabies gE avidin-biotin compounds, passes through Avidin and biotin
Binding force connection magnetic particle and pseudorabies gE albumen.
Wherein, the magnetic particle core is iron oxide.
Wherein, the preparation method of the protein solution of the luminescent label includes the following steps：
S1 takes the albumen combined with pseudorabies gE albumen or with pseudorabies gE 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 gE albumen be selected from pseudorabies gE full length proteins, natural pseudorabies gE protein fragments,
The pseudorabies gE full length proteins of recombinant expression, the pseudorabies gE protein fragments of recombinant expression, pseudorabies gE polypeptides,
One or more combinations in pseudorabies gE 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, the mode of the pseudorabies gE albumen indirect conjugation magnetic particle is any one following：Pig puppet is mad
Dog gE antibody coupling magnetic particles, pseudorabies gE albumen form pig by being incubated on the antibody being trapped on magnetic particle
Pseudo- mad dog gE albumen, magnetic particle combination；Streptavidin is coated on magnetic particle, and biotin is coupled at pseudorabies gE
On albumen, pseudorabies gE albumen and magnetic particle are combined by Streptavidin-biotin effect；Anti-FITC antibody is coated on
On magnetic particle, FITC is crosslinked on pseudorabies gE albumen, and pseudorabies are combined by anti-FITC antibody-FITC interactions
GE albumen and magnetic particle.
Wherein, any one of the luminous marker in acridinium ester, alkaline phosphatase, peroxidase.
Wherein, the albumen of the luminescent label is selected from pseudorabies gE antigens, monoclonal antibody, Anti-TNF-α
One or more combinations 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, the dilution is buffering
Liquid, bovine serum albumin(BSA), blocking agent, monoclonal antibody, mostly anti-, one or more combinations in reaction tube.
Wherein, the quality-control product is pseudorabies gE antibody quality-control product 1 and quality-control product 2；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.
Wherein, calibration object shares 2, respectively pseudorabies gE negative antibodies, positive serum dilution.
The second aspect of the present invention provides a kind of detection method of pseudorabies gE antibody, using above-mentioned detection reagent
Box includes the following steps：
S1, sequentially added into reaction vessel 10-100 μ L samples to be tested or calibration object, pseudorabies gE 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, luminous value is detected with Chemiluminescence Apparatus.
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 gE 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 gE 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 gE 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 existing detection method such as ELISA, latex agglutination experiment, colloidal gold
Etc. detection methods be 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.And current pseudorabies gE antibody
Detection method is limited by its principle, operation, and homogeneous testing inspection result is not difficult to repeat.
Description of the drawings
Fig. 1 is the most suitable pseudorabies gE albumen dosage curves of carboxyl magnetic particle of the present invention；
Fig. 2 is the most suitable pseudorabies gE albumen dosage curves of tosyl magnetic particle of the present invention.
Specific implementation mode
Pseudorabies gE full length proteins contain 579 amino acid, molecular weight 69kD, protein sequence such as SEQ ID NO.:1
It is shown.Porcine pseudorabies gE albumen for use in the present invention or its segment are not particularly limited, may include it is described naturally or again
The overall length or its segment of the gE albumen of group.Preferably, it may include pseudorabies gE protein fragment sequences such as SEQ ID NO.:2 institutes
Show, contains 324 amino acid, molecular weight 38kD；Or using pseudorabies gE polypeptides sequence such as SEQ ID NO.:3 institutes
Show, contains 46 amino acid, molecular weight 5.5kD.
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 pseudorabies gE albumen and magnetic particle in the present invention.For example, described
Direct coupling include being condensed to form amide, magnetic particle amino and histone amino and lead to by magnetic particle carboxyl and histone amino
Cross five carbon bridges of glutaraldehyde cross-linking formation, tosyl magnetic particle is connected with histone amino covalent coupling.Described is indirect
Coupling includes the coupling mediated in the following manner：Streptavidin-the coupling of biotin mediation, anti-FITC antibody-FITC are even
Connection.Preferably mode is：Streptavidin is coated on magnetic particle, and biotin is coupled on pseudorabies gE albumen, is passed through
Streptavidin-biotin effect combines pseudorabies gE albumen and magnetic particle；Anti-FITC antibody is coated on magnetic particle,
FITC is crosslinked on pseudorabies gE albumen, and pseudorabies gE albumen and magnetic are combined by anti-FITC antibody-FITC interactions
Property particle.
Pseudorabies gE antibody assay kits include the magnetic suspension liquid of coating pseudorabies gE albumen, luminescent marking
The protein solution and luminous substrate of substance markers.
A kind of detection method of pseudorabies gE antibody is included the following steps using above-mentioned detection kit：
S1 sequentially adds 10-100 μ L samples to be tested or calibration object into reaction vessel, is coated with pseudorabies gE albumen
Magnetic suspension liquid；
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, luminous value is detected with Chemiluminescence Apparatus.
By taking carboxyl magnetic particle and tosyl magnetic particle as an example, magnetic particle and pseudorabies gE albumen are carried out most
Dosage is tested.
One, carboxyl magnetic particle and pseudorabies gE albumen optimum doses are tested
Using 3 kinds of pseudorabies gE albumen, wherein the addition of pseudorabies gE full length proteins be 10,20,50,100,
200,500 μ g, the amount of tie substance is 0.14,0.29,0.72,1.45,2.90,7.25nmol；Pseudorabies gE protein fragments
Addition be 10,20,50,100,200,500 μ g, the amount of tie substance is 0.26,0.53,1.32,2.63,5.26,
13.16nmol；The addition of pseudorabies gE polypeptides is 1,2,5,10,20,50 μ g, the amount of tie substance is 0.18,
0.36、0.91、1.82、3.64、9.09nmol；The addition of carboxyl magnetic particle is 10mg.
Above-mentioned pseudorabies gE albumen and carboxyl magnetic particle are respectively used to prepare the magnetic of coating pseudorabies gE albumen
Property suspension, by the kit comprising the magnetic suspension liquid be respectively used to detection pseudorabies gE Positive Seras, as a result such as
Shown in table 1, Fig. 1 is coating protein dosage curve corresponding with its luminous value.
The result shows that when albumen dosage is below 0.5nmol/10mg magnetic beads, luminous value increases with albumen dosage on rapid
It rises；More than 0.5nmol/10mg magnetic beads, luminous value amplification slows down, and illustrates magnetic bead binding protein close to saturation；And albumen dosage
When more than 5nnom/10mg magnetic beads, luminous value decreases instead with the increase of albumen dosage, and it is more to illustrate that albumen starts to occur
Self-crosslinking.
Therefore, for carboxyl magnetic particle, the optimum dose of pseudorabies gE albumen is that 0.5-5nmol/10mg magnetism is micro-
Grain；Three kinds of albumen (overall length, segment, polypeptide) are calculated, most although amino acid quantity, molecular weight difference are very big by the amount of substance
Good coating protein dosage is not much different.
Table 1
Two, tosyl magnetic particle and pseudorabies gE albumen optimum doses are tested
Using 3 kinds of pseudorabies gE albumen, wherein the addition of pseudorabies gE full length proteins be 10,20,50,100,
200,500 μ g, the amount of tie substance is 0.14,0.29,0.72,1.45,2.90,7.25nmol；Pseudorabies gE protein fragments
Addition be 10,20,50,100,200,500 μ g, the amount of tie substance is 0.26,0.53,1.32,2.63,5.26,
13.16nmol；The addition of pseudorabies gE polypeptides is 1,2,5,10,20,50 μ g, the amount of tie substance is 0.18,
0.36、0.91、1.82、3.64、9.09nmol；The addition of tosyl magnetic particle is 10mg.
Above-mentioned pseudorabies gE albumen and carboxyl magnetic particle are respectively used to prepare the magnetic of coating pseudorabies gE albumen
Property suspension, by the kit comprising the magnetic suspension liquid be respectively used to detection pseudorabies gE Positive Seras, as a result such as
Shown in table 2, Fig. 2 is coating protein dosage curve corresponding with its luminous value.
The result shows that when albumen dosage is below 0.5nmol/10mg magnetic beads, luminous value increases with albumen dosage on rapid
It rises；More than 0.5nmol/10mg magnetic beads, luminous value amplification slows down, and illustrates magnetic bead binding protein close to saturation；And albumen dosage
When more than 5nnom/10mg magnetic beads, luminous value decreases instead with the increase of albumen dosage, and it is more to illustrate that albumen starts to occur
Self-crosslinking considers cost factor, and albumen dosage is no more than 5nmol/10mg magnetic beads.
Therefore, for tosyl magnetic particle, the optimum dose of pseudorabies gE albumen is 0.5-5nmol/10mg
Magnetic particle；Three kinds of albumen (overall length, segment, polypeptide) press the gauge of substance although amino acid quantity, molecular weight difference are very big
It calculates, best coating protein dosage is not much different.
Table 2
Embodiment one
A kind of kit of pseudorabies gE antibody tests includes the magnetic suspension liquid of coating pseudorabies gE albumen, alkali
The pseudorabies gE 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 gE 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 gE full length proteins (being purchased from hundred million minot bio tech ltd of Hangzhou) 80 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 pseudorabies gE 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 gE 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 gE 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 divided into calibration object 1, calibration object 2.Calibration object 1 is pseudorabies gE negative antibody serum dilutions, calibration
Product 2 are pseudorabies gE Positive Sera dilutions.
The preparation of calibration object：
(1) pseudorabies gE negative antibodies serum, pseudorabies gE Positive Seras are inactivated 1 hour in 60 DEG C of heat；
(2) 0.1% ProClin is added through 0.2 μm of micro-filtrate membrane filtration in the serum after inactivation in (1)TM300；
(3) serum in (2) is demarcated, with the diluted containing 1%BSA.
Quality-control product is proven pseudorabies gE antibody positive Swine serums.It is divided into quality-control product 1 and quality-control product 2.Quality-control product 1
S/CO (ratio of luminous value and critical value) between 1.2-1.8, the S/CO of quality-control product 2 is between 0.6-0.9.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：
(1) 10 parts or more pseudorabies gE Positive Seras are selected, 60 DEG C of heat inactivate 1 hour, through 0.2 μm after mixing
0.1% ProClin is added in micro-filtrate membrane filtrationTM300。
(2) adjustment mixing positive serum makes the S/CO of quality-control product 1 between 1.2-1.8, quality-control product 2 to suitable concentration
S/CO between 0.6-0.9.
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 gE 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 gE 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 gE 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.
Calculate the ratio (S/CO) of sample luminous value and critical value.
Critical value=1 average irradiance of calibration object × 2 average irradiance × 0.2 of 0.8+ calibration objects
Sample S/CO>1.00 are determined as Pseudorabies virus gE negative antibodies, are not required to further detect.
Sample S/CO≤1.00 are determined as Pseudorabies virus gE antibody positives.
The sample of all initial survey positives applies this kit repetition measurement 2 times, if 2 results are all feminine gender, is determined as feminine gender；
If being at least once the positive in two times result, judge the sample for the repetition measurement positive.
Two, sensitivity experiment
Pseudorabies gE 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 gE 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).With CV%<20% maximum dilution multiple is as sensitivity, spirit in the present embodiment
Sensitivity is better than ELISA kit.
ELISA kit S/N >=0.70 is determined as feminine gender, 0.60<S/N ＜ 0.70 are determined as suspicious, the judgements of S/N≤0.60
For the positive.
Table 3 be embodiment one kit and ELISA kit remolding sensitivity compared with
Three, repeated experiment
2 porcine pseudorabies virus gE Positive Seras, 1 part of negative serum are taken to be detected with this kit, per natural gift 2 batches
Detection, 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 detection datas calculate the coefficient of variation, as a result such as table 4.The results show that 3 parts of Virus monitory result repeatability are good.Table 4
Four, coincidence rate is tested
This kit detects more parts of Swine serums, testing result such as table 5 simultaneously with ELISA kit.The results show that this reagent
With ELISA kit positive coincidence rate 96.2%, negative match-rate 96.7%, overall coincidence rate 95.9%.
Table 5
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), pseudorabies gE deleted virus (PRV, gE-), porcine reproductive and respiratory syndrome virus
(PRRSV), porcine circovirus 2 type (PCV2), bovine viral diarrhea virus (BVDV).Testing result S/CO is all higher than 1.0, is
Feminine gender does not find cross reaction.
Table 6
| Correlated virus | CSFV | FMDV-O | PRV, gE- | PRRSV | PCV2 | BVDV |
| S/CO | 1.61 | 1.48 | 1.72 | 1.73 | 1.97 | 1.75 |
Embodiment two
A kind of kit of pseudorabies gE antibody tests includes the magnetic suspension liquid of coating pseudorabies gE 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 gE 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 90 μ g of pseudorabies gE 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 gE 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 gE 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.
Calculate the ratio (S/CO) of sample luminous value and critical value.
Critical value=1 average irradiance of calibration object × 2 average irradiance × 0.9 of 0.1+ calibration objects
Sample S/CO<1.00 being determined as Pseudorabies virus gE negative antibodies, it is not required to further detect.
Sample S/CO >=1.00 are determined as Pseudorabies virus gE antibody positives.
The sample of all initial survey positives applies this reagent repetition measurement 2 times, if 2 results are all feminine gender, is determined as feminine gender；If
It is at least once the positive in two times result, then judges the sample for the repetition measurement positive.
Two, sensitivity experiment
Detect the pseudorabies gE antibody sun of different extension rates simultaneously with the kit and ELISA kit of the present embodiment
Property serum, wherein this reagent does 10 repetitions to every part of blood sample and detects, and calculates the coefficient of variation, as a result such as table 7.The results show that
The sensitivity of the present embodiment is better than ELISA kit.
ELISA kit S/N >=0.70 is determined as feminine gender, 0.60<S/N ＜ 0.70 are determined as suspicious, the judgements of S/N≤0.60
For the positive.
Table 7 be the present embodiment kit and ELISA kit remolding sensitivity compared with
Three, repeated experiment
2 porcine pseudorabies virus gE Positive Seras, 1 part of negative serum are taken to be detected with the kit of the present embodiment, often
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
20 detection datas are respectively obtained, the coefficient of variation is calculated, as a result such as table 8.The results show that 3 parts of Virus monitory result repeatability are good.
Table 8
Four, 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 9.The present embodiment
Kit and ELISA kit positive coincidence rate 96.2%, negative match-rate 94.1%, overall coincidence rate 94.0%.
Table 9
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), pseudorabies gE deleted virus (PRV, gE-), porcine reproductive and respiratory syndrome virus
(PRRSV), porcine circovirus 2 type (PCV2), bovine viral diarrhea virus (BVDV).Testing result S/CO is below 1.0, is
Feminine gender does not find cross reaction.
Table 10
| Correlated virus | CSFV | FMDV-O | PRV, gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.12 | 0.19 | 0.17 | 0.10 | 0.20 | 0.13 |
Embodiment three
A kind of kit of pseudorabies gE antibody tests includes the magnetic suspension liquid of coating pseudorabies gE albumen, peppery
The pseudorabies gE 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 gE 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 gE 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 pseudorabies gE 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 gE 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 gE 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 gE 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 gE 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.
Calculate the ratio (S/CO) of sample luminous value and critical value.
Critical value=1 average irradiance of calibration object × 2 average irradiance × 0.9 of 0.1+ calibration objects
Sample S/CO<1.00 are determined as Pseudorabies virus gE negative antibodies, are not required to further detect.
Sample S/CO >=1.00 are determined as Pseudorabies virus gE antibody positives.
The sample of all initial survey positives applies this reagent repetition measurement 2 times, if 2 results are all feminine gender, is determined as feminine gender；If
It is at least once the positive in two times result, then judges the sample for the repetition measurement positive.
One, sensitivity experiment
Detect the pseudorabies gE antibody sun of different extension rates simultaneously with the kit and ELISA kit of the present embodiment
Property serum, wherein this reagent does 10 repetitions to every part of blood sample and detects, and calculates the coefficient of variation, as a result such as table 11.As a result it demonstrate,proves
Bright, this reagent sensitivity is better than ELISA kit.
ELISA kit S/N >=0.70 is determined as feminine gender, 0.60<S/N ＜ 0.70 are determined as suspicious, the judgements of S/N≤0.60
For the positive.
Table 11
Two, repeated experiment
2 porcine pseudorabies virus gE Positive Seras, 1 part of negative serum are taken to be detected with the kit of the present embodiment, often
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
20 detection datas are respectively obtained, the coefficient of variation is calculated, as a result such as table 12.The results show that 3 parts of Virus monitory result repeatability are good
It is good.
Table 12
Three, coincidence rate is tested
The kit of the present embodiment detects more parts of Swine serums, testing result such as table 13 simultaneously with ELISA kit.This implementation
The kit of example and ELISA kit positive coincidence rate 94.9%, negative match-rate 95.1%, overall coincidence rate 94.4%.
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), pseudorabies gE deleted virus (PRV, gE-), porcine pseudorabies virus gD (PRV-gD), pig are numerous
It grows and breath syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), bovine viral diarrhea virus (BVDV).Testing result
S/CO is below 1.0, is feminine gender, does not find cross reaction.
Table 14
| Correlated virus | CSFV | FMDV-O | PRV, gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.13 | 0.08 | 0.09 | 0.08 | 0.11 | 0.07 |
Example IV
A kind of kit of pseudorabies gE antibody tests includes the magnetic suspension liquid of coating pseudorabies gE albumen, alkali
The pseudorabies gE 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 gE 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 gE 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 gE 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 gE 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) the 0.01mol/L pH PBS buffer solution for being 7.4 of solution in (4) is dialysed 20-24 hours at 2-8 DEG C.
(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 gE 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 gE 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 gE 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.
Calculate the ratio (S/CO) of sample luminous value and critical value.
Critical value=1 average irradiance of calibration object × 2 average irradiance × 0.9 of 0.1+ calibration objects
Sample S/CO<1.00 are determined as Pseudorabies virus gE negative antibodies, are not required to further detect.
Sample S/CO >=1.00 are determined as Pseudorabies virus gE antibody positives.
The sample of all initial survey positives applies this reagent repetition measurement 2 times, if 2 results are all feminine gender, is determined as feminine gender；If
It is at least once the positive in two times result, then judges the sample for the repetition measurement positive.
One, sensitivity experiment
Detect the pseudorabies gE 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
15.The results show that this reagent sensitivity is better than ELISA kit.
ELISA kit S/N >=0.70 is determined as feminine gender, 0.60<S/N ＜ 0.70 are determined as suspicious, the judgements of S/N≤0.60
For the positive.
Table 15
Two, repeated experiment
2 porcine pseudorabies virus gE Positive Seras, 1 part of negative serum are taken to be detected with the kit of the present embodiment, often
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
20 detection datas are respectively obtained, the coefficient of variation is calculated, as a result such as table 16.The results show that 3 parts of Virus monitory result repeatability are good
It is good.
Table 16
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 17.The present embodiment
Kit and ELISA kit positive coincidence rate 94.9%, negative match-rate 94.6%, overall coincidence rate 94.0%.
Table 17
Four, 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), pseudorabies gE deleted virus (PRV, gE-), porcine pseudorabies virus gD (PRV-gD), pig are numerous
It grows and breath syndrome virus (PRRSV), porcine circovirus 2 type (PCV2), bovine viral diarrhea virus (BVDV).Testing result
S/CO is below 1.0, is feminine gender, does not find cross reaction.
Table 18
| Correlated virus | CSFV | FMDV-O | PRV, gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.14 | 0.24 | 0.14 | 0.23 | 0.15 | 0.21 |
The present invention is used as coating carrier using the magnetic particle of specified particle diameter, using specific pseudorabies gE albumen and magnetic
Property the ratio between particle mixed and reacted, obtain coating homogeneous, stable structure pseudorabies gE antibody 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 gE antibody assay kits and its detection method
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> PRT
<213>Artificial sequence
<400> 1
MRPFLLRAAQ LLALLALALS TEAPSLSAET TPGPVTEVPS PSAEVWDDLS TEADDDDLNG 60
DLDGDDRRAG FGSALASLRE APPAHLVNVS EGANFTLDAR GDGAVLAGIW TFLPVRGCDA 120
VAVTTVCFET ACHPDLVLGR ACVPEAPEMG IGDYLPPEVP RLRREPPIVT PERWSPHLSV 180
LRATPNDTGL YTLHDASGPR AVFFVAVGDR PPAPADPVGP ARHEPRFHAL GFHSQLFSPG 240
DTFDLMPRVV SDMGDSRENF TATLDWYYAR APPRCLLYYV YEPCIYHPRA PECLRPVDPA 300
CSFTSPARAR LVARRAYASC SPLLGDRWLT ACPFDAFGEE VHTNATADES GLYVLVMTHN 360
GHVATWDYTL VATAAEYVTV IKELTAPARA PGTPWGPGGG DDAIYVDGVT TPAPPARPWN 420
PYGRTTPGRL FVLALGSFVM TCVVGGAVWL CVLCSRRRAA SRPFRVPTRA RTHMLSPVYT 480
SLPTHEDYYD GDDDDDEEAG VIRRRPASPG GDSGYEGPYA SLDPEDEFSS DEDDGLYVRP 540
EEAPRSGFDV WFRDPEKPEV TNGPNYGVTA NRLLMSRPA 579
<210> 2
<211> 324
<212> PRT
<213>Artificial sequence
<400> 2
PNDTGLYTLH DASGPRAVFF VAVGDRPPAP ADPVGPARHE PRFHALGFHS QLFSPGDTFD 60
LMPRVVSDMG DSRENFTATL DWYYARAPPR CLLYYVYEPC IYHPRAPECL RPVDPACSFT 120
SPARARLVAR RAYASCSPLL GDRWLTACPF DAFGEEVHTN ATADESGLYV LVMTHNGHVA 180
TWDYTLVATA AEYVTVIKEL TAPARAPGTP WGPGGGDDAI YVDGVTTPAP PARPWNPYGR 240
TTPGRLFVLA LGSFVMTCVV GGAVWLCVLC SRRRAASRPF RVPTRARTHM LSPVYTSLPT 300
HEDYYDGDDD DDEEAGVIRR RPAS 324
<210> 3
<211> 46
<212> PRT
<213>Artificial sequence
<400> 3
ADESGLYVLV MTHNGHVATW DYTLVATAAE YVTVIKELTA PARAPG46
Claims (10)
1. a kind of pseudorabies gE antibody assay kits, which is characterized in that including pseudorabies gE albumen couplings or indirectly even
Join the solution of magnetic particle, the protein solution and luminous substrate of luminescent label.
2. pseudorabies gE antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gE 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 gE 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 gE antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gE 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 gE protein bindings biotin
S1 takes pseudorabies gE albumen, dialysis；
Biotin, reaction is added in S2；
Sealer, reaction is added in S3；
S3, dialysis remove unbonded biotin, obtain pseudorabies gE avidin-biotins.
3) magnetic particle-Avidin is mixed with pseudorabies gE avidin-biotin compounds, passes through the knot of Avidin and biotin
Resultant force connection magnetic particle and pseudorabies gE albumen.
4. pseudorabies gE 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 gE albumen or pseudorabies gE 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 gE antibody assay kits as described in claim 1, which is characterized in that the pseudorabies gE albumen
Selected from pseudorabies gE full length proteins, natural pseudorabies gE protein fragments, pseudorabies gE full length proteins of recombinant expression, again
The pseudorabies gE protein fragments of group expression, one in pseudorabies gE polypeptides, pseudorabies gE 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 gE 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 gE antigens, monoclonal antibody, polyclonal antibody, genetic engineering antibody, anti-pig IgG antibody, anti-pig IgM antibody
Kind.
7. pseudorabies gE antibody assay kits as described in claim 1, which is characterized in that the luminous substrate with shine
Marker corresponds.
8. pseudorabies gE 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 gE antibody assay kits as claimed in claim 8, which is characterized in that the quality-control product is that pig puppet is mad
Dog gE antibody quality-control product 1 and quality-control product 2；The cleaning solution is the trishydroxymethylaminomethane that 0.05mol/L pH are 8.0
(Tris) phosphate (PBS) buffer solution that buffer solution or 0.01mol/L pH are 7.0, the Tris buffer solutions and PBS buffer solution
It is middle respectively containing 0.1% Tween-20.
10. a kind of detection method of pseudorabies gE 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 gE 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 detects luminous value with Chemiluminescence Apparatus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019644.9A CN108303540B (en) | 2017-01-11 | 2017-01-11 | Porcine pseudorabies gE antibody detection kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019644.9A CN108303540B (en) | 2017-01-11 | 2017-01-11 | Porcine pseudorabies gE antibody detection kit and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108303540A true CN108303540A (en) | 2018-07-20 |
| CN108303540B CN108303540B (en) | 2023-06-30 |
Family
ID=62871946
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710019644.9A Active CN108303540B (en) | 2017-01-11 | 2017-01-11 | Porcine pseudorabies gE antibody detection kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108303540B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109307772A (en) * | 2018-10-12 | 2019-02-05 | 华南农业大学 | A kind of Pseudorabies virus gE and gB IgG antibody double fluorescent microballoon immunological detection method |
| CN112326971A (en) * | 2020-10-20 | 2021-02-05 | 成都海默云因医学检验实验室有限公司 | Novel method and kit for NMDAR antibody quantitative detection |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101452001A (en) * | 2007-12-06 | 2009-06-10 | 王颖 | Quantitative determination RBP4 kit by chemiluminescence magnetic enzymoimmune method |
| CN102608315A (en) * | 2012-02-21 | 2012-07-25 | 武汉科前动物生物制品有限责任公司 | Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof |
| CN102998442A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit |
| CN103308684A (en) * | 2012-03-14 | 2013-09-18 | 武汉中博生物股份有限公司 | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody |
| CN103353526A (en) * | 2013-07-23 | 2013-10-16 | 河南枫华生物科技有限公司 | Kit for detecting pig pseudorabies and application thereof |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
-
2017
- 2017-01-11 CN CN201710019644.9A patent/CN108303540B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101452001A (en) * | 2007-12-06 | 2009-06-10 | 王颖 | Quantitative determination RBP4 kit by chemiluminescence magnetic enzymoimmune method |
| CN102608315A (en) * | 2012-02-21 | 2012-07-25 | 武汉科前动物生物制品有限责任公司 | Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof |
| CN103308684A (en) * | 2012-03-14 | 2013-09-18 | 武汉中博生物股份有限公司 | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody |
| CN102998442A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit |
| CN103353526A (en) * | 2013-07-23 | 2013-10-16 | 河南枫华生物科技有限公司 | Kit for detecting pig pseudorabies and application thereof |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109307772A (en) * | 2018-10-12 | 2019-02-05 | 华南农业大学 | A kind of Pseudorabies virus gE and gB IgG antibody double fluorescent microballoon immunological detection method |
| CN109307772B (en) * | 2018-10-12 | 2021-12-24 | 华南农业大学 | Double fluorescent microsphere immunological detection method for antibodies of pseudorabies virus gE and gB IgG |
| CN112326971A (en) * | 2020-10-20 | 2021-02-05 | 成都海默云因医学检验实验室有限公司 | Novel method and kit for NMDAR antibody quantitative detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108303540B (en) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104614535B (en) | TMA detection kit and its preparation method and application | |
| CN105548565B (en) | A kind of kit and its preparation and application for being used to detect schizotrypanum cruzi antibody | |
| JPH02276968A (en) | Immunity test using fragment of f(ab')2 | |
| CN106771149B (en) | Infectious bursa of Fabricius virus antigen conjugated magnetic particle and its preparation method and application | |
| CN105699653A (en) | Ultra-sensitive superparamagnetic nano immunization microsphere and GP73 antigen detection method | |
| CN108303530A (en) | A kind of pseudorabies gB antibody assay kits and its detection method | |
| CN110297091B (en) | Multi-tube chemiluminescence detection kit for foot-and-mouth disease O, A, Asia I-type antibody | |
| CN108303540A (en) | A kind of pseudorabies gE antibody assay kits and its detection method | |
| CN110672850A (en) | Hepatitis A virus antibody IgM detection kit and preparation method thereof | |
| CN108303542B (en) | Pig breeding and respiratory syndrome antibody detection kit and detection method thereof | |
| JP2008249534A (en) | Carrier for immunoassay | |
| CN114252591A (en) | Magnetic bead coating and preparation method thereof and detection kit | |
| JP6729995B2 (en) | Immunoassay method and reagent | |
| CN108303541A (en) | A kind of porcine circovirus type 2 antibody testing kit and its detection method | |
| CN108303543A (en) | A kind of swine fever E2 protein antibodies detection kit and its detection method | |
| JP4975601B2 (en) | Reagent kit for HCV antibody measurement and HCV antibody measurement method | |
| CN108303531B (en) | O-type foot-and-mouth disease antibody detection kit and detection method thereof | |
| CN111007248A (en) | Chemiluminescence immunoassay kit for detecting rubella virus IgM antibody | |
| CN106771245B (en) | H9 subtype avian influenza virus antigen conjugated magnetic particle and its preparation method and application | |
| JP2013205408A (en) | Immunity analysis method and reagent | |
| JPH0763761A (en) | Manufacture of fine magnetic particles for fixing physiologically active material | |
| CN111999494B (en) | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof | |
| CN103575892A (en) | Kit for quantitatively detecting CEA (Carcino-Embryonic Antigen) as well as preparation method and application method of kit | |
| CN103364551A (en) | ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit | |
| CN109917124A (en) | A kind of hepatitis C virus antigen-antibody combined detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |