CN105807052B - O-shaped FMDV antibody direct competive ELISA detection kit - Google Patents
O-shaped FMDV antibody direct competive ELISA detection kit Download PDFInfo
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Abstract
A kind of O-shaped FMDV antibody direct competive ELISA detection kit, containing ELISA Plate, cleaning solution, sample diluting liquid, HRP marks rabbit-anti, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera and shrouding film in the detection kit, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens.The kit is loaded using one-step method, and detection time shortens.ELIAS secondary antibody is replaced using HRP mark rabbit-antis, animal species difference is eliminated, can detect the blood serum samples such as pig, ox, sheep.The kit can overcome the shortcomings of existing serodiagnosis, possess it is sensitive, special, quick, prepares the features such as simple, can be widely applied to O-shaped FMD immune effect of vaccine and monitor and epidemiology survey.
Description
Technical field
The present invention relates to a kind of direct competive ELISA kit that can be used for detecting O-shaped FMDV antibody, belong to biotechnology
Field.
Background technology
Aftosa (foot-and-mouth disease, FMD) is that a kind of highly contagious disease, pig, ox, sheep etc. is even
Hoof class animal is most susceptible.Foot and mouth disease virus (foot-and-mouth disease virus, FMDV) shares 7 serum at present
Type, O, A, C, South Africa I (SAT 1), South Africa II (SAT 2), South Africa III (SAT 3) and Asia I (Asia 1) are respectively designated as,
Cross immunogenicity is not produced between different FMDV serotypes.From 2005 to 2014, China defended altogether to world animal
Raw tissue (OIE) reports 115 FMD epidemic situations, wherein 1 type epidemic situations of Asia 46 times, O-shaped epidemic situation 38 times, A types epidemic situation 31 times.
After 2009, China's Major Epidemic is O-shaped and A type FMD epidemic situations, wherein O-shaped FMDV variability is strong, propagates extensive, seriously endangers
Livestock breeding industry develops.
FMDV is a kind of more epitopes, the virus of height variation.Virus without cyst membrane structure, capsid protein by VP1, VP2,
Tetra- kinds of structural proteins compositions of VP3 and VP4.Research shows, O-shaped FMDV surfaces at least 5 Neutralization and crystallizations, wherein 3 tables
Position concentrates on VP1 albumen, and the linear epitope being made up of the 133-157 of G-H rings and the 200-213 amino acid residues of C-terminal is FMDV
Most important antigen site, and influence the critical sites of antigenic variation.VP1 albumen can induce animal and produce neutralizing antibody, be
FMD immunoprophylaxis, the important research object of Genetic evolution.
China takes at present to be immunized with slaughtering the FMD prevention and control strategies being combined, and serology antibody detection is to influence epidemic situation to prevent
The key point of control.FMD serodiagnosis be widely used in foreign trade monitoring, suspected case make a definite diagnosis, epidemic disease purification with
And immune effect of vaccine monitoring.The serodiagnosis that wherein OIE recommends has 3 kinds, including virus neutralization tests (VN), solid phase
Block ELISA and LPB-ELISA.VN experiments are most classical serological diagnostic methods, but this method needs to use carefully
Born of the same parents carry out Virus culture, test period length.Compared with VN is tested, LPB-ELISA and solid phase block ELISA to shorten examination
Test the time, but still needing multiple antibody incubation and washing, detection speed is restricted.With immunology and biotechnology
Development, it is significant to establish prevention and control of sensitive, special, the quick Serology test to FMD.
The content of the invention
It is an object of the invention to provide a kind of direct competive ELISA kit for being used to detect O-shaped FMDV antibody.The examination
Agent box can overcome the shortcomings of existing serodiagnosis, possess it is sensitive, special, quick, cheap, prepare the features such as simple.
The Cleaning Principle of kit of the present invention：Horseradish peroxidase (HRP) mark specific rabbit source antibody with it is to be measured
Coated O-shaped FMDV gene engineering antigens in FMDV antibody competition combination ELISA Plate micropores in blood serum sample.Add nitrite ion,
By HRP Catalytic color reactions, the colour developing depth is in inverse correlation with the antibody content in testing sample.
According to the present invention O-shaped FMDV antibody direct competive ELISA detection kit in containing ELISA Plate, cleaning solution, sample
Product dilution, HRP marks rabbit-anti, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera and shrouding
Film, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens.The antigen protein contains the main neutralizations of O-shaped FMDV and resisted
Former epitope, there is very strong affinity to protectiveness neutralizing antibody caused by vaccine immunity or virus infection, this just determines examination
The specificity and sensitiveness of agent box.
The preparation method of the O-shaped FMDV engineered proteins, comprises the following steps：Transferred from NCBI gene pools O-shaped
FMDV capsid protein gene sequences, codon optimization is carried out to sequence.Synthetic gene, structure recombinant strains pET-32a/
BL21.Bacterial strain is subjected to a large amount of induced expressions, uses affinity chromatography and ion exchange technique purifying protein.
The HRP marks rabbit-anti is obtained by O-shaped FMDV engineered proteins immune rabbit, and its preparation method is as follows：With
The O-shaped FMDV capsid proteins of technique for gene engineering expression be immunogene, and immune rabbit obtains serum, serum through saturated ammonium sulfate with
G posts are coupled with HRP after purification.HRP marks rabbit-anti and envelope antigen to have very high binding activity, while and Sample serum
In O-shaped FMDV antibody there is very strong competition activity.In addition HRP marks rabbit-anti to decompose substrate, produces chromogenic reaction, avoids
The use of enzyme mark secondary antibody, substantially reduces the testing inspection time.
The positive effect of the present invention is
1st, animal species difference can be eliminated by carrying out detection using the kit of the present invention, i.e., only mark rabbit-anti just with HRP
O-shaped FMDV specific antibodies in the blood serum samples such as detectable pig, ox, sheep.
2nd, using the kit of the present invention in detection process, one-step method sample-adding, serum and HRP mark rabbit-anti to be checked is simultaneously
ELISA Plate is added, then develop the color reading, and compared to conventional ELISA method, detection time greatly shortens.
3rd, the present invention is gene engineering expression for coated antigen, compared to traditional totivirus inactivation antigen, has operation
Simply, the advantages that cost is cheap, purity is high, biological safety is good.
4th, using the how anti-progress HRP marks of rabbit anteserum, compared with monoclonal antibody, how anti-the preparation be simple, cost is low,
With multiple epitope binding sites, there is competition activity well with the antibody in serum to be checked.
Brief description of the drawings
Fig. 1 is the PCR qualification results of recombinant bacterium.1 in figure：Recombinant bacterial strain PCR is expanded；2：Escherichia coli negative control；3：
DNA Marker。
Fig. 2 is the SDS-PAGE qualification results of recombinant bacterium induced expression product.1 in figure：Protein Marker；2：Large intestine bar
Bacterium negative control；3：Recombinant bacterial strain induced expression.
Fig. 3 is the SDS-PAGE qualification results after recombinant bacterium induced expression product purification.1 in figure：Protein Marker；2：
Escherichia coli negative control；3：Recombinant bacterial strain expression product purifies.
Embodiment
According to the present invention O-shaped FMDV antibody direct competive ELISA detection kit in containing ELISA Plate, cleaning solution, sample
Product dilution, HRP marks rabbit-anti, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera and shrouding
Film, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens.
Lower mask body introduce the source (or preparation process) of each reagent in kit of the present invention, kit test philosophy and
Test process.
Main raw material(s) source in following embodiments is：O-shaped FMDV, A type FMDV and Asia I type FMDV antibody liquid phases
ELISA kit is blocked to be purchased from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.Other reagents and material are derived from business
Approach.Described experimental method, it is normal experiment method unless otherwise noted.
First, the source of each reagent and preparation process in kit
The preparation of 1.1 ELISA Plates
(1) preparation of envelope antigen (O-shaped FMDV engineered proteins)
O-shaped FMDV O/HKN/10/2004 capsid protein coding sequences (GenBank is transferred from NCBI gene databases：
DQ164887.1), synthetic gene, structure recombinant strains pET-32a/BL21.Recon, upstream are identified using round pcr
Primer sequence is 5 '-TTTCTCGAGACCACCTCTGCGGGTGAG-3 ', downstream primer sequence 5 '-
TTTGAATTCCAGAAGCTGTTTTGCGGG-3’.PCR qualification results are shown in accompanying drawing 1, and PCR stripe sizes are 639bp.Take identification just
True recon, it is inoculated in 1: 100 ratio in the LB liquid medium of the AMP containing 100ug/mL, 37 DEG C of cultures to OD600 reach
During to 0.6, IPTG to final concentration of 0.8mmol/L is added.After induced expression 5h, 4 DEG C of 10000r/min of bacterium solution are centrifuged into 5min,
Precipitation is collected, albumen is extracted and carries out SDS-PAGE identifications, albumen size is about 32KD (accompanying drawing 2).Purifying protein (accompanying drawing 3),
It is placed in -80 DEG C of preservations after packing.
(2) the coating process of ELISA Plate
Envelope antigen is diluted with pH9.6 carbonate buffer solution, ELISA Plate is added with the amount in 100uL/ holes, 4 DEG C
12h is coated with, is washed 3 times with cleaning solution.Add confining liquid, 200uL/ holes, 37 DEG C of closing 2h.Deblocking liquid is got rid of, board-washing 3 times, is put
In 37 DEG C of dryings, sealed with aluminum foil under vacuum.
(3) determination of envelope antigen best effort concentration
The optimal coating concentration of antigen protein is determined using square formation titration.It is anti-with pH9.6 carbonate buffer solution dilution
Former albumen, concentration are respectively 400ng/uL, 200ng/uL, 100ng/uL, 50ng/uL, 25ng/uL, and 12.5ng/uL is laterally added
ELISA Plate, 4 DEG C of coating 12h.Add 10% tryptone, 200uL/ holes, 37 DEG C of closing 2h.Yin and yang attribute control serum is done again simultaneously
Than dilution 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800, longitudinal direction adds ELISA Plate, group
Indirect ELISA is carried out into square formation.Add terminate liquid 50uL per hole, determine OD450nm values.Positive OD values are selected close to 1.0, positive OD
Antigen coat concentration and antibody dilution when value/negative OD values (P/N values) is maximum are best effort concentration.The antigen of determination is most
Good working concentration is 50ng/uL.
(4) determination of confining liquid and off-period
With the most suitable working concentration coated elisa plate of fixed antigen, the PBS containing 3% gelatin is used respectively, containing 10% degreasing
The PBS of milk powder, the PBS containing 10% tryptone are as confining liquid.37 DEG C close off 30min, 1h, 2h and 3h.Substrate is added to show
Color, read OD450nm values.Select confining liquid and off-period conduct most suitable bar of the positive OD values close to 1.0, P/N values when maximum
Part.PBS of the selection containing 10% tryptone is as optimal confining liquid, off-period 2h.
1.2HRP marks the preparation of rabbit-anti
From the adult large ear rabbit of health, blood is taken to collect non-immune serum as control before immune.By vivoexpression
The O-shaped FMDV capsid proteins (i.e. envelope antigen) prepared mix with complete Freund's adjuvant and emulsified, and carry out the subcutaneous multiple spot note of rabbit back
Penetrate, every μ g of rabbit 500.Incomplete Freund's adjuvant, every μ of rabbit 250 are used when 2 weeks booster immunizations 1 time, booster immunization
g.Three exempt from latter 14 days, blood sampling progress bioactivity.When potency reaches more than 1: 10000, arteria carotis sacrificed by exsanguination animal, blood is separated
Clearly.Serum carries out preliminary purification using caprylic acid-ammonium and G-protein post method, then with O-shaped FMDV capsid proteins and label egg
It is white to carry out affinity chromatography, to remove non-specific antibody.Use NaIO4The glycan molecule on HRP surfaces is oxidized to aldehyde radical, Ran Houyu
The amino of serum antibody combines, and obtains HRP mark rabbit-antis.
In order to easy to detect, also the HRP mark rabbit-antis of acquisition can be pre-configured to titer as detection kit
One component uses, and the preparation process of titer is：Tris 2.42g, enzyme stabilizers 0.8g, sodium chloride 8.5g, red 2g are taken,
NBCS 100ml, Proclin300 0.5ml, triton x-100 1.5ml, add distilled water 850ml, add hydrochloric acid
1.65ml, fully mix, adjust pH value 7.0, be settled to 1000ml, add HRP mark rabbit-anti 0.2ml, stir, with 0.22
μm bacterial filter filtering.It is aseptic subpackaged.2~8 DEG C are kept in dark place.
The preparation of 1.3 negative control seras
From 5 week old sodium selenites, ELSIA kits are blocked to be accredited as O-shaped FMDV negative antibodies through O-shaped FMDV liquid phases
Pig, O-shaped FMDV antigen negatives pig is accredited as through PCR method.Arteria carotis sacrificed by exsanguination animal, serum is separated, with octanoic acid-ammonium sulfate
The precipitation method and G post affinity chromatography purified blood serums.The serum of purifying is made into 10 times of dilutions with negative control sera dilution, fully
Mix, as negative control sera, then aseptic subpackaged, 2~8 DEG C preservations degerming with 0.22 μm of membrane filtration.
The preparation of 1.4 positive control serums
From 5 week old sodium selenites, O-shaped FMDV negative antibodies are accredited as through O-shaped FMDV LPB-ELISAs kit
Pig, O-shaped FMDV antigen negatives pig is accredited as through PCR method.O-shaped FMDV inactivated vaccines are immunized, with O-shaped FMDV LPB-ELISAs
Kit carries out titration.When potency reaches more than 1: 2056, arteria carotis sacrificed by exsanguination animal, separating and purifying serum.With sun
Property control serum dilution the serum of purifying is made into 10 times of dilutions, fully mix, it is degerming with 0.22 μm of membrane filtration, as the positive
Control serum, then aseptic subpackaged, 2~8 DEG C of preservations.
The preparation of 1.5 sample diluting liquids
Sodium dihydrogen phosphate 1.28g, disodium hydrogen phosphate 0.41g, sodium chloride 20.09g, indicator 0.2g, NBCS
The 0.5ml of 50ml, Proclin 300, adds distilled water 950ml, fully mixes, and adjusts pH value 6.0, is settled to 1000ml.Then divide
Dress.
The preparation of 1.6 cleaning solutions
Potassium dihydrogen phosphate 4g, disodium hydrogen phosphate 58g, the 0.5ml of sodium chloride 160g, Proclin 300, Tween-20
10ml, distilled water is added to be settled to 1000ml.Then dispense.
The preparation of 1.7 substrate A liquid and substrate B liquid
Substrate A liquid：Citric acid 2.10g, sodium acetate 12.25g is crystallized, urea peroxide 0.6g, adds distilled water 950ml, fully
Mix, adjust pH value 5.0, add distilled water to be settled to 1000ml.Packing.
Substrate B liquid：Citric acid 2.10g, EDTA 0.3g, TMB-HCl 0.6g, adds distilled water 950ml, fully mixes, and adjusts
PH value 3.0 is saved, adds distilled water to be settled to 1000ml.Packing.
The preparation of 1.8 terminate liquids
Distilled water 850ml is taken, is carefully added into sulfuric acid 108.5ml, dissolving is complete, and 1000ml is settled to after cooling.Packing.
2nd, test philosophy and process
2.1 test philosophy
The Cleaning Principle of kit of the present invention：Horseradish peroxidase (HRP) mark specific rabbit source antibody with it is to be measured
Coated O-shaped FMDV gene engineering antigens in FMDV antibody competition combination ELISA Plate micropores in blood serum sample.Add nitrite ion,
By HRP Catalytic color reactions, the colour developing depth is in inverse correlation with the antibody content in testing sample.
2.2 test process
Step 1：Sample diluting liquid 100uL is added on ELISA Plate, then adds testing sample serum, positive control blood
Clearly, each 10uL of negative control sera.The HRP added again to every hole after 100uL dilutions marks rabbit-anti titer, 37 DEG C of incubations
30min.Positive serum, negative serum control respectively set 2 holes.
Step 2：Dry, with cleaning solution board-washing 3 times, 3min/ time, add bottom each 50uL of liquid A, B, 37 DEG C of 10min that develop the color.
Step 3：Add terminate liquid 50uL per hole, determine OD450nm values.
The determination of 2.3 direct competive ELISA optimum reaction conditionses
Indirect ELISA is carried out with the most suitable working concentration of fixed antigen, determines that HRP marks rabbit-anti using square formation titration
Most suitable working concentration.After the completion of closing, with pH7.4 phosphate buffer dilution HRP mark rabbit-antis titer (1: 500,1:
1000th, 1: 2000,1: 4000,1: 8000,1: 10000), ELISA Plate is added with amounts of the 100uL per hole.Show after the completion of 37 DEG C of incubations
Color, read OD450nm values.HRP mark rabbit-anti dilution factor of the positive OD values close to 1.0, P/N values when maximum is selected to be used as most suitable work
Make concentration.The best effort concentration for determining HRP mark rabbit-antis is 1: 2000 dilution.
The determination of 2.4 kit criterion
If positive control mean OD value ＜ 0.4, negative control mean OD value ＞ 0.7, then experiment is judged to effectively, continuing to count
Calculate inhibiting rate.Inhibiting rate is more than 30% for the positive, and inhibiting rate is less than 30% for feminine gender.Inhibiting rate calculation formula：
2.5 kit sensitivity tests
(1) to the sensitivity tests of O-shaped FMDV antibody positives control serum
O-shaped FMDV positive control serums are subjected to gradient dilution, is measured with the kit of the present invention, determines the reagent
Sensitivity of the box to positive control serum, the results are shown in Table 1.Kit detects the positive serum of gradient dilution, and detection potency reaches 1
∶128。
Sensitivity test of the table 1 to the positive control serum of gradient dilution
(2) to the sensitivity tests of known positive serum samples
Identified using O-shaped FMDV LPB-ELISAs kit and obtain 120 parts of positives.Use the reagent of the present invention
Box detects to sample.ELISA kit 109 parts of the positive of detection of the present invention, has 11 parts not detect.As a result this is shown
Kit is 90.8% to the sensitiveness of 120 parts of positives.
2.6 kit specific tests
(1) to the specific test of O-shaped FMDV negative antibodies serum
Detect 7 parts of O-shaped FMDV negative antibodies serum with the kit of the present invention, the results are shown in Table 2, kit of the invention with
These antiviral antibody no cross reactions, particularly there is no cross reaction with A types FMDV and Asia I type FMDV antibody yet.Wherein
N1 is A type FMDV Positive Seras, and N2 is Asia I type FMDV Positive Seras, above serum respectively with A types FMDV and
Asia I type FMDV antibody liquids mutually block ELISA kit to identify.N3 is antibody against swine fever virus positive serum, and N4 is pig blue-ear disease
Virus antibody positive serum, N5 are pig parvoviral Positive Sera, and N6 is porcine pseudorabies virus Positive Sera, N7
For swine influenza virus Positive Sera, above serum is identified with AGP test immunity test.
Specific test of the table 2 to known negative blood serum sample
(2) to the specific test of known negative blood serum sample
Identified using O-shaped FMDV LPB-ELISAs kit and obtain 120 parts of negative samples.Use the liquid phase of the present invention
ELISA kit is blocked to detect sample.ELISA kit 112 parts of the negative sample of detection of the present invention, has 8 parts not examine
Go out.As a result it is 93.3% to the specificity of 120 parts of negative samples to show this kit.
2.7 direct competive ELISA replica tests
Using the ELISA Plate of 3 different batches, batch interior repetition is carried out on one block of plate and is tested, is carried out on different plates between criticizing
Repeat to test, determine OD450 values, calculate the coefficient of variation.Coefficient of variation ＜ 10%, illustrate that kit repeatability and stability are good.3
The ELISA Plate of individual different batches, batch in batch between testing result it is consistent, the equal ＜ 7% of the coefficient of variation, kit repeatability very well.
The coefficient of variation (CV) calculation formula：
3rd, the assembling of direct competive ELISA kit
Each reagent is assembled into kit, takes each component to be sealed respectively by table 3, pastes inner packing label, assembling examination
Agent box.
The kit component of table 3
The kit assembled is placed in 4 DEG C of preservations, interval determines its Sensitivity and Specificity in 2 months, to determine reagent
The storage life of box.4 DEG C of the kit of the present invention is kept in dark place, and storage life is 6 months.
It will be appreciated by those skilled in the art that above-described embodiment is only used for explaining that the present invention is any not for it is made
Limitation.In the case where spirit of the invention is guided, the present invention can be implemented using other alternative solutions.
Claims (5)
1. a kind of O-shaped FMDV antibody direct competive ELISA detection kit, ELISA Plate, cleaning solution, sample are contained in the kit
Dilution, HRP marks rabbit-anti, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera and shrouding
Film, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens；
The gene order of wherein described O-shaped FMDV gene engineering antigens is the O-shaped FMDV O/HKN/10/ of NCBI gene databases
2004 capsid protein coding sequences GenBank:DQ164887.1；
As immunogene, immune rabbit obtains the O-shaped FMDV capsid proteins that wherein described HRP marks rabbit-anti is expressed using technique for gene engineering
Serum is obtained, serum carries out preliminary purification using caprylic acid-ammonium and G-protein post method, then with O-shaped FMDV capsid proteins and mark
Sign albumen and carry out affinity chromatography, to remove non-specific antibody；Use NaIO4The glycan molecule on HRP surfaces is oxidized to aldehyde radical, so
Combined afterwards with the amino of serum antibody, obtain HRP mark rabbit-antis.
2. O-shaped FMDV antibody direct competive ELISA detection kit according to claim 1, wherein the ELISA Plate bag
Process by O-shaped FMDV gene engineering antigens is：With coating buffer solution by antigen diluent, enzyme mark is added with the amount in 100uL/ holes
Plate, 4 DEG C of coating 12h, is washed 3 times with cleaning solution；Add confining liquid, 200uL/ holes, 37 DEG C of closing 2h；Get rid of deblocking liquid, board-washing 3
It is secondary, 37 DEG C of dryings are placed in, are sealed with aluminum foil under vacuum.
3. O-shaped FMDV antibody direct competive ELISA detection kit according to claim 2, the coating buffer solution are
PH9.6 carbonate buffer solution.
4. O-shaped FMDV antibody direct competive ELISA detection kit according to claim 2, the confining liquid be containing
The PBS of 10% tryptone.
5. O-shaped FMDV antibody direct competive ELISA detection kit according to claim 1, wherein HRP mark rabbit-anti quilts
Titer is pre-configured to, configuration process is：Tris 2.42g, enzyme stabilizers 0.8g, sodium chloride 8.5g, red 2g are taken, newly
Raw cow's serum 100ml, Proclin 300 0.5ml, triton x-100 1.5ml, add distilled water 850ml, add hydrochloric acid
1.65ml, fully mix, adjust pH value 7.0, be settled to 1000ml, add HRP mark rabbit-anti 0.2ml, stir, with 0.22
μm bacterial filter filtering.
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| RU2811996C1 (en) * | 2023-08-08 | 2024-01-22 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Test system for determining titer of antibodies against 146s particles of foot-and-mouth disease virus genotype sat-2/iv using elisa liquid-phase |
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| RU2811996C1 (en) * | 2023-08-08 | 2024-01-22 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Test system for determining titer of antibodies against 146s particles of foot-and-mouth disease virus genotype sat-2/iv using elisa liquid-phase |
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