CN109799341A - A kind of kit and preparation method detecting 2 type antibodies against foot-and-mouth disease virus of South Africa - Google Patents

Abstract

The present invention discloses a kind of detection kit and preparation method using the preparation of 2 type foot and mouth disease virus sample particle (VLPs) of South Africa.The preparation method of kit of the invention is marked directly on HRP in the competitive antibody with being detected antibody while effect, and the interior coated 2 type foot and mouth disease virus sample particle of South Africa of ELISA Plate is assembled by structural proteins VP0, VP1 and VP3 of 2 type foot and mouth disease virus of South Africa.The VLPs that the present invention uses can be assembled efficiently, and not influence its diagnosis efficiency, advantageously reduce the cost of diagnostic preparation.The detecting step of the method for the present invention compared with the prior art is reduced, and experimental implementation is simple, and workload is small, and the used time is short, and the specificity and sensibility of diagnostic reagent can be improved.

Description

A kind of kit and preparation method detecting 2 type antibodies against foot-and-mouth disease virus of South Africa

Technical field

The present invention relates to a kind of 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection kits of South Africa.

Background technique

Aftosa has generation in Asia, Africa and the Middle East and South America, causes huge economic loss, and the world defends Aftosa is classified as and legal reports infectious disease by raw tissue.2 type foot and mouth disease virus (SAT2) is Hostis in South Africa, small One of the member of RNA virus section, the virus are mainly broken out in African south and west area, seriously threaten surrounding area animal husbandry Development.Nearest decades, there is the report of 2 type foot and mouth disease virus of South Africa in succession in the surrounding area other than the African continent, this is just Illustrating that the virus has had already appeared the danger of cross-border propagation, there is the epidemic situation of SAT2 aftosa in Kuwait in 2000 and Saudi Arabia, Also there are the appearance of SAT2 aftosa case in Pakistan in 2012 and Bahrein Island.It can be seen that just to threaten the world each by SAT2-FMDV A country, so prevention, the diagnosis for SAT2 aftosa are just particularly important.

Virus-like particle (Virus like particles, VLPs) is as closest to natural virion but without disease The viruslike particle of virus gene shows its advantage fully in multiple research fields.The capsid protein or memebrane protein of virus are assembled into VLPs Afterwards, it can be used as antigen to detect antibody.VLPs after assembling can be formed by the interaction between each subunit The three-dimensional conformation that single albumen does not have, therefore relative to single virus albumen, carrying out detection to antibody using VLPs has more High sensitivity and specificity.And VLPs has the structure of similar natural viral, has very strong immunogenicity, is exempted from VLPs Epidemic disease animal can obtain the antibody of high titre, and exempt from that this antibody can be used for corresponding virus detects.

The serodiagnosis of aftosa is the technology hand being most widely used in epidemiological survey technical system Section, it can not only break out in aftosa and combine other serological methods to predict and judge epidemic situation when popular, and And main function can be played in the assessment of aftosa vaccine immune efficacy.At present, China's aftosa structural proteins antibody test It is mainly used for the assessment of aftosa vaccine immune efficacy.There are three types of the aftosa serum detection methods that OIE in 2004 is announced: disease Malicious neutralization test (VNT), Liquid-phase blocking ELISA method (LBE) and solid phase competitive ELISA (SCE).Virus neutralization tests is three Method the most classical in kind method, is the gold standard for evaluating other methods；1986, by aftosa world reference laboratory Sensibility, specificity and the stability for the Liquid-phase blocking ELISA method set up gain public acceptance always in the world, are current The whole world uses most wide aftosa serum detection method.It is special for the solid phase competitive ELISA method to grow up after 2001 Property it is good, the operating time is short, operate it is more easy, be more suitable for high-volume serum detect, aftosa blood was determined as by OIE in 2004 It is clear to learn detection method.

Most of competitive ELISA kits are all by HRP label on the antibody of anti-competitive antibody at this stage, this just leads Cause their experimental procedure more, with duration, mostly in 1 h or more, referring to Chinese patent application CN105445457A, The disclosures such as CN107064501A.

Summary of the invention

The present invention provides one kind and can overcome the shortage of prior art, for detecting the competition of 2 type antibodies against foot-and-mouth disease virus of South Africa ELISA detection kit and preparation method thereof.

Include in 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection kit of South Africa of the invention: coating 2 type of South Africa It the ELISA Plate of foot and mouth disease virus sample particle, the rabbit-anti of HRP label, the sample diluting liquid containing tween and phosphate buffer and washes Wash the terminate liquid that liquid, tmb substrate, positive control serum, negative control sera and the concentrated sulfuric acid are mixed with water.

2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa of the invention is directly to mark HRP Remember that preparation process includes: with 2 type foot and mouth disease virus sample particle of South Africa with being detected in the competitive antibody that antibody acts on simultaneously Immune rabbit after being immunized, is taken a blood sample from rabbit heart, is separated serum and is isolated and purified to obtain with Protein A affinity chromatography HRP is tagged on IgG by IgG using sodium periodate method.

Preferably, 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa of the invention, in The coated 2 type foot and mouth disease virus sample particle of South Africa of ELISA Plate be by 2 type foot and mouth disease virus of South Africa structural proteins VP0, VP1 and VP3 assembles, and the sequence of VP0, VP1 and VP3 are respectively SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.

Preferably, 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa of the invention, enzyme Target preparation is with 0.05%(pH=9.6) carbonate be coated with buffer 2 type FMDV VLPs of South Africa be diluted to 0.4 μ ɡ/mL, ELISA Plate is added with the amount of every 100 μ L of hole, 4 DEG C of coatings are stayed overnight, and cleaning solution board-washing 3 times；What addition was prepared with ELISA Plate stabilizer Confining liquid containing 1%BSA, every 120 μ L of hole, 37 DEG C of insulating boxs close 30 min, board-washing 3 times, dry, vacuum packaging.

The present invention has the advantage that

1. the present invention uses South Africa 2 type (SAT2) foot and mouth disease virus sample particle (VLPs) as envelope antigen for the first time, make full use of VLPs is highly-safe, immunogenicity is strong, can substitute the characteristics of natural viral plays a significant role in immunology detection, establishes The competitive ELISA method of detectable SAT2-FMDV antibody, quickly examines the antibody level of the SAT2 aftosa in serum It surveys.Due to the efficient assembling of VLPs, both without influencing diagnosis efficiency, and the cost of diagnostic preparation is reduced.

2. the method for the present invention uses the IgG of HRP label rabbit-anti SAT2-FMDV VLPs, during making kit, enzyme mark Plate is the finished product being coated with, so practical operation step only needs two steps, simple experiment is easy to operate, and the used time is shorter, this method used time About in 45min.(action time of most of competitive ELISA kits is 1h or so, see patent CN105445457A, CN107064501A etc.).

3. after being assembled into VLPs due to capsid protein, passing through each Asia in terms of the specificity and sensibility that improve test Interaction between unit can form the three-dimensional conformation that single albumen does not have, therefore be detected using VLPs to antibody With higher sensitivity and specificity, and avoid the problem that host type.

4. the present invention combines demand of China port to the detection of SAT2 foot-and-mouth disease antibody, monitoring, to SAT2-FMDV VLPs Competitive ELISA antibody detection method is studied, and establishes criterion.

Specific embodiment

The present invention is explained below in conjunction with example.

1. the preparation of 2 type foot and mouth disease virus VLPs of South Africa

(1) by the 2 type foot and mouth disease virus of South Africa saved by this laboratory (with reference to AJ251473 strain in GenBank) positive restructuring Plasmid pSMK-VP0VP3 and pSMK-VP1 cotransformation to competent cell BL21 (DE3)-RIL, picking monoclonal colonies press 1: 100 ratio is inoculated into going out through high pressure containing 10 μ g/mL kanamycins, 50 μ g/mL ampicillins and 25 μ g/mL chloramphenicol In the fresh LB liquid medium of bacterium, in 37 DEG C, 220r/min shaker overnight culture.VP0, VP1 and VP3 and gene order point It Wei not SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.

(2) the expression bacterium being incubated overnight is inoculated into the LB culture medium containing above-mentioned 3 kinds of antibiotic, 220 r at 37 DEG C Culture to OD600 value is 0.6-0.7 or so under conditions of/min, and 1 ml that keeps sample is as control before induction.

(3) IPTG of final concentration of 0.5mmol/L is added, and induces 16h under the conditions of 16 DEG C.

(4) 4750 r/min of bacterium solution after inducing collects thallus after being centrifuged 20 min, then will by 1:50 concentration ratio Thallus be suspended in Buffer A (500mmol/L NaCl, 20 mmol/L Tris-HCl, 20 mmol/L Imidazole, 2mmol/L DTT, 0.05%TritonX-100, pH8.4), 6 min of ultrasonication on ice after mixing well, then Supernatant is collected after being centrifuged 15 min at 10000 4 DEG C of r/min.

(5) supernatant collected is according to His label protein purification kit specification purified fusion albumen.

(6) it collects eluent and carries out 10% SDS-PAGE electrophoresis, be then transferred to recombinant protein using wet robin poly- inclined On vinyl fluoride hybond membrane (pvdf membrane), 1h is closed with 37 DEG C of items of confining liquid (PBS, 5% skimmed milk power, pH 7.0), is then distinguished Respectively with horseradish mistake after sufficiently being washed with anti-His primary antibody (1:3000) and 37 DEG C of anti-FLAG primary antibody (1:1000) incubation 1h, PBST 37 DEG C of incubation 1h of rabbit anti-mouse igg (1:6000) and goat anti-rabbit igg (1:4000) of oxide enzyme label, after PBST is sufficiently washed 3min is reacted in luminous substrate is added in darkroom, is placed under Kozak film and exposes, after developing and fixing is fixed, observes purpose egg White expression.It is saved backup after measurement protein concentration in -80 DEG C.

(7) His-SUMO label protein endonuclease reaction carries out in the bag filter of 8000 MWCO.The SUMO of purifying is merged Albumen and SUMO enzyme are placed in bag filter according to the ratio of 100:1, and bag filter is placed in assembling buffer (40 mmol/L Tris-HCl、500 mmol /L NaCl、1 mmol /L CaCl₂, pH 7.4) in 4 DEG C of 14 h of dialysis, entire buffer system It should be placed on stirring instrument, to buffer mild agitation.

(8) structural proteins for cutting off SUMO label are voluntarily assembled into VLPs.VLPs after collecting assembling carries out SDS-PAGE It is identified with WB.

(9) VLPs is analyzed and identified using transmission electron microscope (TEM), 25 μ g VLPs room temperatures after taking sucrose density gradient centrifugation It is adsorbed onto 2.5min on the coated copper mesh of carbon film, with the liquid that it is extra on copper mesh that filter paper is gone out, with 2%~3% phosphoric acid tungsten negative staining After 2.5 min, filter paper removes surplus liquid, and 100 kV observe sample.

2. the preparation of hyper-immune serum

(1) animal selects: 1.8-2Kg or so health male rabbit 4 is divided into two groups, experimental group 3, control group 1.

(2) antigen: being diluted to 0.2mg/mL with PBS for SAT2 aftosa VLPs after purification, mixed with isometric adjuvant Merging is emulsified into 1mL completely, and spine's subdermal muscle multi-point injection immunizing dose 1mL/ times/, carries out being immunized for 4 times altogether, between each Every 14d.Initial immunity Freund's complete adjuvant, the 2nd, 3 immune incomplete Freund's adjuvant are helped for last 1 time completely with Freund Agent.

(3) antibody test: the immune preceding blood sampling at auricular vein every time, with Liquid-phase blocking ELISA kit measurement potency. Last time is immune to terminate 14d, takes a blood sample from rabbit heart, separates serum, after the fragments of tissue such as cell are removed in centrifugation, packing to With.

3. Protein A affinity chromatography isolates and purifies IgG

(1) preparation of samples: rabbit anteserum is mixed with combination buffer 1:1, filtering.

(2) it balances pillar: crossing Protein A column with the phosphate buffer of 5-10 times of volume.

(3) loading: the ready blood serum sample of 1mL is injected with syringe.

(4) it elutes foreign protein: rinsing pillar with phosphate buffer, be free of albumen in liquid until combining.

(5) it collects antibody: eluting IgG with the citrate buffer of 0.1mol/L, PH=3.0 of 6 times of volumes, collect sample The Tris-HCL buffer of 1mol/L, PH=9.0 of 50 μ L is added in product, every pipe 0.5mL in every pipe.It measures in each collecting pipe Protein content merges protein pipe.

(6) antibody that PBS dialysis is collected.

4. sodium periodate method prepares SAT2 aftosa VLPs IgG-HRP

(1) 5mgHRP is weighed to be dissolved in 1mL distilled water.

(2) the 0.1M NaIO that 0.2ml newly matches is added in upper liquid₄Solution is protected from light stirring 20 minutes at room temperature.

(3) above-mentioned solution is fitted into bag filter, is dialysed to the sodium-acetate buffer of 1mM PH4.4,4 DEG C overnight.

(4) add 20 μ l 0.2M pH9.5 carbonate buffer solutions, so that the pH of the above hydroformylation HRP is increased to 9.0~9.5, then 10mg IgG is added immediately in 1ml 0.01M carbonate buffer solution, room temperature, which is protected from light, to be gently mixed 2 hours.

(5) the 4mg/mL NaBH for adding 0.1ml newly to match_{4 is molten}Liquid mixes, and 4 DEG C stand 2 hours.Then above-mentioned liquid is packed into saturating It analyses in bag, dialyses to 0.15M pH7.4 PBS, 4 DEG C overnight.

(6) above-mentioned dialyzate being centrifuged 30min in 10000r/min, removal precipitating, supernatant is enzyme conjugates, point Dress, -20 DEG C of preservations.

5. the foundation of competitive ELISA method

(1) testing principle that this method is established

Using competition law, the VLPs of SAT2-FMDV is coated in microwell plate, is then closed ELISA Plate with 1%BSA, is added simultaneously Enter sample to be tested and enzyme labelled antibody.Coated VLPs competition in SAT2-FMDV antibody and enzyme labelled antibody and 96 orifice plates in sample Reaction, SAT2-FMDV antibody and enzyme labelled antibody participate in the competition in conjunction with epitope jointly.Then, horseradish peroxidase bottom is added Object TMB colour developing, terminate liquid terminate reaction, through microplate reader under 450nm wavelength, measure each hole absorbance value, the size of OD value (depth of color after color development stopping reaction) and the content of SAT2-FMDV antibody in sample to be tested are inversely proportional.

(2) determination of antigen coat concentration and serum dilution

Using Checkerboard titration method using the carbonate buffer solution of the 0.05M of pH=9.6 as coating buffer, by the SAT2- of purifying FMDV VLPs is diluted to 0.2,0.3,0.4,0.5,0.6,0.7 μ g/mL, and each gradient respectively adds two column, is added by 100 holes μ L/ micro- In orifice plate.Overnight, next day discards coating buffer to 4 DEG C of coatings, and cleaning solution pats dry after washing 3-4 times, and 1%BSA is added by 120 holes μ L/ Confining liquid, after 37 DEG C of standing 30min, washing drying.Positive and negative standard serum is subjected to doubling dilution, concentration is 1:2~1: 521, one gradient yin and yang attribute of every row respectively adds 1 hole, every 50 μ L of hole, while every hole that same volume is added under different antigen concentrations The enzyme labelled antibody of working concentration, concussion mixes 30s at room temperature, is placed in 37 DEG C of 30 min of effect, and every hole is added 50 μ L's after washing TMB, after being placed in 37 DEG C of 15 min of colour developing, 100 μ L terminate liquids are added in every hole, and microplate reader detects 450nm absorbance value.According to P/N It is worth and determines that the best peridium concentration of antigen is 0.4 μ g/mL, serum optimum dilution degree is 1:64.

(3) enzyme labelled antibody optimum dilution degree determines

After determining peridium concentration, enzyme labelled antibody is diluted, range 1:10000,1:12000,1:14000,1:16000, 1:18000,1:20000,1:22000,1:24000.Square matrix titration is utilized in the case where other conditions are fixed, according to P/N It is worth and determines that enzyme labelled antibody optimum dilution degree is 1:20000.

(4) optimization of antigen coat time and temperature

By antigen with best packet concentration respectively at 4 DEG C of overnight coatings, 37 DEG C of 1 h of coating, 37 DEG C of 1.5 h of coating, 37 DEG C of coatings 2 H, 37 DEG C of 2.5 h of coating, 37 DEG C of 3 h of coating calculate P/N value, determine that antigen the best use time and temperature is 4 DEG C and was coated with Night.

(5) determination of confining liquid and off-period

It selects 1%BSA, 1% gelatin, 5% fetal calf serum and 5% skimmed milk power to be at war with ELISA respectively, selects best confining liquid； 37 DEG C close off 30,45,60 min and are at war with ELISA, determine off-period.The results show that 37 DEG C 30 of 1% BSA Min sealing effect is best.

(6) determination of serum to be checked and enzyme labelled antibody most suitable action time

In the case where other conditions most have, by serum to be checked and enzyme labelled antibody respectively at 37 DEG C of 30,45,60 min of effect into Row competitive ELISA, to determine the most suitable action time of the two.It is determined according to P/N value, serum to be checked and the most suitable work of enzyme labelled antibody It is determined as 37 DEG C of 30 min with the time.

(7) optimization of TMB action time and temperature

By acting on respectively after fixed step addition TMB at 37 DEG C, 10 min, 15 min, 20 min, 25 min, 30 min are aobvious Color.Determine that TMB the best use time and temperature is 37 DEG C of 10 min of effect according to P/N value.

(8) determination of competitive ELISA yin-yang critical value

200 parts of SAT2 negative serums are selected, are at war with ELISA with the condition optimized.Take the average OD of each sample₄₅₀ Value, calculates inhibiting rate (PI)=(1-OD of sample₄₅₀Sample/OD₄₅₀Negative control) × 100%.It determines, it, can when blood-serum P I≤45% It is determined as the positive；When blood-serum P I≤40%, it can determine that as feminine gender；40% < PI > 45% is suspicious.

(9) specificity and repetitive test identification

With O-shaped, Asia1 type and A type FMDV yin and yang attribute serum, PCV2 yin and yang attribute serum, PRRS yin and yang attribute serum and ASFV yin-yang Property serum be that serum to be checked is at war with ELISA, calculate PI% value judgement specificity.The results show that only SAT2 aftosa is positive Serum can block reacting for enzyme labelled antibody and antigen, remaining serum is feminine gender, show the competitive ELISA high specificity established. The serum of 4 parts of different potency and yin and yang attribute control serum is selected to be at war with ELISA, every part of serum carries out 3 repetitions, selects same Block elisa plate repeat in plate, carries out repeating between plate between different elisa plates.According to value for coefficient of variation, the weight of evaluation test Renaturation effect.Its coefficient of variation is respectively less than 10% respectively as the result is shown, shows the monoclonal antibody competitive ELISA repeatability established preferably.

(10) sensitivity tests is identified

VLPs is coated with by best coating quality concentration.To 100 parts of immune pig anteserum sample, 100 parts of Swine serum is not immunized Be at war with ELISA.Being computed positive rate is 97%, and negative recall rate is 98%.

(11) configuration of working reagent

Serum dilution: the phosphate buffer of 0.01 mol/L and pH=7.2~7.4 containing 0.1% Tween-20 of volumetric concentration (PBS)；Cleaning solution: add 1 mL Tween-20 (Tween-20) in the 0.01 M PBS solution of 1000 mL；Substrate buffer solution (pH5.0 phosphate citrate acid): 0.2M Na₂HPO₄25.7mL, 0.1M citric acid 24.3mL, add distilled water 50mL；TMB (tetramethyl Benzidine) use liquid: TMB (10mg/5mL dehydrated alcohol) 0.5mL, substrate buffer solution 10mL, 0.75%H₂O₂32μL；Terminate liquid (2M H₂SO₄): taking 54.34mL concentration is that 98% concentrated sulfuric acid adds distilled water to 1000 mL.

Sequence table

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>a kind of kit and preparation method for detecting 2 type antibodies against foot-and-mouth disease virus of South Africa

<160> 3

<170> SIPOSequenceListing 1.0

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<211> 912

<212> DNA

<213>2 type Foot-and-mouth disease (VP0) of South Africa

<400> 1

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accacgcgcc acggtaccac gaccagtacg acccagagct ctgtgggcat cacctatggt 360

tacgcggata gcgactcttt tcgtagcggc ccgaatacgt ctggtctgga aacccgtgtt 420

gaacaagccg aacgcttttt caaagaaaag ctgttcgatt ggacgagtga caaaccgttt 480

ggtaccctgt atgtgctgga actgccgcgt gatcataaag gcatttacgg caagctgacg 540

gactcctata cctacatgcg caacggctgg gatgtccaag tgagcgccac gtctacccaa 600

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gcacacctgc aggtcccgta tctgggcgtg aaccgtcatg atcagggtaa acgccaccaa 780

agttggtccc tggtggttat ggtgctgacc ccgccgacga ccgaagcaca gatgaatagc 840

ggtaccgttg aagtctatgc gaacattgcc ccgaccaatg tgtacgttgc gggtgaactg 900

ccgggcaaac ag 912

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acgcatgtgc acaccaataa gaccacattt gaggttgatc tgatggacac caaagaaaag 180

gcactggttg gtgctatcct gcgctctgcg acctattact tctgcgatct ggaagttgcc 240

tgtgtcggca aacataagca cgtgttttgg cagccgaacg gtgcgccgcg tacgacccaa 300

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agtctgccgt ccacgtttaa tttcggcttt gtgaccgcag ataaaccggt tgacgtctat 540

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<210> 3

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<212> DNA

<213>2 type Foot-and-mouth disease (VP3) of South Africa

<400> 3

ggtatcgtcc cggtggcatg cgctgatggc tatggcggtt ttcaaaacac cgatccgaag 60

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ccgtatgtcg tgaccaaaaa caatggtgat aaggttatgg cggccttcga cgtcgccttt 240

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gttgtcgtga gcgtgtctgc tggcccggat ctggaatttc gtttcccgat tgacccggtg 660

cgtcag 666

Claims (4)

1. a kind of 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection kit of South Africa, it is characterised in that include in detection kit Have: the ELISA Plate of coating 2 type foot and mouth disease virus sample particle of South Africa, the rabbit-anti of HRP label, containing tween and phosphate buffer The termination that sample diluting liquid and cleaning solution, tmb substrate, positive control serum, negative control sera and the concentrated sulfuric acid are mixed with water Liquid.

2. 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa described in claim 1, feature It is that HRP is marked directly in the competitive antibody with being detected antibody while effect, preparation process includes: with 2 type mouth of South Africa Rabbit is immunized in aphtovirus sample particle, after being immunized, takes a blood sample from rabbit heart, separates serum and with Protein A affinity chromatography It isolates and purifies to obtain IgG, HRP is tagged on IgG using sodium periodate method.

3. 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa according to claim 2, The coated 2 type foot and mouth disease virus sample particle of South Africa of the ELISA Plate being characterized in that in kit is by 2 type foot and mouth disease virus of South Africa Structural proteins VP0, VP1 and VP3 assemble, and the sequence of VP0, VP1 and VP3 are respectively SEQ ID No.1, SEQ ID No.2 With SEQ ID No.3.

4. 2 type antibodies against foot-and-mouth disease virus competitive ELISA detection reagent box preparation method of South Africa according to claim 2 or 3, It is characterized in that ELISA Plate preparation therein is to be coated with buffer for 2 type FMDV of South Africa with the carbonate of 0.05% pH=9.6 VLPs is diluted to 0.4 μ ɡ/mL, and ELISA Plate is added with the amount of every 100 μ L of hole, and 4 DEG C of coatings are stayed overnight, and cleaning solution board-washing 3 times；It is added With the confining liquid containing 1%BSA that ELISA Plate stabilizer is prepared, every 120 μ L of hole, 37 DEG C of insulating boxs are closed 30 min, board-washing 3 times, are done It is dry, vacuum packaging.