CN103499693A - Competitive Alpha LISA (linked immuno sorbent assay) detection kit for classical swine fever virus (CSFV) antibody and detection method thereof - Google Patents

Abstract

The invention discloses a competitive Alpha LISA (linked immuno sorbent assay) detection kit for a classical swine fever virus (CSFV) antibody and a detection method thereof. The detection kit comprises donor microspheres, receptor microspheres, a swine fever virus E2 protein monoclonal antibody and an E2 protein antigen with a His label. A competitive Alpha LISA detection method for the CSFV antibody is created by optimizing test reaction conditions such as the donor microspheres, the receptor microspheres, the monoclonal antibody, the antigen and serum. The kit for detecting the CSFV antibody is good in specificity, high in sensitivity, low in usage amount of the serum, low in detection cost and short in detection time, does not need to be washed and can not be influenced by hemolysis.

Description

Antibody against swine fever virus competition AlphaLISA detection kit and detection method

Technical field

The present invention relates to the immunology detection of antibody against swine fever virus, be specifically related to a kind of antibody against swine fever virus competition AlphaLISA detection kit and detection method.

Background technology

CSFV (CSFV) belongs to the member of Flavivirus.Due to highly pathogenicity and the high fatal rate of CSFV, it has caused huge loss to aquaculture.Pig can be caused infection in latent period after infecting the highly pathogenicity strain, experiences pig a large amount of toxin expellings before symptom occurring of acute swine fever or subacute swine fever clinical symptoms, and in anti-pig body later, obvious CSFV antibody is arranged, and toxin expelling no longer.The pig that infects the mildness strain is chronic infection, can be for a long time before dying of illness or toxin expelling intermittently.Farrowing sow can cause pig a little less than miscarriage, mummification or output, monster etc. by the placental infection fetus.The result of the low virulence virus of congenital infection is the piglet of output persistent infection, and immune tolerance appears in piglet, does not show the outside toxin expelling of any symptom.Pig also likely infects BVDV or BDV.Although these infection usually are gentle process and are self limitings, it is very important effectively they being distinguished with CSFV.China since being separated to first this virus, successively in Guangdong, economize such as Shanghai, Fujian break out with popular more, this disease is in China's ubiquity at present.The main virulent separation evaluation of existing detection method, immunoperoxidase assay, immune colloidal gold technique, the Immunofluorescence assay technology, ELISA, PCR and real time PCR etc., excellent lacking respectively arranged, and specificity difference is larger.Simultaneously, method having relatively high expectations to sample and personnel.Because pig-pig infection pestivirus or immune hog cholera vaccine all can produce corresponding antibody, and antibody capable reacts preferably its immunity or infects viral situation, therefore, can react by the antibody that detects CSFV its immunoinfective virus situation.

The AlphaLISA technology mainly depends on the interaction of Alpha donor microballon and acceptor microballon.When biological respinse makes donor microballon and acceptor microballon when close to each other, the laser excitation cascade reaction, thus produce very big amplifying signal.Specifically, under the Ear Mucosa Treated by He Ne Laser Irradiation of 680nm, the photosensitizer on the donor microballon is more active free oxygen by the oxygen conversion in surrounding environment.Free oxygen diffuses to the acceptor microballon, produces a series of chemiluminescence reaction, finally transfers energy to europium, and emission wavelength is 615nm.When there is not special interaction in biomolecule, free oxygen can't be diffused into the acceptor microballon, does not have the generation of signal.The AlphaLISA technology relies on a kind of proprietary technology based on microballon, traditional ELISA is converted into to the detection of " without the washing " of homogeneous phase.

The rare earth elements europium that the AlphaLISA technology is used (Eu), emission wavelength is very sharp-pointed, is 615nm, thereby has greatly reduced the interference that detects impurity in sample, but the biomolecule in direct-detection complex sample (serum and body fluid).Clinically, this very is applicable to measuring serum and the body fluid sample of humans and animals.And the high flux of this technology, high sensitivity, easy and simple to handle, can be more accurately, more the biomarkers such as cell factor are measured in timesaving.At present, be widely used in the detection of drug screening, cell factor, pathology gene, serum antibody etc.

Chinese invention patent application (application number is: 201210031802.X, 201210047016.9) discloses respectively the method for employing AlphaLISA technology for detection enterovirns type 71 capsid protein (Anti-EV71 VP1 IgM) and the sick IgM of hepatitis A.But, there is no at present the kit that utilizes AlphaLISA technology for detection antibody against swine fever virus and the report of detection method.

Summary of the invention

The first purpose of the present invention is to provide a kind of antibody against swine fever virus competition AlphaLISA detection kit, and gene and this gene primer used that increases thereof of antigen in the coding kit.The second purpose is to provide a kind of competition of the antibody against swine fever virus for food safety detection AlphaLISA detection method.

For realizing that first purpose the technical solution used in the present invention is antibody against swine fever virus competition AlphaLISA detection kit, comprise donor microballon, acceptor microballon, CSFV E 2 protein monoclonal antibody and with the CSFV E 2 protein antigen of His label.

Wherein, the donor microballon that described donor microballon is chelating nickel, PE, AS101D (being Nickel chelate Donor beads (PE, AS101D)), concentration is 80 μ g/mL, 500 μ L; Described acceptor microballon is anti-mouse IgG acceptor microballon, PE, AL105C(is Anti-mouse IgG Acceptor beads (PE, AL105C)), concentration is 80 μ g/mL, 500 μ L; The amino acid sequence of the described antigen of the CSFV E 2 protein with the His label is as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed amino acid sequence had with CSFV E 2 protein antigen same function.

Aforesaid kit, comprise donor microballon 80 μ g/mL, acceptor microballon 80 μ g/mL, CSFV E 2 protein monoclonal antibody 100nM/L and with the CSFV E 2 protein antigen 1 80nM/L of His label; During use, in cumulative volume 20 μ L, every hole addition is: acceptor microballon 4 μ L, donor microballon 5 μ L, 180nM/L are with the anti-E2 protein monoclonal antibody 5 μ L of CSFV E 2 protein antigen 5 μ L, the 100nM/L CSFV of His label and testing sample 1 μ L.

Particularly, the gene of the above-mentioned antigen of the CSFV E 2 protein with the His label of encoding is the CSFV raq gene, and its nucleotides sequence is classified SEQ ID NO.2 as.The CSFV raq gene obtains by the RT-PCR amplified reaction; The nucleotides sequence of the upstream primer of RT-PCR amplified reaction is classified SEQ ID NO.3 as, and the nucleotides sequence of downstream primer is classified SEQ ID NO.4 as.

For realizing that the technical scheme that the second purpose of the present invention adopts is: a kind of mentioned reagent box that utilizes carries out antibody against swine fever virus competition AlphaLISA detection method, comprises the steps:

1) reaction plate treat that in gaging hole, every hole adds 1 μ L sample to be tested, add 1 μ L 1 * dilution buffer liquid in control wells;

2) every hole adds CSFV E 2 protein antigen and the 5 μ L 100nM/L CSFV E 2 protein monoclonal antibodies of 5 μ L 180nM/L with the His label, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 1h;

3) every hole adds 80 μ g/mL acceptor microballon 4 μ L, 80 μ g/mL donor microballon 5 μ L, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 30 min;

4) result reads: reaction plate is put to Enspire(PE) value of reading in the AlphaLISA detection system.

Adopt technique scheme, the present invention at least has following advantages and beneficial effect:

Utilize kit of the present invention to carry out the detection of antibody against swine fever virus, specificity is good, highly sensitive, and amount of serum is few, does not need washing, not affected by haemolysis.

Advantage of the present invention is (1), simple to operate, does not need washing; (2), high specific: the specific binding of antigen and antibody, the reaction of homogeneous phase, required sample volume is little, only needs 1 L; (3), detection time is short: detection time is shorter than traditional ELISA time, only needs 1.5 h can complete detection; (4), highly sensitive: the common ELISA of lowest detection limiting proportion is high 5～10 times; The recall rate of pattern detection reaches 97.5%; (5), background is low, anti-cancellation ability is strong: adopt long-wavelength excitation, short wavelength's emission, do not have the background of autofluorescence; And utilize the detecting pattern of time-resolved fluorescence, further reduced background, guarantee the authenticity of result, the emission wavelength of 615nm, very sharp-pointed, basic not from the cancellation interference of sample, be to carry out the ideal chose that the complex samples such as serum, blood plasma, body fluid detect; (6), purposes: the fast detecting that can be used for antibody against swine fever virus in serum and correlated samples thereof.

The accompanying drawing explanation

The nucleic acid amplification electrophoretogram that Fig. 1 is CSFV raq gene total length; The amplified production that wherein the 1st swimming lane is the CSFV raq gene, the M swimming lane is DL 2000 DNA marker;

Fig. 2 is that recombinant expression plasmid pET-CSFV-E2 is through Xho Ι, Kpn Ι double digestion rear electrophoresis figure; In figure the 1st swimming lane be plasmid pET-CSFV-E2 after enzyme is cut, the 2nd swimming lane is empty carrier, the 3rd swimming lane is that before plasmid pET-CSFV-E2 enzyme is cut, the M swimming lane is 15000 DL Marker;

The SDS-PAGE gel electrophoresis analysis result that Fig. 3 is CSFV E2 albumen, the M swimming lane is for dying in advance the molecular weight of albumen standard, and molecular weight is followed successively by 97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD from top to bottom; The contrast of the 1st swimming lane for not inducing; The 2nd and 3 swimming lanes are CSFV E2 albumen total length 1-1122, and molecular size range is about 56 KD, consistent with expected results;

The Western blot qualification result of Fig. 4 His-label C SFV E2 full-length proteins; The 1st swimming lane is that blank, the 2nd and 3 swimming lanes are CSFV E2 total length 1-1122, molecular size range is about 56KD, the M swimming lane is Western blot protein molecular standard, and molecular weight is followed successively by 170KD, 95KD, 72 KD, 55 KD, 43KD, 34KD, 26KD, 17KD, 10KD from top to bottom.

Embodiment

Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.

The CSFV attenuated vaccine strain used in below implementing is commercially available prod, is stored in Chongqing Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center.

One, structure and the evaluation of CSFV E2 recombinant protein prokaryotic expression plasmid

1, purpose fragment amplification

1.1, RT-PCR amplification

The genome of CSFV attenuated vaccine strain CSFV (GenBank No.:NC_002657.1) of take is template, carry out inverse transcription polymerase chain reaction (RT-PCR) the required purpose fragment CSFV raq gene that increases, its nucleotide sequence is as shown in SEQ ID No.2, and the amino acid sequence of coding is as shown in SEQ ID No.1.

The upstream and downstream primer is introduced respectively Kpn I and Xho I restriction enzyme site, and design of primers adopts Primer Preier5 (a kind of primer-design software), and primer is synthetic by precious biological (Dalian) company, and primer sequence is in Table 1.

Table 1 primer sequence

RT-PCR reaction system (cumulative volume 25 μ L) as shown in table 2.

Table 2 RT-PCR reaction system

Reaction system	Volume (μ L)
		PrimeScript 1 Step Enzyme Mix	1
2×1 Step Buffer	12.5
		Upstream primer (20 μ M)	0.5
Upstream primer (20 μ M)	0.5
		Template ribonucleic acid (or positive quality control RNA)	3
Rnase Free dH2O	7.5

Reaction conditions is: 50 ℃ of 30min, 94 ℃ of 2min, 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 1min; 30 circulations; 72 ℃ are extended 10min.

The PCR product of amplification, after 2% agarose gel electrophoresis is identified, cuts the Ago-Gel piece that contains the genes of interest fragment in ultraviolet transilluminator, and it is put in the centrifuge tube of 1.5mL, reclaims kit with the nucleic acid gel and reclaims purifying.The amplification electrophoresis result as shown in Figure 1.

1.2 T-A clone

The pcr amplification product reclaimed is connected and composed to recombinant plasmid pMD19-CSFV-E2 with (cloning vector) pMD19-T-vector respectively, coupled reaction system following (cumulative volume 10 μ L, 4 ℃ of connections are spent the night).

Table 3 T carrier linked system

Reaction system	Volume (μ L)
		pMD19-T	1
The DNA fragmentation reclaimed	4
		SolutionⅠ	5
Add up to	10

1.3 double digestion reaction

Adopt restriction enzyme Xho I, Kpn I to carry out double digestion to positive colony plasmid pMD19-CSFV-E2 and the expression vector pET-32-a (+) built, cumulative volume 30 L, 37 ℃ of water-bath enzymes are cut 3h.

Table 4 double digestion reaction system

Reaction system	Volume (μ L)
		The positive colony plasmid	8
10 * M damping fluid	3
		Kpn I	1
Xho I	1
		ddH2O	17

Enzyme is cut to product adds 10 * DNA sample-loading buffer at the enterprising row agarose gel electrophoresis of 1.2% Ago-Gel.After purpose product and T carrier are separated, cut the purpose fragment and reclaim.

2, the structure of the expression vector of E2 total length 1-1122

To be connected with the genes of interest fragment of double enzyme site reaction system following (cumulative volume 20 μ L connect 2h) with the histidine of cutting through same enzyme (His) tag fusion protein expression vector PET-32-a (+) (for commercially available prod).

Table 5 coupled reaction system

Reaction system	Volume (μ L)
		10 * T4 DNA connects damping fluid	2
The T4 DNA ligase	2
		After the pET-32a(double digestion)	3
The genes of interest fragment	13

The connection product that will be connected with pET-32-a (+) prokaryotic vector is transformed into BL21(DE3) in competent cell, adopt conventional method for transformation, get 200 μ L and be applied to (containing 50 μ g/mL Amp) on solid LB agar culture plate, 37 ℃ of incubators are inverted and are cultivated 12 h～16h.The single bacterial clump of picking, be placed in the liquid LB nutrient culture media of 5mL containing 100 μ g/mL Amp, and 37 ℃, 220rpm shakes overnight incubation, extracts plasmid DNA, and send the order-checking of precious biological (Dalian) company limited by plasmid DNA.

3, enzyme is cut evaluation

By Kpn I, the Xho I double digestion for pET-32a-CSFV (1-1122) plasmid that build, total length 1-1122 is 1122bp altogether, send the order-checking of precious biological (Dalian) company limited to confirm.Marker is followed successively by shown in 10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp, 250bp(Fig. 2 from top to bottom).

4, a small amount of expression identification of CSFV E2 total length recombinant protein

To check order respectively and identify that correct monoclonal bacterium liquid is with 1:1000(v/v) ratio be inoculated into the 5 mL LB fluid nutrient mediums that contain Amp, put into 37 ℃ of shaking tables, 170 rpm concussion overnight incubation, again with 1:100(v/v) ratio be forwarded in the 5 mL LB fluid nutrient mediums that contain Amp, 37 ℃, 170 rpm concussions are cultivated 1.5～2h to bacterium liquid optical density value 600(OD600) reach 0.6～0.8, then add the isopropyl-β-D-thiogalactoside (IPTG) that final concentration is 0.5 mmol/L, wherein another pipe does not add IPTG as the contrast of not inducing, 37 ℃, 170rpm continues to cultivate 3～4h.By each sucking-off 1000 μ L bacterium liquid to 1.5 mL centrifuge tubes of every pipe, centrifugal 1 min of 12000 rpm, collect bacterium liquid, use again resuspended precipitation of 25 μ L phosphate buffers (PBS), then add 25 μ L 2 * albumen sample-loading buffers fully to mix latter 100 ℃ and boil 10 min, more centrifugal 1 min of 12000 rpm.Get 15 μ L loadings, routine is carried out 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).After electrophoresis finishes, use coomassie brilliant blue staining, according to before inducing with induce rear comparative analysis protein electrophoresis result, whether observation has protein expression, choose positive bacterial classification be stored in-80 ℃ standby.

5, the purifying of CSFV E2 total length recombinant protein

5.1 reagent is prepared

(1) sex change liquid: 6M guanidine hydrochloride, 20mM dibastic sodium phosphate, 500mM sodium chloride, Ph7.8;

(2) buffer A: 20mM Tris-HCl (pH8.5), 500mM KCl, 20mM imidazole, 5mM β-ME, 10% (v/v) glycerine, 8M Urea;

(3) buffer B: 20mM Tris-HCl (pH8.5), 1M KCl, 5mM β-ME, 10% (v/v) glycerine, 8M Urea;

(4) damping fluid C:20mM Tris-HCl (pH8.5), 100mM KCl, 200mM imidazole, 5mM β-ME, 10% (v/v) glycerine, 8M Urea;

(5) dialysis buffer liquid: 8M Urea is dissolved in 1 * PBS damping fluid;

(6)0.2mM?Ni ₂SO ₄；

(7) 2%(w/v) NaHCO ₃with 1mM EDTA, pH8.0;

(8)1Mm?EDTA，pH8.0。

5.2 test procedure

5.2.1 protein induced expression

(1) the positive expression bacterial classification is with 1:1000(v/v) ratio add 5 mL containing in the liquid LB nutrient culture media of 50 g/mL Amp, 37 ℃, 150 rpm concussion overnight incubation;

(2) by culture in 1:100(v/v) ratio add 100 mL to contain Amp liquid LB nutrient culture media in, 37 ℃, 150 rpm concussions are cultivated 2～3h and are reached 0.6～0.8 to the OD600 of bacterium liquid;

(3) after adding the IPTG that final concentration is 0.5mmol/L, 150 rpm, 37 ℃ of abduction delivering 3 h;

(4) collect nutrient solution, centrifugal 10 min of 10000 rpm, abandon supernatant, collecting precipitation (taking the weight of collecting centrifuge tube front and back centrifugal bottle, to calculate the wet bacterium weight of precipitation);

(5) with the resuspended bacterial sediment of 20 mL precooling PBS, 4 ℃ of 10000 centrifugal 10 min of rpm, this step repeats twice;

(6) the wet bacterium of every 1g adds 250 μ L Cocktail II and 250 μ L Triton-100 (a kind of detergent), ultrasonication bacterium in ice bath, and every ultrasonic 5 sec, intermittently 10 sec, be total to ultrasonic 20 min, and ultrasonic power is 300W.Ultrasonic rear 4 ℃ with centrifugal 10 min of 10000 rpm;

(7) supernatant is first deposited in 4 ℃ of refrigerators, and the 1g precipitation should add 7 mL sex change liquid, resuspended precipitation, and room temperature jog sex change 1 h～2 h, 4 ℃ with centrifugal 10 min of 10000 rpm;

(8) get supernatant and be combined with the nickel post of handling well, normal temperature is in conjunction with 2h.

5.2.2 protein purification (centrifugal centrifugal 3 min of 3000 rpm that are)

(1) buffer A is washed (10 times of column volumes) 6 times; Each 4 ℃, 3 min.

(2) buffer B is washed (10 times of column volumes) 6 times; Each 4 ℃, 3 min.

(3) buffer A is washed (10 times of column volumes) 2 times; Each 4 ℃, 3 min.

(4) damping fluid C wash-out.At least wash 10 times.Each 1～2 min.

5.1.3 albumen dialysis

According to the volume of protein solution, choose the bag filter of suitable interception and length, after cleaning with sterile distilled water, first with the bag filter clamp, live an end, will contain protein solution and pack into after bag filter and seal again the other end.Under 4 ℃ of conditions, first with the PBS(pH8.0 containing 8 mol/L urea) dialysis 4h, use again PBS (pH8.0) progressively to dilute, make the concentration of urea take every 4h and successively decrease the speed of 2 mol/L until ultimate density is 0 mol/L, wherein dialysed overnight when 4 mol/L.With centrifugal 10 min of 10000rpm, get supernatant at 4 ℃, with BCA protein quantification kit measurement protein concentration.

6, the Western Blot of CSFV E2 total length recombinant protein identifies

6.1 method of operating:

(1) sample pipetting volume pre-treatment: the CSFV E2 protein 10 0 μ L got after purifying adds isopyknic 1 * SDS albumen sample-loading buffer, mixes 100 ℃ of sex change 5min;

(2) preparation of SDS-PAGE gel: prepare 12% separation gel and 5% concentrated glue.At concentrated glue 80V, electrophoresis under separation gel 110V voltage conditions, until the indicator bromophenol blue migrates to the glue bottom;

(3) electrotransfer of albumen: gel is put into to electricity and turn damping fluid balance 10 min, according to the gel size, cut out out formed objects PVDF membrane (pvdf membrane), after it is first infiltrated to the 5min activation in methanol solution, then put electricity and turn balance 10min in damping fluid; Simultaneously, prepare 3 onesize metafiltration paper, put electricity and turn damping fluid balance 10min, place successively 3 metafiltration paper, pvdf membrane, gel and 3 metafiltration paper on the anode plate of electroporation, drive each interlayer bubble away, press negative plates, carry out electricity with the 1.5mA/cm2 current stabilization and turn 90 min;

(4) sealing of pvdf membrane, the primary antibodie effect: shift completely, pvdf membrane is with 10mL containing the confining liquid of 5% skimmed milk, and 4 ℃ of sealings are spent the night;

(5) primary antibodie is hatched: abandon confining liquid, 1 * TBST room temperature rinsing 3 times, each 5min.Add the Anti-CSFV E2 monoclonal antibody after 5mL dilutes, by Western blot primary antibodie dilution 1:1000 dilution Anti-CSFV E2 monoclonal antibody.37 ℃ are slowly shaken on shaking table, hatch 1 h.

(6) two anti-hatching: take out pvdf membrane, 1 * TBST room temperature rinsing 3 times, each 5min.Pvdf membrane is put into to hybridization bag, add the sheep anti-mouse igg (two anti-) of horseradish peroxidase (HRP) mark of (1:2500) after dilution, with the anti-dilution 1:2500 of Western blot bis-, doubly dilute sheep anti-mouse igg-HRP, 37 ℃ are shaken and hatch 1h;

(7) colour developing: take out pvdf membrane, 1 * TBST washing lotion is washed 3 times, each 5 min.Add freshly prepared nitrite ion (nitrite ion A liquid 200uL, C liquid 20uL, mix).Pvdf membrane is placed in to nitrite ion, the colour developing of room temperature lucifuge.Observe after 10min, after showing to protein band, use the distilled water cessation reaction.Take pictures after colour developing to print and file.

6.2 result

The albumen of wash-out is carried out to the SDS-PAGE gel electrophoresis analysis, use on the one hand coomassie brilliant blue staining, the molecular weight of albumen size is similar to expection.Coomassie brilliant blue the results are shown in Figure 3, Western Blot qualification result and sees Fig. 4.

Two, Anti-CSFV E2 IgG AlphaLISA reaction conditions gropes

1, test principle

In the method, with the CSFV E2 antigen of His label can with anti-CSFV E2 monoclonal antibody reactive, with anti-mouse IgG Acceptor beads and Nickel chelate Donor beads, interact, Ear Mucosa Treated by He Ne Laser Irradiation by 680nm, photosensitizer on the donor microballon is more active free oxygen by the oxygen conversion in surrounding environment, free oxygen diffuses to the acceptor microballon, produce a series of chemiluminescence reaction, finally transfer energy to europium, by the emission wavelength of 615nm, measure absorbance.If there is CSFV antibody in sample, can compete CSFV E2 antigen with anti-CSFV E2 monoclonal antibody, the detected value reduction (is compared with the control wells of acceptor microballon and donor microballon, value reduces more than 2 times), if there is not CSFV antibody in sample, detected value higher (compare with the control wells of acceptor microballon and donor microballon, difference is not obvious).When if there is not special interaction in biomolecule, free oxygen can't be diffused into the acceptor microballon, does not have signal and produces.

This analysis of experiments thing is CSFV E2 antibody positive contrast (from the ELISA kit).The present invention has selected to carry out groping of system through each 16 parts of the serum sample of ELISA method proof CSFV E2 antibody positive and negative serum samples.

2, method of operating

1) treat that in gaging hole, every hole adds 1 μ L serum sample, control wells adds 1 * dilution buffer liquid;

2) every hole adds CSFV E 2 protein antigen and the 5 μ L 100nM/L CSFV E 2 protein monoclonal antibodies of 5 μ L 180nM/L with the His label, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 1h;

3) every hole adds 80 μ g/mL acceptor microballon 4 μ L, 80 μ g/mL donor microballon 5 μ L, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 30 min;

4) being placed in the Enspire plate reading machine is detected.

Be totally under the condition of 20 μ L, obtaining a more stable detection system.The final concentration that is the acceptor microballon is 16 μ g/mL, every hole 4 μ L; The final concentration of donor microballon is 20 μ g/mL, every hole 5 μ L; 180nM/L is with the every hole 5 μ L of the CSFV E 2 Antigen amount of His label; The CSFV E 2 protein monoclonal antibody 5 μ L of 100nM/L; Serum 1 μ L; Be totally 20 μ L.

Three, the Preliminary Applications of Anti-CSFV E2 IgG AlphaLISA detection method

1, with ELISA method primary dcreening operation some positive serum

The validity of the Anti-CSFV E2 IgG AlphaLISA detection method of having set up for effectively evaluating.The present invention uses business-like hog cholera antibody detection kit (IDEXX) to carry out preliminary screening to 32 parts of pig serum samples.

1.1 detection principle

The IDEXX kit adopts the indirect enzyme-linked immunosorbent assay principle.The CSFV antigen of pre-coated restructuring on capillary strip, when detected sample is hatched in coated hole, if there is specific CSFV antibody, the specific antibody of CSFV can form antigen antibody complex with coated antigen, washes away unconjugated composition; The anti-pig antibody that adds horseradish peroxidase (HRPO) mark, can be combined by the pig antibody in hole, while having CSFV antibody in sample, forms " anti-CSFV antibody-antigen-enzyme labelled antibody " compound, washes away unconjugated composition; Add developer TMB, the HRPO catalysis developer reaction connected on compound, the production blue product, add stop buffer, cessation reaction, color becomes yellow.

1.2 method of operating

(1) preparation of washing lotion: by distilled water or 10 times of dilutions of deionized water for concentrated cleaning solution;

(2) sample number into spectrum: the corresponding microwell plate of sample is numbered according to the order of sequence, and every plate is established negative control 2 holes, positive control 2 holes and blank 1 hole;

(3) dilution of sample and application of sample: every hole adds sample diluting liquid 50 μ L, and sample 50 μ L, add the positive control of 50 μ L and negative control in corresponding control wells respectively, and light shaking mixes.With after shrouding film shrouding, incubated at room 2 h;

(4) wash plate: carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button as far as possible is dry.

(5) adding two resists: every hole adds the anti-pig IgG antibody 100 μ L of horseradish peroxidase (HRPO) mark, and light shaking mixes, incubated at room 30min ± 2min;

(6) wash plate: carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button as far as possible is dry;

(7) colour developing: every hole adds tmb substrate nitrite ion 100 μ L, and light shaking mixes, room temperature lucifuge colour developing 15min;

(8) stop: every hole adds stop buffer 100 μ L, and light shaking mixes, measurement result in 10min.

(9) measure: set the microplate reader ripple and be longer than the 450nm place, measure each hole A(450) value.

1.3 result

CSFV IgG positive sample 24 examples after testing, be mainly the pig of vaccine immunity and the pig of clinical onset symptom arranged.Simultaneously, use 20 parts of not immune vaccines and without the pig serum of clinical symptoms in contrast, result shows that 20 increments are originally all negative.

2, the Preliminary Applications of competition AlphaLISA detection method

The IgG positive sample of the 24 routine CSFV samples that above-mentioned ELISA method is set up and the serum (IgG feminine gender) of 20 parts of health pig are as the preliminary assessment of AlphaLISA detection method.Detecting sample is 1 μ L serum, and the 20 μ L detection system of setting up according to the test condition of optimizing are before carried out the detection of sample.

Testing result is found, the sample of the 24 routine CSFV IgG positives, and the result that the AlphaLISA detection method detects is also positive, and the serum of 20 parts of health pig also is shown as feminine gender by AlphaLISA detection method testing result, and coincidence rate reaches 100%.For further understanding the susceptibility that AlphaLISA detects, test will be through the detection of nucleic acids positive, and the serum of the CSFV of IgG testing result feminine gender has carried out the AlphaLISA detection, and result detects 3 parts of positive sample (table 6)

Table 6 sample results relatively

Six, the evaluation of Anti-CSFV E2 IgG AlphaLISA method

1, pattern detection

In order to estimate built Anti-CSFV E2 IgG AlphaLISA method, we are defined as the IgG positive sample by CSFV nucleic acid and positive sample of ELISA testing result while.Filter out 24 parts of positive sample by the method.Simultaneously, estimate Sample Storehouse (table 7) with this CSFV IgG of negative Sample Establishing 124 increments of the sample of the import boar of 100 parts of health

Table 7 CSFV IgG Sample Storehouse forms

Sample forms	Sample size (part)	Detection of nucleic acids	ELISA（IgM ）	AlphaLISA(IgM)
					Positive sample	49	49（+）	24（+）	23（+）
Healthy sample	100	100（-）	Nothing	2（+）

2, the evaluation of Anti-CSFV E2 IgG AlphaLISA method

Use above-mentioned sample to be estimated the Anti-CSFV E2 IgG AlphaLISA method of setting up.Detecting sample is 1 μ L serum, sets up 20 μ L detection system according to the test condition of having optimized and is detected.Testing result shows: the sample of the 24 routine CSFV IgG positives, the testing result of AlphaLISA method have 23 parts positive.The serum of 100 parts of health pig is shown as feminine gender, 2 parts positive (table 8) by 98 parts of Anti-CSFV E2 IgG AlphaLISA detection method testing results.

Table 8 Anti-CSFV E2 IgG AlphaLISA method evaluation

Testing result shows: the Anti-CSFV E2 IgG AlphaLISA method of foundation, its sensitivity=92.0%, specificity=99.0%, false positive rate=8.0%, false negative rate=1.0%, positive predictive value=95.8%, negative predictive value=98.0%, accuracy=97.5%.

SEQUENCE?LISTING

<110 > Chongqing Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center

<120 > antibody against swine fever virus competition AlphaLISA detection kit and detection method

<160> 4

<210> 1

<211> 372

<212> PRT

<213 > CSFV E 2 protein antigen

<400>?SEQ?ID?NO.1

Arg?Leu?Ala?Cys?Lys?Glu?Asp?Tyr?Arg?Tyr?Ala?Ile?Ser?Ser?Thr?Asn

1 6 11 16

Glu?Ile?Gly?Leu?Leu?Gly?Ala?Gly?Gly?Leu?Thr?Thr?Thr?Trp?Lys?Glu

21 26 31

Tyr?Ser?His?Asp?Leu?Gln?Leu?Asn?Asp?Gly?Thr?Val?Lys?Ala?Ile?Cys

36 41 46

Val?Ala?Gly?Ser?Phe?Lys?Val?Thr?Ala?Leu?Asn?Val?Val?Ser?Arg?Arg

51 56 61

Tyr?Leu?Ala?Ser?Leu?His?Lys?Gly?Ala?Leu?Leu?Thr?Ser?Val?Thr?Phe

66 71 76

Glu?Leu?Leu?Phe?Asp?Gly?Thr?Asn?Pro?Ser?Thr?Glu?Glu?Met?Gly?Asp

81 86 91 96

Asp?Phe?Gly?Phe?Leu?Cys?Pro?Phe?Asp?Thr?Ser?Pro?Val?Val?Lys?Gly

101 106 111

Lys?Tyr?Asn?Thr?Thr?Leu?Leu?Asn?Gly?Ser?Ala?Phe?Tyr?Leu?Val?Cys

116 121 126

Pro?Ile?Gly?Trp?Thr?Gly?Val?Ile?Glu?Cys?Thr?Ala?Val?Ser?Pro?Thr

131 136 141

Thr?Leu?Arg?Thr?Glu?Val?Val?Lys?Thr?Phe?Arg?Arg?Glu?Lys?Pro?Phe

146 151 156

Pro?His?Arg?Met?Asp?Cys?Val?Thr?Thr?Thr?Val?Glu?Asn?Glu?Asp?Leu

161 166 171 176

Phe?Tyr?Cys?Lys?Leu?Gly?Gly?Asn?Trp?Thr?Cys?Val?Lys?Gly?Glu?Pro

181 186 191

Val?Val?Tyr?Thr?Gly?Gly?Gln?Val?Lys?Gln?Cys?Lys?Trp?Cys?Gly?Phe

196 201 206

Asp?Phe?Asn?Glu?Pro?Asp?Gly?Leu?Pro?His?Tyr?Pro?Ile?Gly?Lys?Cys

211 216 221

Ile?Leu?Ala?Asn?Glu?Thr?Gly?Tyr?Arg?Ile?Val?Asp?Ser?Thr?Asp?Cys

226 231 236

Asn?Arg?Asp?Gly?Val?Val?Ile?Ser?Ala?Glu?Gly?Ser?His?Glu?Cys?Leu

241 246 251 256

Ile?Gly?Asn?Thr?Thr?Val?Lys?Val?His?Ala?Ser?Asp?Glu?Arg?Leu?Gly

261 266 271

Pro?Met?Pro?Cys?Arg?Pro?Lys?Glu?Ile?Val?Ser?Ser?Ala?Gly?Pro?Val

276 281 286

Arg?Lys?Thr?Ser?Cys?Thr?Phe?Asn?Tyr?Ala?Lys?Thr?Leu?Lys?Asn?Lys

291 296 301

Tyr?Tyr?Glu?Pro?Arg?Asp?Ser?Tyr?Phe?Gln?Gln?Tyr?Met?Leu?Lys?Gly

306 311 316

Glu?Tyr?Gln?Tyr?Trp?Phe?Asp?Leu?Asp?Val?Thr?Asp?Arg?His?Ser?Asp

321 326 331 336

Tyr?Phe?Ala?Glu?Phe?Val?Val?Leu?Val?Val?Val?Ala?Leu?Leu?Gly?Gly

341 346 351

Arg?Tyr?Val?Leu?Trp?Leu?Ile?Val?Thr?Tyr?Ile?Val?Leu?Thr?Glu?Gln

356 361 366

Leu?Ala?Ala?Gly

372

<210> 2

<211> 1122

<212> DNA

<213 > E 2 gene of Classical Swine Fever

<400> SEQ?ID?NO.2

cgtctggcgt?gtaaagaaga?ttaccgttat?gctatcagct?ccaccaacga?aatcggtctg 60

ctgggcgcag?gcggtctgac?cactacctgg?aaagaataca?gccacgacct?gcagctgaac 120

gatggcaccg?tcaaagccat?ctgcgtagct?ggctctttca?aagttactgc?actgaacgtt 180

gtgagccgtc?gctatctggc?ttctctgcac?aaaggtgcgc?tgctgacctc?tgttaccttt 240

gagctgctgt?tcgacggtac?taacccgtct?accgaggaaa?tgggtgacga?ctttggtttc 300

ggcctgtgcc?cgtttgatac?cagcccggtt?gttaaaggca?agtacaacac?caccctgctg 360

aatggcagcg?ctttttatct?ggtatgcccg?attggctgga?ctggtgtgat?tgaatgtacc 420

gcagtgtctc?cgacgaccct?gcgtaccgag?gtggtgaaaa?ccttccgtcg?tgagaaaccg 480

ttcccacatc?gtatggattg?cgtcaccact?actgtcgaaa?acgaggatct?gttctactgc 540

aaactgggcg?gtaactggac?ctgtgtaaaa?ggtgaaccgg?tagtttacac?gggtggtcag 600

gtgaagcagt?gtaaatggtg?cggtttcgat?tttaatgaac?cggatggcct?gccgcactac 660

cctatcggca?aatgcatcct?ggcgaacgaa?actggctacc?gcatcgtaga?ctccactgac 720

tgcaaccgtg?acggcgtcgt?gatctctgca?gaaggttccc?atgaatgtct?gattggtaac 780

acgaccgtaa?aagttcacgc?atccgatgaa?cgcctgggcc?cgatgccttg?tcgtccgaaa 840

gaaatcgttt?cttctgctgg?tccggttcgc?aaaacctcct?gcactttcaa?ctacgcgaag 900

acgctgaaga?acaaatatta?cgaaccacgc?gactcctatt?tccagcagta?catgctgaaa 960

ggtgagtatc?agtactggtt?cgacctggac?gtcaccgatc?gtcacagcga?ctacttcgcg 1020

gaattcgttg?tgctggtagt?tgttgccctg?ctgggtggcc?gttatgttct?gtggctgatc 1080

gtgacttaca?ttgttctgac?ggaacaactg?gccgcgggtt?aa 1122

<210> 3

<211> 49

<212> DNA

<213 > artificial sequence

<400> SEQ?ID?NO.3

cgcggtaccg?acgacgacga?caagcgtctg?gcgtgtaaag?aagattacc 49

<210> 4

<211> 32

<212> DNA

<213 > artificial sequence

<400> SEQ?ID?NO.4

cgcctcgagt?taacccgcgg?ccagttgttc?cg 32

Claims (5)

1. antibody against swine fever virus competition AlphaLISA detection kit is characterized in that: comprise donor microballon, acceptor microballon, CSFV E 2 protein monoclonal antibody and with the CSFV E 2 protein antigen of His label;

Wherein, the donor microballon that described donor microballon is chelating nickel, PE, AS101D; Described acceptor microballon is anti-mouse IgG acceptor microballon, PE, AL105C; The amino acid sequence of the described antigen of the CSFV E 2 protein with the His label is as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed amino acid sequence had with CSFV E 2 protein antigen same function.

2. antibody against swine fever virus competition AlphaLISA detection kit according to claim 1 is characterized in that: described donor microballon 80 μ g/mL, acceptor microballon 80 μ g/mL, CSFV E 2 protein monoclonal antibody 100nM/L and with the CSFV E 2 protein antigen 1 80nM/L of His label;

During use, in total system 20 μ L, every hole addition is: 80 μ g/mL donor microballon 5 μ L, 80 μ g/mL acceptor microballon 4 μ L, 180nM/L are with the anti-E2 protein monoclonal antibody 5 μ L of CSFV E 2 protein antigen 5 μ L, the 100nM/L CSFV of His label and testing sample 1 μ L.

3. according to the described antibody against swine fever virus competition of claim 1 or 2 AlphaLISA detection kit, it is characterized in that: the gene of the described antigen of the CSFV E 2 protein with the His label of encoding is the CSFV raq gene, and its nucleotides sequence is classified SEQ ID NO.2 as.

4. antibody against swine fever virus is competed the AlphaLISA detection kit according to claim 3, and it is characterized in that: described CSFV raq gene obtains by the RT-PCR amplified reaction; The nucleotides sequence of the upstream primer of RT-PCR amplified reaction is classified SEQ ID NO.3 as, and the nucleotides sequence of downstream primer is classified SEQ ID NO.4 as.

5. utilize the described antibody against swine fever virus competition of claim 1 AlphaLISA detection kit to detect the detection method of the non-medical diagnosis on disease purpose of antibody against swine fever virus, comprise the following steps:

1) reaction plate treat that in gaging hole, every hole adds 1 μ L sample to be tested, add 1 μ L 1 * dilution buffer liquid in control wells;

2) every hole adds CSFV E 2 protein antigen and the 5 μ L 100nM/L CSFV E 2 protein monoclonal antibodies of 5 μ L 180nM/L with the His label, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 1h;

3) every hole adds 80 μ g/mL acceptor microballon 4 μ L, 80 μ g/mL donor microballon 5 μ L, centrifugal 10 sec of 1000rpm, and 23 ℃ of lucifuges of room temperature are hatched 30 min;

4) result reads: reaction plate is put to Enspire(PE) value of reading in the AlphaLISA detection system.

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