CN107064502B - A kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection method - Google Patents
A kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection method Download PDFInfo
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Abstract
The invention discloses a kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection methods, belong to biological field, to solve the problem of that existing serum antibody ELISA kit is to be only suitable for the antibody test that single cause of disease generates not detecting the serum antibody that a variety of cause of diseases generate simultaneously.The present invention has the biological function of all IgG antibodies in specific binding Swine serum using SPA albumen.SPA albumen can specifically bind all types IgG antibody in pig anteserum sample, if there is the antibody of 4 kinds of albumen of anti-three kinds of cause of diseases coding in sample to be tested, also can be specifically bound with SPA albumen, realize a kind of antigen and meanwhile capture 3 kinds of pathogen infections or it is immune after the purpose of 4 serum IgG antibodies (gB the and gE protein antibodies of the E2 protein antibodies of anti-CSFV, the Cap protein antibody of anti-PCV2 and anti-PrV) that generates.
Description
Technical field
The invention belongs to biological fields, and in particular to a kind of multiple ELISA detection of pig virus infectious disease serum IgG antibody
Kit and its detection method.
Background technique
Viral infectious has become the serious hindrance for threatening China's pig-breeding to develop in a healthy way, outburst and popular provisions
Pig dealer causes very big economic loss.Currently, including that swine fever virus (CSFV), porcine circovirus 2 type (PCV2) and pig puppet are mad
Dog disease poison (PrV) is the viral blight of pig breeding industry emphasis prevention and control.In the prevention work of live pig disease, by real-time detection and
Serum Antibody level and differentiation antibody sources are the passes evaluated vaccine inoculation effect, protect swinery health after evaluation vaccine immunity
Key link.The Serum Antibody Detection method of pig virus communicable disease mainly includes enzyme-linked immunosorbent assay (ELISA), cream
Glue agglutination test, hemagglutination test, virus neutralization tests, single layer enzyme-linked immunosorbent assay etc..Since ELISA method operates letter
It is single, low in cost, require experimental condition that low, sensibility and stability are good, can evaluate antibody level and type, are suitble to scale
Change detection serum sample and result judgement can standardize, is to monitor and comment using animal epidemic antibody level of serum at present therefore
Most widely used detection means in valence system.
Common Serum Antibody Detection ELISA method basic principle is to be coated in microwell plate using preparatory to be detected
The antigen (recombinant antigen or the complete cause of disease particle of inactivation) of epidemic disease combines the specific IgG antibodies in serum sample, and anti-igg is added
Enzyme (chemiluminescent substance, fluorescent material etc.) label secondary antibody reacted with the IgG antibody with antigen binding, it is to be checked to detect
Antibody content or type in test sample sheet.According to the difference of antigen-antibody reaction mode and operation sequence, ELISA method can divide
Being includes indirect ELISA, blocking ELISA and competitive ELISA.The common serum antibody ELISA reagent of animal epidemic detection field
Box is the antibody test for being only suitable for single cause of disease and generating, there is not yet a kind of kit can detect what a variety of cause of diseases generated simultaneously
The product of serum antibody.
Summary of the invention
The object of the present invention is to provide a kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody, with
Solving existing serum antibody ELISA kit is the antibody test for being only suitable for single cause of disease and generating, and can not detect a variety of diseases simultaneously
The problem of originating in raw serum antibody.
It is a further object to provide a kind of with the multiple ELISA detection of pig virus infectious disease serum IgG antibody
The method that kit is detected.
Technical solution of the present invention is as follows: a kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody,
Detection plate, serum samples diluted liquid, cleaning solution, developing solution, terminate liquid including pre-coated SPA albumen, the yin-yang of four kinds of cause of diseases
The E2 antigen of CSFV that property standard control serum and HRP are marked respectively, the Cap antigen of PCV2, PrV gB and gE antigen.
Preferably, the package amount of the be coated with SPA albumen of detection plate of the pre-coated SPA albumen is the hole 2ng~28ng/,
The carbonate buffer solution that the pH value that coating dilution is 50mmol/L is 9.6.The purpose for preparing antigen coated microplate is capture serum
All types of IgG antibodies in sample.Different concentration directly influences the accuracy of testing result.
Preferably, the group of every milliliter of coating carbonate buffer solution becomes in the detection plate of the pre-coated SPA albumen
1.59mg Na2CO3With 2.93mg NaHCO3, deionized water is stored in after completely dissolution at a temperature of 4 DEG C.Carbonate buffer solution item
Under part, SPA antigen, which can be stablized, to be adsorbed on ELISA Plate.
Preferably, the confining liquid group used in the detection plate of the pre-coated SPA albumen is divided into mass concentration for the sea 1-5%
Algae sugar, mass concentration 0.5-2.0% gelatin, volumetric concentration 1-8% horse serum, 7.3,200 hole μ l/ 1 × PBST, pH.Confining liquid
In gelatin, horse serum and trehalose can make entire hole bottom in conjunction with the not adsorbable rest position of SPA antigen in ELISA Plate
All covered by albumen.First is that the activity of SPA antigen can be kept, second is that blocking albumen or antibody present in sample to be tested
In conjunction with rest position in ELISA Plate, lead to the generation of nonspecific reaction, and the trehalose added is with heat-resisting
Feature can significantly extend the pot-life of antigen coated microplate.
Preferably, the cleaning solution is the phosphorus that the pH value of the 0.1mol/L for the Tween-20 that volumetric concentration is 0.5% is 7.2
Phthalate buffer, the function of cleaning solution are the compositions washed away in unbonded and non-specific binding confining liquid.Decontamination washing
Liquid is the phosphate buffer that mass concentration 1%EDTA adds that the pH value of 50mmoL/L is 7.2, and 100 μ L/ hole room temperatures act on 1-
15min.The purpose of decontamination cleaning solution is the IgM for removing non-specific adsorption in serum sample, avoids generating nonspecific reaction.
Preferably, in the detection plate of the pre-coated SPA albumen, standard positive and negative serum and serum sample to be detected are equal
It is added in corresponding hole with 100 hole μ l/ of former times concentration, room temperature acts on 60min.
Preferably, the E2 antigen of CSFV of HRP label, the Cap antigen of PCV2, PrV the gE antigen of gB and PrV make
It is respectively 1: 4000,1: 4000,1: 4000,1: 2000 with concentration, corresponding mass concentration is 0.1 μ g/mL-15 μ g/mL, 37
DEG C be incubated for 60min.The detection antigen of HRP- label can realize detection target antibody in conjunction with corresponding specific IgG antibodies
Purpose.
Preferably, in the detection plate preparation of the pre-coated SPA albumen, tmb substrate buffer group used becomes, substrate
Solution A: TMB DMSO solution, concentration 2-6mg/mL；Substrate solution B:15-30g/L sodium acetate, citric acid 1-9g/L, 30%
Hydrogen peroxide 0.1-1ml, distilled water are dissolved to 1L.TMB reacts with HRP, according to IgG antibody content to be detected in serum sample
Difference, deep mixed blue is presented, can use 450nm excitation wavelength, reads reaction result.
Preferably, the terminate liquid is 1.5mol/L H2SO4。H2SO4Reaction is terminated, to read absorbance value.
A method of it is detected with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody, including
Following steps:
(1) serum sample is added: by serum sample to be detected, the positive and negative control sera with 100 μ L/ of former times concentration
Hole is added in corresponding aperture, and positive and negative control serum does multiple holes；Sealing, room temperature act on 60min；
(2) nonspecific reaction is removed: with the phosphate of decontamination cleaning solution and 50mmoL/L that mass concentration is 1%EDTA
Buffer acts on 5min with 100 μ L/ hole room temperatures, discards liquid in hole, and the pH value of phosphate buffer is 7.2；
(3) board-washing: abandoning remnants ePBS liquid in clear opening, continuously washed 4 times with the hole 200-300 μ L/ 1 × PBST cleaning solution,
Last time abandons liquid in clear opening, pats dry；
(4) HRP- labelled antigen is added: the antibody of detection cause of disease, addition have titrated HRP labelled antigen as needed,
100 holes μ L/, 37 DEG C of effect 1h, washing；
(5) substrate colour developing is added: tmb substrate buffer is added with the dosage in 100 holes μ L/, room temperature is protected from light colour developing 15min,
Terminate liquid is added with the dosage in 100 holes μ L/；
(6) result judgement:
It tests establishment condition: calculating the mean absorbance values (OD450nm) of positive control, each hole of negative control sera, when
Positive standard serum is averaged OD450 >=1.0, negative standards' serum OD450≤0.20, is determined as that test result is set up；
Determine: when sample to be tested OD450/ negative standards' serum OD450 >=2.10, determining the blood serum sample to detect cause of disease
Antibody positive；As sample to be tested OD450/ negative standards serum OD450 < 2.10, determine the blood serum sample for the cause of disease of detection
Negative antibody.
Staphylococcus aureus protein A (StaphyLococcaL protein A, SPA) be cell wall antigen it is main at
Point, it is successively made of five structural domains of S, E, D, A, B, C, X from N-terminal to C, molecular weight and 42kD or so, nothing in amino acid composition
Cysteine, so isoelectric point 5.1 belongs to a kind of acidic protein, and natural structure is highly stable, adds without disulfide bond pattern
The extreme conditions such as heat, formalin processing, 4moL/L urea, 6moL/L guanidine hydrochloride, hydrochloric acid (pH2.5) and thiocyanates acid not shadow
Ring its activity.Compatibility of the SPA in conjunction with variety classes IgG has differences, binding ability by by force to it is weak be successively pig, dog, rabbit,
People, monkey, mouse, rat, sheep, in conjunction with IgG hypotype be IgG1, IgG2 and IgG4 (adsorption rate 90%~98%), not with
IgG3 combine, can with IgM (adsorption rate 2%~30%) a small amount of in serum, IgA (adsorption rate 1.5%~20%), IgGE and
B cell antigen receptor combines, and binding site is Fabric sections of the functional areas VH.Each SPA molecule has the binding ability of bivalent,
It can be in combination with 2 IgG molecules.SPA has and Fc sections of non-specific bindings of people and a variety of mammalian serum IgG molecules
Characteristic, and the specific binding capacity of IgG molecule Fab segment and antigen is not interfered with, this special immunological properties,
The unique useful tool of immunological investigation aspect is become, has in terms of aetology and Serologic detection and potentially answers
With value.
The present invention utilizes staphylococcus aureus protein A using the reaction pattern of double antigens sandwich
(StaphyLococcaL protein A, SPA) combines all IgG molecules in pig anteserum sample to be detected, with horseradish peroxidating
IgG molecule reaction of the recombinant antigen of object enzyme (HRP) label in conjunction with SPA, resisting in sample to be tested can be detected simultaneously by establishing
The multiple ELISA method of the gB and gE protein antibodies of the E2 protein antibodies of CSFV, the Cap protein antibody of anti-PCV2 and anti-PrV, system
Standby HRP-E2 enzyme-labelled antigen belongs to the E2 antigen with complex function, can be in conjunction with swine fever virus antiserum, simultaneously also
It can react with tmb substrate, directly the result of instruction detection.It realizes an ELISA reaction while detecting four kinds of epidemic diseases
Antibody (detects swine fever virus (CSFV), porcine circovirus 2 type (PCV2) and porcine pseudorabies virus infection (PrV) or immune swine
Serum IgG antibody) purpose, sensibility and specificity is suitable with existing single pathogenic autoantibody detection kit, but significantly mentions
Risen detection efficiency, for above-mentioned four kinds of disease vaccines are immune and the Serum Antibody Detection of clinical infection pig provide it is a kind of effectively,
Efficiently detection reagent.
A variety of detection antibody are concentrated in an ELISA reaction carrying out by the present invention, can significantly improve detection efficiency, section
About detection time and reduction testing cost.
Multiple ELISA detection method provided by the invention be it is a kind of have good sensibility, specificity, accuracy, when
Effect property, stability can be completed at the same time simple, the quick side of four kinds of serum IgG antibodies detection of three kinds of swine diseases by once testing
Method, the detection work suitable for scale clinical serum sample.
Detailed description of the invention
Fig. 1 is a kind of experiment flow figure of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody；
Fig. 2 is a kind of schematic diagram of multiple ELISA detection method of pig virus infectious disease serum IgG antibody.
Specific embodiment
The present invention will be further described below.
Illustrate: the buffer solution of 1 × concentration refers to when preparing solution, stores and reduce workload for convenience,
10 times (10x) can generally be prepared to the concentrated solution of 1000 times of (1000x) working concentrations as storage.It takes when in use a small amount of
Storing liquid is diluted to working concentration (being exactly so-called 1X) again.
" material and method "
1.1 antigens, standard yin and yang attribute serum, reagent and SPA
Known CSFV, PCV2 and PrV (gE Gene deletion mutation) positive serum and negative serum, E.coLi expression
Recombinant antigen includes CSFV E2 antigen (GST label), PCV2Cap antigen (His label), PrV gE and gB antigen (His mark
Label), it is existing product.Horseradish peroxidase (HRP), SPA are purchased from Sigma company；Sepharose 4B, nickel column GE
HeaLthcare product；Ultrafiltration chromatographic column (10kD) is purchased from MiLLipore；Other related reagents are that domestic analysis is pure.
1.2 antigens are prepared and purified
1.2.1 prepared by CSFV E2 recombinant antigen
Soluble recombinant C SFV E2 antigen is carried out according to existing method to prepare and purify.
1.2.2 prepared by PCV2Cap recombinant antigen
Soluble recombination PCV2Cap antigen is carried out according to existing method to prepare and purify.
1.2.3 PrV gE and the preparation of gB recombinant antigen
1.2.3.1 the clone of PrV gE gene, expression and purifying.
(1) building of gE gene cloning and recombinant expression carrier:
Pair for amplification gE gene expression primer is synthesized with reference to existing GenBank:AF207700 strain gene order, is used for table
Up to gE albumen.Primer sequence, gE109:ACTGGATCCATGgaggtcccgagtc；GE858:
TCACTCGAGgtggtagatgcaggg, restriction enzyme site are respectively BamHI and Xhol, and amplified fragments size is 780bp.
PCR amplification, digestion target fragment after the recovery are subcloned on pGEX-6p-1 prokaryotic expression, convert DH5a competent cell, mirror
Fixed correct recombinant plasmid is named as 6p-E, converts BL-21 (DE3) competent cell, carries out recombinant protein expression.
(2) expression, purifying of gE and protein function identification:
5 monoclonal colonies of picking respectively in 5mL LB liquid medium test tube, train by 220r/min, 37 DEG C of overnight shakings
It supports；Respectively from drawing 50 μ L bacterium solutions in 5 pipes in new LB liquid medium test tube, 220r/min2~3h to OD600≈0.5
~0.6, the final concentration 1.0mol/L of IPTG is added in 4 pipes wherein, remaining 1 pipe is as negative control, 37 DEG C of 200r/
4~6h of min shaken cultivation.SDS-PAGE identification recombination E2 albumen: take 1mL recombinant bacterium into Ep pipe, 10000r/min centrifugation
1min discards supernatant, 100 μ LTris pH6.8 is added, 100 DEG C are boiled 5min, and 10000r/min is centrifuged 15min.Prepare 15%
SDS-PAGE protein electrophorese gel, each sample add 10 μ L supernatants, standard protein Marker 5 μ L, 80v are added, glue is concentrated,
120v separation gel, blob of viscose coomassie brilliant blue staining observed gE albumen successful expression after decoloration.Immunoblot experiment
(Western Blotting, WB) the result shows that, recombination E2 albumen can identify by pseudorabies gE positive serum, have well
Reactionogenicity.Then using sepharose 4B purifying resin gE albumen as the envelope antigen of ELISA antibody test.
1.2.3.2 the identification of the clone of PrV gB gene, expression, purifying and reactionogenicity:
Pair for amplification gB gene expression primer is synthesized with reference to existing PCV2 genome sequence (GenBank:KJ526439), is used
In expression gB albumen.Primer sequence, gB1627:ACTGGATCCATGgcgcacgtgaacgacat；GB2214:
GTCAAGCTTCgagcgcgtgcagctggtt, introduces restriction enzyme site BamHI and HindIII respectively, and amplification 580bp is big
Small gB gene.PCR amplification, digestion target fragment after the recovery are subcloned on PET-28a prokaryotic expression, conversion DH5a impression
State cell identifies that correct recombinant plasmid is named as 28a-B, converts BL-21 (DE3) competent cell, carries out recombinant protein table
It reaches.The part expression process reference " " 1.2.3.1 of gB recombinant protein ", the recombinant protein of acquisition are carried out with nickel affinity chromatography filler
Purifying, by 4B purifying resin gE albumen, WB experiment shows that the identification of B Positive Sera can be crossed by pig by recombinating gB albumen, can
Using the envelope antigen as ELISA antibody test.
The coated ELISA Plate preparation of 1.3 SPA
The group of every milliliter of coating carbonate buffer solution becomes 1.59mg Na2CO3With 2.93mg NaHCO3, deionized water
It is stored at a temperature of 4 DEG C after completely dissolution.
SPA albumen is diluted with 50mmol/L carbonate buffer solution (pH 9.6), 100 holes μ L/ are coated with 96 hole elisa Plates, SPA
Package amount is the hole 4ng/, and sealing is placed in room temperature and acts on 16~20 hours overnight.Liquid in clear opening is abandoned, with 1 × PBST of cleaning solution
(cleaning solution is the phosphate buffer that the pH value of the 0.1mol/L for the Tween-20 that volumetric concentration is 0.5% is 7.2) 200-300 μ
The hole L/ is continuously washed 4 times, and last time abandons liquid in clear opening.Be added 200 holes μ L/ confining liquid (5% trehalose of mass concentration,
The gelatin of mass concentration 2%, the horse serum of volumetric concentration 2.5%, 1 × PBST, pH 7.3) room temperature effect 30min；Use washing
(cleaning solution is the phosphate-buffered that the pH value of the 0.1mol/L for the Tween-20 that volumetric concentration is 0.5% is 7.2 to 1 × PBST of liquid
Liquid) washing.The standard positive and negative of four kinds of epidemic diseases and 100 hole μ L/ of blood serum sample to be detected are added in detection hole, room temperature is incubated
Educate 60min.
With decontamination cleaning solution ePBS (mass concentration 1%EDTA, the phosphate buffer of 50mmoL/L, pH 7.2) 100 μ
The hole L/ room temperature acts on 5min, removes the IgM antibody of non-specific binding, discards liquid in hole, with 1 × PBST (0.05%
The phosphate buffer of Tween-20,50mmoL/L, pH 7.2) hole 200-300 μ L/ continuously washs 4 times, and last time abandons clear opening
Interior liquid.The type of antibody is detected as needed, and corresponding HRP- antigen, 100 holes μ L/, 37 DEG C of incubations are added in corresponding aperture
60min, washing are same as above.Tmb substrate buffer (substrate solution A:TMB DMSO solution, concentration 2mg/mL is added；Substrate solution
B:27.2g/L sodium acetate, citric acid 3.2g/L, 30% hydrogen peroxide 0.6ml, distilled water are dissolved to 1L, and substrate solution A and B are kept away
Light saves, and equivalent mixes when use.), 100 holes μ L/ are incubated at room temperature 30min, and the H of 100 μ L/ hole 1.5mol is added2SO4Eventually
Only liquid, OD450Nm wavelength measures absorbance value, calculated result.
1.4 horseradish peroxidases (HRP) mark recombinant antigen:
The experiment of HRP- antigenic mark is carried out using existing improvement Over-voltage protection, is marked using AGP test experimental identification HRP
Remember the activity identification of antigen.Enzyme mark rate=OD403/OD280, the absorbance (OD of hematin prothetic group in enzyme403) and antigen egg
Absorbance (the OD of tryptophan, tyrosine in white280) the ratio between indicate HRP ratio shared in antigen-enzyme, wherein Percentage bound
(%) ﹥ 30 be it is best, preferably, ﹥ 7 is general by ﹥ 9~10.The HRP- antigen of label is divided into 200 μ L/ pipe, and the nothing of final concentration 33% is added
Bacterium glycerol is stored in -20 DEG C, until titration working concentration experiment.
The working concentration of 1.5 titration HRP labelled antigens
1.5.1 the best effort concentration of the CSFV E2 antigen (HRP-E2) of titration HRP label
Respectively by CSF antibody V seropositivity known to CSFV serum antibody feminine gender known to 3 parts and 3 parts with 100 μ of former times concentration
The hole L/ is added to the Kong Zhongzhong of the closed SPA antigen coated microplate of " 1.3 " preparation, and every part of serum does diplopore detection, and 37 DEG C
It is incubated for 45min, liquid in clear opening is abandoned, is continuously washed 4 times with 200-300 μ L/ 1 × PBST of hole, last time abandons liquid in clear opening,
It pats dry.By the HRP-E2 antigen of enzyme label with 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000 dilution, 100 holes μ L/ add
Enter in corresponding aperture, each dilution is repeated once, 37 DEG C of effect 60min, and 100 hole μ L/ of tmb substrate buffer, room is added in washing
Temperature is protected from light colour developing 15min, and 100 hole μ L/ terminate liquid (2M H are added2SO4).Absorbance value is detected at wavelength OD450nm, is calculated
The absorbance ratio of positive serum and negative serum selects positive serum OD value (Pos) 1.0~1.6, negative serum OD value
(Neg) diluted concentration of the HRP-E2 of < 0.2 is optimum dilution degree.
1.5.2 titration HRP label PCV2-Cap (HRP-Cap) PPV-VP2 (HRP-VP2), PrV-gE (HRP-gE) and
The working concentration of gB (HRP-gB)
Specific experiment operating procedure and result judgement method are the same as described in " 1.5.1 ".
1.6 HRP-E2, HRP-Cap, the determination of HRP-gB and HRP-gE best effort concentration
According to enzyme-labelled antigen titration experiments as a result, HRP-E2, HRP-Cap, HRP-gB and HRP-gE in Swine serum IgG
Working concentration in the multiple ELISA detection kit of antibody is as shown in table 1:
1 enzyme-labelled antigen titration experiments result of table
| As a result antigen | HRP-E2 | HRP-Cap | HRP-gB | HRP-gE |
| *Pos/OD450 | 1.35 | 1.43 | 1.26 | 1.39 |
| *Neg/OD450 | 0.15 | 0.12 | 0.15 | 0.18 |
| Pos/Neg | 9 | 11.92 | 8.4 | 7.72 |
| Best effort concentration | 1∶4000 | 1∶4000 | 1∶4000 | 1∶2000 |
Remarks: * Pos/OD450With * Neg/OD450Part is not 3 parts of CSFV positive serums and 3 parts of negative serum OD450 each
Average OD under corresponding enzyme-labelled antigen concentration450Value.
The foundation of the 1.7 multiple ELISA detection methods of pig virus infectious disease serum IgG antibody
1.7.1 SPA antigen coat: it is to being measured with 50mmoL/L carbonate buffer solution (pH 9.6) dilution SPA albumen
The hole 4ng/, 100 holes μ L/ are coated with 96 hole elisa Plates, are placed in 4 DEG C overnight, discard liquid, wash, pat dry, close, and washing pats dry.
1.7.2 serum sample is detected: with serum dilution to positive and negative control sera with 100 hole μ L/ of former times concentration,
Positive and negative control serum does multiple holes；Blood serum sample to be checked is added in corresponding aperture with former 100 hole μ L/ of times concentration, sealing, room temperature
Act on 60min.
1.7.3 the decontamination cleaning solution ePBS (phosphate buffer (pH7.2) of mass concentration 1%EDTA, 50mmoL/L is used
100 μ L/ hole room temperatures act on 5min, remove the IgM antibody of non-specific binding, discard liquid in hole, 1.7.4 board-washing.
Remnants ePBS liquid in clear opening is abandoned, is continuously washed 4 times with the hole 200-300 μ L/ 1 × PBST cleaning solution, last time
Liquid in clear opening is abandoned, is patted dry.
1.7.5 HRP- labelled antigen is added
The antibody of detection cause of disease as needed, addition have titrated HRP labelled antigen, 100 holes μ L/, 37 DEG C of effects
60min, washing.(such as: CSFV antibody is detected, HRP-E2 antigen is added in corresponding aperture.To detect 4 kinds of albumen simultaneously
Antibody, the correspondence antigen that HRP label is added in corresponding hole can be realized.)
1.7.6 substrate colour developing is added
Tmb substrate buffer is added in 100 holes μ L/, and room temperature is protected from light colour developing 15min, and terminate liquid is added in 100 holes μ L/.
1.7.7 result judgement
(1) it tests establishment condition: calculating the mean absorbance values (OD450nm) of positive control, each hole of negative control sera,
When positive standard serum is averaged OD450 >=1.0, negative standards' serum OD450≤0.20 is determined as that test result is set up.
(2) result judgement
When sample to be tested OD450/ negative standards' serum OD450 >=2.10, determine the blood serum sample for the anti-of detection cause of disease
Body is positive；
As sample to be tested OD450/ negative standards serum OD450 < 2.10, determine that the blood serum sample is anti-for the cause of disease of detection
Body is negative.
1.8 specific detection
With the clinical common viral infectious of the multiple ELISA detection method pig of pig virus infectious disease serum IgG antibody
Positive serum carries out cross detection, studies the specificity of the detection method.Coated in this ELISA is SPA antigen, can be with
Association reaction occurs for the IgG antibody in pig anteserum sample.Therefore, the purpose of specific detection is to identify the correspondence that HRP is marked to resist
Non-specific binding (i.e. cross reaction) occurs for the former IgG antibody that whether can be generated with the virus of other common pig infection, from
And lead to false positive results, interference experiment result.So the blood serum sample of selection should not contain when carrying out specificity experiments
Carry out special Journal of Sex Research, HRP labelled antigen antibody.Detection method, according to operation sequence described in " 1.7 " into
Row, specific result of study are as shown in table 2.
The specificity experiments of the multiple serum antibody ELISA detection kit of table 2
NegbIndicate that testing result is negative, PosaIndicate that testing result is positive
The repeated experiment of 1.9 multiple ELISA antibody detection methods
3 batches of SPA antigen coat ELISA Plates are prepared, every kind of viral antigen selects 5 parts of antibody positive pig anteserum samples and 5 parts of yin
Property pig anteserum sample carry out batch in and batch between repetitive test, calculate the coefficient of variation, verify the repeatable of ELISA detection method
Property.The result shows that 3 different batches SPA antigen coat ELISA Plates 5 parts of yin and yang attribute serum of detection, variation within batch coefficient < 3.1%,
Interassay coefficient of variation < 2.5%, it was demonstrated that this method has good repeatability.
1.10 clinical sample test experience
50 parts of acquisitions are used for the control experiment of method involved in the present invention from the pig anteserum sample on Gansu pig farm.Acquisition
When peripheral blood, 21 days after being inoculated with hog cholera lapinised virus vaccine (cell source), but never it is inoculated with PCV2 vaccine, PrV vaccine
With PPV vaccine.Result judgement is carried out according to operating method described in " 1.7 " and standard.By with commercially available commercial reagents
Box compares experiment, the positive rate of kit and commercial kit more involved in the present invention.
1.10.1 the multiple ELISA detection method of pig virus infectious disease serum IgG antibody-swine fever virus Serum Antibody Detection
It selects to block ELISA kit (IDEXX using the CSFV serum antibody that more universal IDEXX is produced in China
CSFV Ab kit) as the method for referring to.CSFV Serum Antibody Detection commercial kit and IgG blood involved in the present invention
The clear multiple ELISA detection method comparison result of antibody is shown in Table 3.From comparison result as can be seen that HRP-E2 hog cholera antibody detection side
Method positive rate is 82% (41/50), and negative verification and measurement ratio is 18% (9/50)；Import IDEXX CSFV Ab kit detection examination
Agent box positive detection rate is 68% (34/50), and negative verification and measurement ratio is 32% (16/50).The positive coincidence rate of two kinds of methods is
68.29%, negative match-rate 33.33%, overall coincidence rate reaches 62%.Due to detection swine fever virus used in the present invention
The E2 antigen of antibody comes from CSFV attenuated vaccine, is better than import using the reactivity of the serum sample after attenuated vaccine with field
Detection kit.In the multiple ELISA that the present invention researches and develops, HRP-E2CSFV antibody assay kit positive rate compares import
Be higher by 14%, this result meets the clinical rule of China's hog cholera antibody.In recent years, China took hog cholera vaccine to force always
Immune policy, immunisation coverage nearly reaches 100%, so the positive rate of group's antibody against swine fever virus remains at
80% or more.
3 hog cholera antibody of table detects clinical serum sample detection result
1.10.2 the multiple ELISA detection method-PCV2 Serum Antibody Detection of pig virus infectious disease serum IgG antibody
PCV2 Serum Antibody Detection method (HRP-Cap) of the present invention is compared with indirect immunofluorescence (IF) is tested
Compared with.IF experimental implementation process abstract: PK-15 cell in 6 orifice plates it is long to 75% when, the PCV2 strain that laboratory separates is connect
Kind, it is incubated for 1h, discards virus liquid, sterile PBS washing is three times；MEM cell maintenance medium culture 48h (5%CO is added2, 37 DEG C), it abandons
Remove culture solution；The 4% fixed 15min of paraformaldehyde is added, discards liquid, sterile PBS washing is three times；It is added 0.1%
TritonX-100 permeabilization 10min discards surplus liquid, and sterile PBS washing is three times；By standard PCV2 positive serum, negative serum
1:50 dilution, the hole 1.5ml/ are done with blood serum sample to be detected, serum, 1 × PBST (pH7.2) washing 4 is sucked out in 4 DEG C of overnight incubations
It is secondary；The rabbit-anti pig IgG monoclonal antibody of FITC label is added, 37 DEG C of incubation 5h discard liquid, 1 × PBST (pH7.2) washing 4
It is secondary, inverted fluorescence microscope observation experiment result.
PCV2 serum antibody comparison result is shown in as shown in table 4 in the multiple ELISA detection method of IgG serum antibody.From comparing
As a result as can be seen that HRP-Cap antibody detection method positive rate is 86% (43/50), negative verification and measurement ratio is 14% (7/
50).In IF experiment, the positive detection rate of PCV2 serum antibody is 76% (38/50), and negative verification and measurement ratio is 24% (12/50).Two
The positive coincidence rate of kind method is 81.39%, and negative match-rate 57.14%, overall coincidence rate reaches 78%.Present invention research and development
Multiple ELISA in, HRP-Cap PCV2 antibody assay kit positive rate ratio IF experiment be higher by 10%.Two methods
Comparison result explanation, ELISA method ratio IF experimental sensitivity is high, and operating process, consuming time and experimental expenses are remote low
It is tested in IF, therefore, ELISA method is clinically to detect the most common method of PCV2 serum antibody.
Table 4:PCV2 detects clinical serum sample detection result
Although the never inoculated PCV2 vaccine in the pig farm, PCV2 antibody positive rate can reach 81.39% in 50 parts of serum,
Illustrate that pig farm PCV2 persistent infection situation is commonplace, but entire swinery does not observe clinic caused by doubtful PCV2 infection
Symptom, piglet survival ratio are steady always.It is found according to massive epidemiology investigation result, PCV2 persistent infection phenomenon is almost
Throughout China or even global all pig farms, effective ways assessment PCV2 persistent infection there is no to lose journey caused by swinery
Degree also lacks effective means and purifies PCV2 swinery.Preventing PCV2 infection is a great problem that world's pig breeding industry faces.
1.10.3 the multiple ELISA detection method of pig virus infectious disease serum IgG antibody --- porcine pseudorabies virus HRP-gE
With HRP-gB Serum Antibody Detection
PrV-gE and gB Serum Antibody Detection kit is purchased from Wuhan Ke Qian biological products Co., Ltd, and method is indirect
ELISA (iELISA:iELISA-gE, iELISA-gB).The comparison result of three kinds of kits is as shown in table 5.
Swinery of 50 parts of blood serum samples from never immune PrV vaccine, which never occurred pseudo- mad dog epidemic situation.At this
The antibody of the anti-mad dog E protein of puppet is 3 positive pigs in experiment, shows as health, does not observe any clinical symptoms.Foundation
This experimental result illustrates that the positive pig for picking up from gE and gB antibody once infected Pseudorabies virus, rehabilitation at present.Institute of the present invention
The total coincidence rate of iELISA-gE and iELISA-gB for the HRP-gE and HRP-gB Serum Antibody Detection method and commercialization being related to reaches
To 92%, illustrate that this method and commercialization have similar sensibility and specificity.
Table 5:PrV gE and gB detect clinical serum sample detection result
The antibody of anti-PrV gE albumen is 3 positive pigs in this experiment, shows as health, does not observe any face
Bed symptom.According to this experimental result, illustrate that the positive pig for picking up from gE and gB antibody once infected Pseudorabies virus, at present health
It is multiple.The iELISA-gE and iELISA-gB of HRP-gE and HRP-gB Serum Antibody Detection method according to the present invention and commercialization
Total coincidence rate reaches 92%, illustrates that this method and commercialization have similar sensibility and specificity.
Claims (7)
1. a kind of method detected with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody, the side
Method is non-disease diagnostic method, it is characterised in that: uses the multiple ELISA detection reagent of pig virus infectious disease serum IgG antibody
Box, the yin of detection plate, serum samples diluted liquid, cleaning solution, developing solution, terminate liquid including pre-coated SPA albumen, four kinds of cause of diseases
The E2 antigen for the CSFV that positive criteria control serum and HRP are marked respectively, the Cap antigen of PCV2, the gB and gE of PrV are anti-
It is former；
The E2 antigen of CSFV of HRP label, the Cap antigen of PCV2, PrV the gE antigen of gB and PrV distinguished using concentration
It is 1: 4000,1: 4000,1: 4000,1: 2000, corresponding mass concentration is 0.1 μ g/mL-15 μ g/mL；
Specifically includes the following steps:
(1) serum sample is added: serum sample to be detected, yin and yang attribute standard control serum are added with 100 hole μ L/ of former times concentration
Enter in corresponding aperture, yin and yang attribute standard control serum does multiple holes；Sealing, room temperature act on 60min；
(2) remove nonspecific reaction: the phosphate with decontamination cleaning solution and 50 mmoL/L that mass concentration is 1% EDTA is slow
Fliud flushing acts on 5 min with 100 μ L/ hole room temperatures, discards liquid in hole, and the pH value of phosphate buffer is 7.2；
(3) board-washing: remnants EDTA and PBS liquid in clear opening is abandoned, continuously washs 4 with 1 × PBST cleaning solution in the hole 200-300 μ L/
Secondary, last time abandons liquid in clear opening, pats dry；
(4) HRP- labelled antigen is added: the antibody of detection cause of disease, addition have titrated HRP labelled antigen, 100 μ L/ as needed
Hole, 37 DEG C of effect 1h, washing；
(5) substrate colour developing is added: tmb substrate buffer is added with the dosage in 100 holes μ L/, room temperature is protected from light 15 min of colour developing, with
Terminate liquid is added in the dosage in 100 holes μ L/；
(6) result judgement:
It tests establishment condition: calculating the mean absorbance values OD450nm of positive control, each hole of negative standards' control serum, work as sun
Property standard serum be averaged OD450 >=1.0, negative standards' serum OD450≤0.20 is determined as that test result is set up；
Determine: when sample to be tested OD450/ negative standards' serum OD450 >=2.10, determining the blood serum sample for the anti-of detection cause of disease
Body is positive；As sample to be tested OD450/ negative standards serum OD450 < 2.10, determine the blood serum sample for the pathogenic autoantibody of detection
It is negative.
2. according to claim 1 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: tmb substrate buffer group used becomes, substrate solution A:TMB DMSO solution, concentration 2-
6mg/mL；Substrate solution B:15-30g/L sodium acetate, citric acid 1-9g/L, 30% hydrogen peroxide 0.1-1ml, distilled water are dissolved to 1L.
3. according to claim 1 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: the package amount of the be coated with SPA albumen of detection plate of the pre-coated SPA albumen be 2ng~
The hole 28ng/, the carbonate buffer solution that the pH value that coating dilution is 50 mmol/L is 9.6.
4. according to claim 3 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: the composition of every milliliter of coating carbonate buffer solution in the detection plate of the pre-coated SPA albumen
For 1.59mg Na2CO3With 2.93mg NaHCO3, deionized water is stored in after completely dissolution at a temperature of 4 DEG C.
5. according to claim 4 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: the confining liquid group used in the detection plate of the pre-coated SPA albumen is divided into mass concentration for 1-
5% trehalose, mass concentration 0.5-2.0% gelatin, volumetric concentration 1-8% horse serum, 7.3,200 hole μ l/ 1 × PBST, pH.
6. according to claim 5 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: the cleaning solution is that the pH value of the 0.1mol/L for the Tween-20 that volumetric concentration is 0.5% is 7.2
Phosphate buffer.
7. according to claim 6 examined with the multiple ELISA detection kit of pig virus infectious disease serum IgG antibody
The method of survey, it is characterised in that: the terminate liquid is 1.5mol/L H2SO4。
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| CN103033614A (en) * | 2012-12-19 | 2013-04-10 | 北京伟嘉人生物技术有限公司 | ELISA kit for porcine circovirus type 2 antibody detection |
| CN104634961A (en) * | 2013-11-07 | 2015-05-20 | 珠海出入境检验检疫局检验检疫技术中心 | Preparation method and detection method of rabies virus homogeneous fluorescent composite microsphere |
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