CN108931644A - A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection - Google Patents
A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection Download PDFInfo
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- CN108931644A CN108931644A CN201810797244.5A CN201810797244A CN108931644A CN 108931644 A CN108931644 A CN 108931644A CN 201810797244 A CN201810797244 A CN 201810797244A CN 108931644 A CN108931644 A CN 108931644A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a kind of evaluations of foot and mouth disease virus immune antiboidy and infection to diagnose bigeminy test strips with Immune dctection, the detection line 1 that the test strips are printed including the use of FMDV VP1 epitope polypeptide artificial antigen, and the detection line 2 using the printing of foot and mouth disease virus non-structural protein epitope polypeptide artificial antigen.Artificial antigen prepared by the present invention, which has, to be easy to get, stable structure, purity is at low cost up to 99%, can rapid, high volume production the advantages that, it is low to solve tradition expression expressing quantity, it is difficult to ensure natural space structure, renaturation is difficult, it is difficult to remove mycoprotein, the disadvantages of influencing the specificity of testing result, also solve the risk incomplete there are inactivation of virus using inactivation of viruses.The problems such as present invention also pre-processes gold-labelled pad, is more advantageous to the release of gold-labelled pad water suction and gold mark albumen, and it is slow and incomplete effectively to solve the mark protein delivery of gold existing for existing test paper, influences test paper product testing accuracy, sensitivity and shelf-life.
Description
Technical field
The present invention relates to a kind of evaluations of foot and mouth disease virus immune antiboidy and infection to diagnose bigeminy test strips with Immune dctection, belongs to
In technical field of immunoassay.
Background technique
Aftosa is to cause a kind of strong infectiousness epidemic disease disease artiodactylous by foot and mouth disease virus.Foot and mouth disease virus has
The extremely strong property of polymorphism, mutability, host's popularity, contagiousness, so once morbidity is i.e. in popular, great outburst.It is more tight
Weight, aftosa is a kind of deadly infectious disease, according to there is extremely strong infectiousness, for the same group of affected animal and contact cause of disease
Animal, it is necessary to be strictly isolated, be blocked, forbid animal movement and livestock products listing etc., therefore, Infected regions is caused even to be sent out
The livestock products foreign trade of sick country stops, and causes huge economic loss.
The main policies of developing country's control aftosa are vaccine immunities, and it is dynamic that animal population vaccine immunity is immunized in aftosa
The formulation of object immune effect, maternal antibody and immune programme requires to rely on antibody test.Antibodies against foot-and-mouth disease virus water at present
The serological method of flat detection is mainly Liquid-phase blocking ELISA (Liquid phase blocking ELISA, LPBE) and resists
Body test strip.Antigen used in these methods is mainly inactivation of viruses or expression albumen, and there are viruses to go out for inactivation of viruses
The problem of incomplete, purification difficult living；Expressing albumen, there are this expressing quantities that low, mycoprotein is difficult to the problems such as removing.Antibody
Test strip has quick, easy, low-cost advantage, and gold-labelled pad is the important component of test paper, it determines gold
The activity and release property of albumen are marked, therefore very big to the influential effect of colloidal gold immune chromatography test.Currently, the preparation of gold-labelled pad
Method is albumen/antibody of direct spraying or immersion colloid gold label on cellucotton, or cellucotton is impregnated using treatment fluid
After processing, then spray or impregnate the albumen of colloid gold label.But it finds in use, because of gold-labelled pad processing method and processing
Formula of liquid is improper, it is often slow in the presence of gold mark protein delivery and not exclusively, to influence test paper product testing accuracy, sensitivity
And the problems such as shelf-life.
Summary of the invention
In order to overcome in the prior art expression expressing quantity it is low, it is difficult to guarantee natural space structure, renaturation is difficult, bacterium
Body protein influences the specificity and inactivation of viruses the disadvantages of there are inactivation of virus infull risks and existing examination of testing result
The problems such as mark protein delivery of gold existing for paper is slow and incomplete, influences test paper product testing accuracy, sensitivity and shelf-life, this
Invention combine existing aftosa detection ELISA method and test strips advantage and disadvantage, using polypeptide epitope preparation artificial antigen and
Gold-labelled pad processing method invents a kind of evaluation of foot and mouth disease virus immune antiboidy and infection with Immune dctection and diagnoses bigeminy test strips.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection, including the use of mouth hoof
The detection line 1 of epidemic disease virus VP 1 epitope polypeptide artificial antigen printing, and it is artificial using foot and mouth disease virus non-structural protein epitope polypeptide
The detection line 2 of antigen printing.
VP1 epitope polypeptide artificial antigen is the 142nd~158 amino acids polypeptide on artificial synthesized FMDV VP1, and
The artificial antigen that coupling carrier albumen is formed；The 142nd~158 amino acids polypeptide is foot and mouth disease virus on FMDV VP1
Upper 142nd~158 amino acids sequence NNVRGDLQVLAQKAERA of O/GX/09-7 strain VP1；Foot and mouth disease virus O/HN/CHA/
Upper 142nd~158 amino acids sequence SNVRGDLQVLAQKAERA of 93 strain VP1；Foot and mouth disease virus O/TAW/97 strain VP1
Upper 142nd~158 amino acids sequence NNVRGDLQVLAQKAERT；Foot and mouth disease virus O/MYA/98 strain VP1 the upper 142nd~
At least one of 158 amino acids sequence TNVRGDLQVLAQKAARP.
Preferably, foot and mouth disease virus O/GX/09-7 strain, O/HN/CHA/93 strain, O/TAW/97 strain, O/MYA/98
Strain VP1 upper 142nd~158 amino acids polypeptide 1:1:1:1 in mass ratio mixing.
Non-structural protein epitope polypeptide artificial antigen be artificial synthesized foot and mouth disease virus NS2 Protein B the upper 1096th~
1106 amino acids RTPEDLERAEK, 2C upper 1414th~1425 amino acids HEKVSSHPIFKQ, 3B the upper 1602nd~1613
At least one of amino acids GPYAGPMERQKP, and in its-NH4A cysteine, then coupling carrier albumen shape is added in end
At artificial antigen.
Preferably, upper 1096th~1106 amino acids polypeptide preparation of foot and mouth disease virus NS2 Protein B is artificial anti-
Original, the artificial antigen of upper 1414th~1425 amino acids polypeptide preparation of 2C, upper 1602nd~1613 amino acids polypeptide system of 3B
The mixing of standby artificial antigen, in mass ratio 1:1:1.
Test strips further include utilizing the control line for resisting two anti-igg of animal species to be checked to print.
Test strips include sample pad, gold-labelled pad, absorption pad and bottom plate；Detection line and control line are printed on nitrocellulose filter
Pad.
Gold-labelled pad is pre-processed, treatment fluid is sprayed on fiber mat, it is dry, obtain pretreated gold-labelled pad.
Treatment fluid is made of following raw material: BSA5%, PVP-10 0.1%, Triton X-100 0.1%, surplus are
The Na of 0.02mol/L2B4O7·10H2O solution；Spraying method are as follows: along the center spray treatment liquid of fiber mat length direction, and spray
There is gap between treatment fluid both sides of the edge line after painting on fiber mat and the lateral edge of fiber mat respective side.
Dry condition are as follows: 37-42 DEG C, 10-70min.
The invention has the advantages that:
1, detection line antigen used in test paper of the present invention uses the epitope polypeptide of synthesis, artificial anti-after coupling carrier albumen
Original has and is easy to get, and stable structure, purity is at low cost up to 99%, can rapid, high volume production the advantages that, it is this artificial anti-
It is low that former use solves tradition expression expressing quantity, it is difficult to guarantee natural space structure, renaturation is difficult, it is difficult to remove bacterium
Body protein, also solves the risk incomplete there are inactivation of virus using inactivation of viruses at the disadvantages of influencing the specificity of testing result.
2, in the test paper mode of multi-joint antibody test, gold mark albumen can only select can be in conjunction with the albumen of mammal IgG
(such as SPA) or antiantibody.In delivery system of the invention, control line is two anti-igg for resisting animal species to be detected, secondary antibody
IgG can ensure that control line develops the color in conjunction with gold mark thing and the IgG- gold mark thing for combining not tested survey line antigen protein to intercept
Stability.
3, be formulated in gold-labelled pad treatment fluid of the present invention it is simple, without sugar, be more advantageous to gold-labelled pad water suction and gold mark albumen
Release.Wherein, BSA and PVP-10 can close cellucotton and NC film, while protect the stability of gold mark albumen；Na2B4O7·
10H2O can provide alkali ion environment, more conducively antigen-antibody reaction and the release of gold mark albumen；Triton X-100 is used for
The processing of gold-labelled pad can accelerate the release of gold mark albumen, and can reduce the non-specificity of product.Treatment fluid selection of the present invention
The reason of not sugaring: sugar is easy to crystallize during product storage, and the release that will affect gold mark albumen and gold particle are in film
On movement speed, to influence the shelf life and detection accuracy of product.
4, method disclosed by the invention is different from the existing method for impregnating gold-labelled pad in treatment fluid, but direct spraying
On glass fibre cotton, the mode of spraying is selected to substitute dipping pretreatment gold-labelled pad, can be effectively improved and impregnate gold-labelled pad generation
Edge effect；The mode of spraying can make treatment fluid in local fixation, and gold-labelled pad rest part can be allowed to keep fluffy state, increase
Add the water imbibition and adhesiveness of gold-labelled pad, applicability is more extensive；The mode of spraying is fixed on one after can making gold mark albumen spraying
Determine to spread in range without large area, be more advantageous to the stabilization and release of gold mark albumen, greatly improves colloidal gold immunochromatographimethod examination
The detection sensitivity and stability of paper.
5, easy to operate, quick: using in test strips detection process of the present invention without adding any other instrument and reagent,
As long as its test lead is inserted into 30s or so in serum to be checked, then testing result can determine that in 5min or so.
6, reduce investment and reduce testing cost: using Rapid detection test strip of the present invention, do not need separately to match Other Instruments,
Equipment and reagent save big measuring appratus, equipment and additive reagent expense；One test paper once both can detecte aftosa and group be immunized
The antibody level of body, and can detecte infection of foot-and-mouth disease or with malicious animal, and profession and layman can be whenever and wherever possible
Real-time online detection is carried out, without paying expert diagnosis Laboratory Fee and its correlative charges.Therefore, testing cost can be saved, is dropped
Low testing cost.
7, have a wide range of application, convenient for popularization and use: Rapid detection test strip of the present invention is easy to operate at " single step " or " stupid
Melon formula ", and be convenient for carrying and save, it is able to satisfy the needs of different levels personnel, including professional chemical examination, customs quarantine control, health
Epidemic prevention, quality-monitoring, livestock products processing, intensive culture to individual cultivation etc., have a vast market foreground and biggish warp
Ji, social benefit.
Detailed description of the invention
Below in conjunction with attached drawing, specific embodiments of the present invention will be described in further detail.
Fig. 1: the evaluation of foot and mouth disease virus immune antiboidy and infection and Immune dctection diagnosis bigeminy test strips specific assay knot
Fruit.
Fig. 2: the evaluation of foot and mouth disease virus immune antiboidy and infection and Immune dctection diagnosis bigeminy test strips sensitivity test knot
Fruit.
Fig. 3: the effect of the processed gold-labelled pad of embodiment 1 and not pretreated gold-labelled pad spraying gold mark albumen.
Fig. 4: the gold-labelled pad situation of control methods processing.
Fig. 5: the service condition of embodiment 2, the gold-labelled pad of control methods processing.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.Unless otherwise specified, originally
" % " in invention specific embodiment is quality percent by volume (g/mL).
Embodiment 1
It prepares the evaluation of foot and mouth disease virus immune antiboidy and infection and diagnoses bigeminy test strips with Immune dctection: first according to aftosa
B cell epitope, artificial synthesized foot and mouth disease virus structure egg on B cell epitope and foot and mouth disease virus non-structural protein on virus VP 1
Epitope polypeptide and upper 3 epitope polypeptides of foot and mouth disease virus NS2 Protein B, 2C, 3B on white VP1, difference coupling carrier albumen,
Detection line 1 (T1), the detection line 2 (T2) being used to prepare on cellulose membrane pad, while preparation resists the secondary antibody of animal species to be checked
IgG, the control line (C) being used to prepare on cellulose membrane pad；Gold-labelled pad treatment fluid and gold-labelled pad pretreatment are prepared, for spraying gold
Mark albumen；Finally assemble test strips.
1, the synthesis of Foot-and-mouth disease and non-structural protein epitope polypeptide:
Synthesizing the 142nd~158 amino acids sequence on FMDV VP1, (amino acid sequence is according to examined aftosa poison
Strain is different and different), in its-NH4A cysteine is added in end；Synthesis foot and mouth disease virus NS2 Protein B the upper 1096th~
1106 amino acids (amino acid sequence are as follows: RTPEDLERAEK), 2C the upper 1414th~1425 be amino acid (amino acid sequence are as follows:
HEKVSSHPIFKQ), upper 1602nd~1613 amino acids sequence of 3B (amino acid sequence are as follows: GPYAGPMERQKP), exists respectively
Its-NH4A cysteine is added in end, and all polypeptide sequences are synthesized and purified by commercial company.
2, the preparation of Foot-and-mouth disease and non-structural protein epitope polypeptide artificial antigen:
With Heterobifunctional reagents Sulfo-SMCC (MW:436.37, Spacer Arm Length:Pierce)
- NH on activated carrier protein B SA (or OVA)2, specific steps are as follows:
(1) coupling buffer (50mL): 0.15M NaCl, 0.1M PB buffer (pH 7.2), 1 μM of EDTA (ethylenediamine
Tetraacethyl)；
(2) BSA (bovine serum albumin(BSA)) solution: the carrier protein BSA for weighing 8mg is dissolved in 1.0mL coupling buffer；
(3) it Sulfo-SMCC solution: weighs 2mg Sulfo-SMCC and is added in the DMSO (dimethyl sulfoxide) of 100 μ L, instead
Multiple piping and druming, dissolves it sufficiently；
(4) BSA solution and Sulfo-SMCC solution are mixed, after mixing well, at room temperature, react 1h or 37 DEG C,
30min, and mixing frequently；
(5) dialysis 48h is carried out to the solution of step (4) under the conditions of 4 DEG C with coupling buffer, every 6h is changed the liquid once, gone
Except extra coupling agent (Sulfo-SMCC) and DMSO；
(6) with coupling buffer adjustment carrier protein BSA concentration to 5mg/mL, which is SMCC activated carrier albumen
(SMCC-BSA), -20 DEG C freeze it is spare.
- NH on carrier protein2It is connected after activated with the-SH of polypeptide (Pep) N-terminal cysteine (Cys), forms people
Work combination antigen (BSA-Pep).Coupling step are as follows: weigh 4mg polypeptide, with the PBS buffer solution of 0.01M after completely dissolution (for
Dissolubility relatively low polypeptide DMF or DMSO dissolves)；300 μ L 0.01M PB buffers are added, and (pH 7.2 contains 5mM
EDTA), polypeptide storing liquid, concentration 10mg/mL are prepared.When coupling, 20 μ L polypeptide storing liquids are taken, is added and contains 5mM in equal volume
In the 0.01M PB buffer (pH 7.2) of EDTA, adds 40 μ L SMCC-BSA solution and be sufficiently mixed, after reacting at room temperature 4h, 4
DEG C be incubated overnight.Dialysis 48h is carried out to above-mentioned solution under the conditions of 4 DEG C with physiological saline, every 6h is changed the liquid once, and it is extra to remove
Coupling agent；Its protein concentration is measured with ultraviolet specrophotometer.
3, goat-anti or rabbit-anti are detected the preparation of the secondary antibody of animal species IgG:
With the 50 μ g IgG/kg of μ g~100 weight subcutaneously with intramuscular injection negative healthy sheep or rabbit 3~4 times, last is exempted from
Venous blood collection after epidemic disease 20 days measures its serum antibody titer in 1:2000 or more, Culling heart blood or arteria carotis bloodletting with ELISA,
Collect its hyper-immune serum.It takes 1 volume of serum that 2 volume PBS buffer solution (pH 7.2) is added to mix, adds isometric saturated ammonium sulfate liquid mixed
It is even, 2h in 4 DEG C of refrigerators is set, 15min is centrifuged in 4 DEG C, 10000r/min, abandons supernatant；It is molten with appropriate PBS buffer solution (pH7.2)
Solution precipitating, adding saturated ammonium sulfate liquid to its ultimate density is 33%, 2h in 4 DEG C of refrigerators is set, under the conditions of 4 DEG C, 10000r/min
It is centrifuged 15min, abandons supernatant, is dissolved and is precipitated with a small amount of PBS buffer solution (pH7.2), set in 4 DEG C of refrigerators and use PBS buffer solution
(pH7.2) it is dialyzed overnight, changes liquid 2~3 times, 15min is centrifuged under the conditions of 4 DEG C, 10000r/min, supernatant is collected, with ultraviolet
Its protein concentration of spectrophotometric determination.
4, the preparation of gold mark albumen:
Aurosol is prepared with reduction of sodium citrate method: i.e. in 0.01~0.05% chlorine of mass fraction of 50~100mL boiling
2~4mL mass fraction, 0.5~2% citric acid three sodium solution is added in auric acid aqueous solution, obtains the colloid of diameter 15nm or so
Gold.With the K of 0.1mol/L2CO3Colloidal gold pH to 8.5~9.5 is adjusted, with the label of 1:1000~1300 than by albumen to be marked
SPA is added in the colloidal gold of pH8.5~9.5, after marking 10min, adds the mass fraction 20%PEG-10000 to be to its ultimate density
0.05%, 4 DEG C, 1500~3000r/min centrifugation 20min remove unbonded colloid gold particle, 4 DEG C, 15000r/min centrifugation
1h abandons supernatant, obtains colloid gold label albumen.
5, the preparation of gold-labelled pad:
Gold-labelled pad preprocess method: treatment fluid is sprayed on fiber mat with the amount of 8 μ L/cm using spraying apparatus, 42 DEG C
50min is dried, pretreated gold-labelled pad is obtained.Gold-labelled pad treatment fluid is made of following raw material: BSA 5%, PVP-10 0.1%,
Triton X-100 0.1%, surplus are the Na of 0.02mol/L2B4O7·10H2O solution.Spraying method are as follows: along fiber mat length
The center spray treatment liquid in direction, and after spraying the lateral edge of the treatment fluid both sides of the edge line on fiber mat and fiber mat respective side it
Between there is gap.Then the colloid gold label albumen of preparation is uniformly sprayed in gold-labelled pad, 42 DEG C of baking 40min.
6, the assembling of test strips
It is sprayed on Foot-and-mouth disease epitope polypeptide artificial antigen on nitrocellulose filter, forms detection print
Foot and mouth disease virus non-structural protein epitope polypeptide artificial antigen is sprayed on nitrocellulose filter by mark/line 1 (T1), forms inspection
It surveys trace/line 2 (T2), the secondary antibody that goat-anti or rabbit-anti are detected animal species IgG is sprayed on nitrocellulose filter, formation pair
According to trace/line (C)；Sample pad, gold-labelled pad, cellulose membrane pad, absorption pad are successively pasted on bottom plate, suitable dimension is cut into
Obtain test paper.
It is printed with red samples mark line in sample pad surface layer corresponding with gold-labelled pad intersection, and is printed on max printed words,
The label alerts at the side 1.1-1.2cm of linear distance sample pad top.
The principle of Rapid detection test strip examinations of the present invention:
After Rapid detection test strip test lead of the present invention is inserted into serum to be detected, serum to be checked is driven to be checked by siphon
Gold mark albumen in serum antibody and gold mark albumen mineral wool eventually penetrates handle end filter together to nitrocellulose membrane diffusion
In paper, antibody can be combined with gold mark albumen in diffusion process, which detects with the artificial antigen on cellulose membrane in turn
Trace combines, to show the detection trace (T1 and/or T2) of brownish red；And control line trace can be in conjunction with gold mark thing, shape
Trace (C) is compareed at brownish red.If not having antibodies against foot-and-mouth disease virus and/or vaccine antibody in serum to be checked, test strips are only shown
One (a) brownish red control trace (C) is shown；If anti-containing Anti-FMDV antibody and/or vaccine in serum to be checked
Body is then first marked protein binding with its gold, then in conjunction with artificial antigen detection trace T1 and/or T2 on cellulose membrane, is shown
The detection trace of brownish red is positive mark；If there is no any brownish red trace to show on cellulose membrane, show test strips
It has failed or operation error.
The detection operating method of test strips:
(1) preparation of test sample liquid: the sterile blood for taking tested animal, and serum is separated, 1:200 is made with physiological saline
Times dilute serum is to be measured；Rapid detection test strip test lead of the present invention is inserted into serum to be detected, insertion depth is no more than mark
Remember line, take out test strips after about 30s, is horizontally arranged about 1~5min, while observing result.
(2) result judges: if only showing (a) brownish red control print in test strip cellulose film layer
Mark (C) indicates that testing result is negative, and illustrates not detecting antibodies against foot-and-mouth disease virus and/or aftosa epidemic disease in tested serum
Seedling antibody, i.e., tested animal are immune without mouth disease virus infection and/or aftosa vaccine；If the cellulose in test strip
Occur the control trace (C) of brownish red and detection trace 1 (T1) in film layer, indicates that testing result is positive, i.e., in serum to be checked
In detect aftosa vaccine antibody, i.e., it is immune that tested animal has carried out aftosa vaccine；If detecting trace T1, T2 to go out simultaneously
Existing, expression detects mouth disease virus infection antibody in serum to be checked, i.e., tested animal has infected or carried foot and mouth disease virus；
If there is no any brownish red trace to show on cellulose membrane band, show that test strips have failed or operated wrong.
Embodiment 2
The evaluation of foot and mouth disease virus immune antiboidy and infection and the preparation method of Immune dctection diagnosis bigeminy test strips are such as implemented
Example 1 sprays upper 142nd~158 amino acids sequence of foot and mouth disease virus O/GX/09-7 strain VP1 in detection line 1
NNVRGDLQVLAQKAERA (SEQ ID NO.1), its end-NH4 be added a cysteine, coupling carrier protein B SA or
The artificial antigen of OVA preparation；Foot and mouth disease virus non-structural protein artificial antigen is sprayed in detection line 2, which selects simultaneously
With upper 1096th~1106 amino acids (amino acid sequence are as follows: RTPEDLERAEK, SEQ of foot and mouth disease virus NS2 Protein B
ID NO.5) polypeptide, the 1414th~1425 amino acids (amino acid sequence are as follows: HEKVSSHPIFKQ, SEQ on Nonstructural protein 2C
ID NO.6) polypeptide, upper 1602nd~1613 amino acids (amino acid sequence are as follows: GPYAGPMERQKP, SEQ of non-structural protein 3B
ID NO.7) polypeptide, the artificial antigen of coupling carrier protein B SA or OVA preparation, three kinds of artificial antigen 1:1:1 in mass ratio are mixed
It closes.
Embodiment 3
The evaluation of foot and mouth disease virus immune antiboidy and infection and the preparation method of Immune dctection diagnosis bigeminy test strips are such as implemented
Example 2, difference be, in detection line 1 artificial antigen polypeptide be foot and mouth disease virus O/HN/CHA/93 strain VP1 the upper 142nd~
158 amino acids sequence SNVRGDLQVLAQKAERA (SEQ ID NO.2).
Embodiment 4
The evaluation of foot and mouth disease virus immune antiboidy and infection and the preparation method of Immune dctection diagnosis bigeminy test strips are such as implemented
Example 2, difference are that artificial antigen polypeptide is foot and mouth disease virus O/TAW/97 strain VP1 upper 142nd~158 in detection line 1
Amino acid sequence NNVRGDLQVLAQKAERT (SEQ ID NO.3).
Embodiment 5
The evaluation of foot and mouth disease virus immune antiboidy and infection and the preparation method of Immune dctection diagnosis bigeminy test strips are such as implemented
Example 2, difference are that artificial antigen polypeptide is foot and mouth disease virus O/MYA/98 strain VP1 upper 142nd~158 in detection line 1
Amino acid sequence TNVRGDLQVLAQKAARP (SEQ ID NO.4).
Verify example
1, specific detection
Test paper detection infection of foot-and-mouth disease serum, aftosa immune serum, aftosa negative serum and swine fever of the invention
Virus, PRRS virus, pig circular ring virus, porcine pseudorabies virus, Latex agglutination test positive serum, the results show that this
The specificity of invention test paper is 100%, with aftosa negative serum and other virus-positive serum no cross reactions (such as Fig. 1).
2, sensitivity technique
The standard Schweineseuche immune serum of doubling dilution is detected with test paper of the invention, as the result is shown test strips of the present invention
1:3200 and 1:6400 (such as Fig. 2) are not less than respectively to the sensitivity of infection of foot-and-mouth disease and immune standard positive serum.
Comparative experiments
1, gold mark albumen (spraying position and treatment fluid spraying position phase are sprayed in the processed gold-labelled pad of embodiment 1
Together), gold mark albumen is fixed in gold-labelled pad at a line of aggregation；And gold mark albumen is sprayed in not pretreated gold-labelled pad
(spraying position is identical as processed gold-labelled pad position), gold mark albumen are saturated with entire gold-labelled pad at disperse shape, illustrate the present invention
The gold-labelled pad of processing can allow the proteopexy of gold mark to be more advantageous to the stabilization and release (Fig. 3) of gold mark albumen in a certain range.
2, as a comparison with the preprocess method of other gold-labelled pads, compared with the pretreating effect of gold-labelled pad of the present invention
Compared with.The preprocess method of comparison gold-labelled pad is that fiber mat is immersed in 15min in treatment fluid, then takes out and dries at 37 DEG C, obtains
To pretreated gold-labelled pad.Treatment fluid is made of following raw material: BSA 3%, trehalose 8%, Tween-20 2%, and surplus is
0.01mol/L phosphate buffer.As a result it known to (Fig. 4), is unevenly distributed after the gold-labelled pad of control methods processing is dry, edge
There is processed material precipitating jaundice, and quality is harder, can not adhere on the supporting plate, cut because containing Tween-20 in treatment fluid
When be easy to happen gold-labelled pad obscission, this disadvantage can be improved using the method for spray treatment.
3, the SPA of colloid gold label is sprayed in embodiment 2 and the pretreated gold-labelled pad of control methods, and test paper inspection is made
Infection of foot-and-mouth disease serum is surveyed, is observed after 10min.Gold mark protein delivery rate is 95% or more in 2 gold-labelled pad of embodiment, control methods
Gold-labelled pad on gold mark SPA do not discharge completely, and test strips colour developing be weaker than embodiment 2 (Fig. 5).
The foregoing is merely the optimal embodiments of the present invention, and for those skilled in the art, the present invention can have
Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all
It is included within protection scope of the present invention.
Sequence table
<110>Henan Academy of Agricultural Sciences
<120>a kind of foot and mouth disease virus immune antiboidy evaluation and infection and Immune dctection diagnosis bigeminy test strips
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 1
Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Ala
<210> 2
<211> 17
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 2
Ser Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Ala
<210> 3
<211> 17
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 3
Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Thr
<210> 4
<211> 17
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 4
Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg
1 5 10 15
Pro
<210> 5
<211> 11
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 5
Arg Thr Pro Glu Asp Leu Glu Arg Ala Glu Lys
1 5 10
<210> 6
<211> 12
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 6
His Glu Lys Val Ser Ser His Pro Ile Phe Lys Gln
1 5 10
<210> 7
<211> 12
<212> PRT
<213>foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 7
Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln Lys Pro
1 5 10
Claims (10)
1. a kind of foot and mouth disease virus immune antiboidy evaluation and infection diagnose bigeminy test strips with Immune dctection, which is characterized in that institute
The detection line 1 that test strips are printed including the use of FMDV VP1 epitope polypeptide artificial antigen is stated, and non-using foot and mouth disease virus
The detection line 2 of structural proteins epitope polypeptide artificial antigen printing.
2. test strips according to claim 1, which is characterized in that VP1 epitope polypeptide artificial antigen is artificial synthesized mouth hoof
142nd~158 amino acids polypeptide on epidemic disease virus VP 1, and the artificial antigen that coupling carrier albumen is formed；FMDV VP1
Upper 142nd~158 amino acids polypeptide is upper 142nd~158 amino acids sequence of foot and mouth disease virus O/GX/09-7 strain VP1
NNVRGDLQVLAQKAERA；Upper 142nd~158 amino acids sequence of foot and mouth disease virus O/HN/CHA/93 strain VP1
SNVRGDLQVLAQKAERA；Upper 142nd~158 amino acids sequence of foot and mouth disease virus O/TAW/97 strain VP1
NNVRGDLQVLAQKAERT；Upper 142nd~158 amino acids sequence of foot and mouth disease virus O/MYA/98 strain VP1
At least one of TNVRGDLQVLAQKAARP.
3. test strips according to claim 2, which is characterized in that preferred, foot and mouth disease virus O/GX/09-7 strain, O/
Upper 142nd~158 amino acids polypeptide in mass ratio 1 of HN/CHA/93 strain, O/TAW/97 strain, O/MYA/98 strain VP1:
1:1:1 mixing.
4. test strips according to claim 1, which is characterized in that non-structural protein epitope polypeptide artificial antigen is artificial closes
At upper 1096th~1106 amino acids RTPEDLERAEK of foot and mouth disease virus NS2 Protein B, 2C upper 1414th~1425
At least one of upper 1602nd~1613 amino acids GPYAGPMERQKP of amino acid HEKVSSHPIFKQ, 3B, and in its-NH4
A cysteine, then the artificial antigen that coupling carrier albumen is formed is added in end.
5. test strips according to claim 4, which is characterized in that preferred, foot and mouth disease virus NS2 Protein B upper the
The artificial antigen of 1096~1106 amino acids polypeptides preparation, upper the artificial of 1414th~1425 amino acids polypeptide preparation of 2C resist
Original, the artificial antigen of upper 1602nd~1613 amino acids polypeptide preparation of 3B, the mixing of 1:1:1 in mass ratio.
6. test strips according to claim 1, which is characterized in that further include utilizing two anti-igg for resisting animal species to be checked
The control line of printing.
7. test strips according to claim 1, which is characterized in that the test strips further include sample pad, gold-labelled pad, absorption
Pad and bottom plate；Detection line and control line are printed on nitrocellulose filter pad.
8. test strips according to claim 7, which is characterized in that pre-processed to gold-labelled pad, treatment fluid is sprayed on
It is dry on fiber mat, obtain pretreated gold-labelled pad.
9. test strips according to claim 7, which is characterized in that treatment fluid is made of following raw material: BSA5%, PVP-
100.1%, Triton X-100 0.1%, surplus are the Na of 0.02mol/L2B4O7·10H2O solution；Spraying method are as follows: along fibre
The center spray treatment liquid of dimension pad length direction, and treatment fluid both sides of the edge line and fiber mat respective side after spraying on fiber mat
Lateral edge between there is gap.
10. test strips according to claim 8, which is characterized in that dry condition are as follows: 37-42 DEG C, 10-70min.
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| CN201810797244.5A CN108931644B (en) | 2018-07-19 | 2018-07-19 | Foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip |
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