CN108931644A - A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection - Google Patents

Abstract

The invention discloses a foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip. Protein epitope polypeptide artificial antigen printed detection line 2. The artificial antigen prepared by the present invention has the advantages of easy acquisition, stable structure, purity up to 99%, low cost, rapid mass production, etc., which solves the problem of low expression of traditionally expressed proteins, difficulty in ensuring natural spatial structure, difficulty in renaturation, and difficulty in The removal of bacterial proteins affects the specificity of the test results and other shortcomings, and also solves the risk of incomplete virus inactivation when using inactivated viruses. The invention also pre-treats the gold-labeled pad, which is more conducive to the gold-labeled pad’s water absorption and the release of gold-labeled protein, effectively solving the slow and incomplete release of gold-labeled protein in the existing test paper, which affects the detection accuracy, sensitivity and issues such as shelf life.

Description

Evaluation of immune antibody against foot-and-mouth disease virus and dual test for differential diagnosis of infection and immunity note

technical field

The invention relates to a foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip, which belongs to the technical field of immune detection.

Background technique

Foot-and-mouth disease is a highly contagious disease of artiodactyls caused by the foot-and-mouth disease virus. Foot-and-mouth disease virus has polymorphism, variability, wide range of hosts, and strong infectivity, so once it occurs, it will be epidemic and explosive. What's more serious is that foot-and-mouth disease is a severe infectious disease, which is said to be extremely contagious. Strict isolation, blockade, prohibition of animal movement and listing of livestock products must be carried out for diseased animals and animals in the same group exposed to the pathogen. , leading to the suspension of the import and export trade of livestock products in the affected areas and even the affected countries, resulting in huge economic losses.

The main strategy for controlling foot-and-mouth disease in developing countries is vaccine immunization. The animal immune effect of vaccine immunization, maternal antibody and the formulation of immunization procedures for foot-and-mouth disease immunized animal populations all rely on antibody detection. Currently, the serological methods for detecting FMD antibody levels are mainly liquid phase blocking ELISA (Liquid phase blocking ELISA, LPBE) and antibody detection test strips. The antigens used in these methods are mainly inactivated viruses or expressed proteins. Inactivated viruses have the problems of incomplete virus inactivation and difficult purification; expressed proteins have problems such as low protein expression and difficult removal of bacterial proteins. Antibody detection test strips have the advantages of fast, simple and low cost. The gold label pad is an important part of the test paper, which determines the activity and release of the gold label protein, so it has a great influence on the effect of the colloidal gold immunochromatography test paper . At present, the preparation method of the gold label pad is to directly spray or soak the colloidal gold-labeled protein/antibody on the fiber cotton, or soak the fiber cotton in a treatment solution, and then spray or soak the colloidal gold-labeled protein. However, in the process of use, it was found that due to the inappropriate treatment method of the gold standard pad and the formula of the treatment solution, there were often problems such as slow and incomplete release of the gold standard protein, which affected the detection accuracy, sensitivity and shelf life of the test strip product.

Contents of the invention

In order to overcome the disadvantages of low protein expression in the prior art, difficulty in ensuring the natural spatial structure, difficulty in renaturation, bacterial protein affecting the specificity of the test results, and the risk of incomplete virus inactivation of the inactivated virus, and existing test strips The existing gold standard protein is released slowly and incompletely, which affects the detection accuracy, sensitivity and shelf life of test paper products. The present invention combines the advantages and disadvantages of the existing ELISA method for the detection of foot-and-mouth disease and test strips, and uses artificial antigens prepared by polypeptide epitopes and Gold standard pad treatment method, invention of a foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip.

In order to achieve the above object, the technical solution adopted in the present invention is:

A foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip, including the detection line 1 printed using the foot-and-mouth disease virus VP1 epitope polypeptide artificial antigen, and the foot-and-mouth disease virus non-structural protein epitope polypeptide artificial antigen printed The detection line 2.

The VP1 epitope polypeptide artificial antigen is an artificial antigen formed by artificially synthesizing the 142-158th amino acid polypeptide on VP1 of foot-and-mouth disease virus and coupled with a carrier protein; the 142-158th amino acid polypeptide on VP1 of foot-and-mouth disease virus is foot-and-mouth disease virus O/GX/09 The 142nd to 158th amino acid sequence NNVRGDLQVLAQKAERA on the VP1 of the -7 strain; the 142nd to 158th amino acid sequence SNVRGDLQVLAQKAERA on the VP1 of the foot-and-mouth disease virus O/HN/CHA/93 strain; The 158th amino acid sequence NNVRGDLQVLAQKAERT; at least one of the 142nd to 158th amino acid sequence TNVRGDLQVLAQKAARP on the foot-and-mouth disease virus O/MYA/98 strain VP1.

Preferably, the 142nd to 158th amino acid polypeptide on the VP1 of foot-and-mouth disease virus O/GX/09-7 strain, O/HN/CHA/93 strain, O/TAW/97 strain, O/MYA/98 strain is according to Mass ratio 1:1:1:1 mixed.

The non-structural protein epitope polypeptide artificial antigen is at least one of the 1096-1106th amino acid RTPEDLERAEK on the artificially synthesized foot-and-mouth disease virus non-structural protein 2B, the 1414-1425th amino acid HEKVSSHPIFKQ on the 2C, and at least one of the 1602-1613th amino acid GPYAGPMERQKP on the 3B, And add a cysteine to its -NH ₄ end, and then couple the artificial antigen formed by the carrier protein.

Preferably, the artificial antigen prepared by the 1096-1106 amino acid polypeptide on the non-structural protein 2B of foot-and-mouth disease virus, the artificial antigen prepared by the 1414-1425 amino acid polypeptide on 2C, and the artificial antigen prepared by the 1602-1613 amino acid polypeptide on 3B, Mix according to mass ratio 1:1:1.

The test strip also includes a control line printed with a secondary antibody IgG against the animal species to be tested.

The test strip includes a sample pad, a gold standard pad, an absorbent pad and a bottom plate; the detection line and the control line are printed on the nitrocellulose membrane pad.

The gold standard pad is pretreated, and the treatment liquid is sprayed on the fiber pad, and dried to obtain a pretreated gold standard pad.

The treatment solution is composed of the following raw materials: BSA 5%, PVP-10 0.1%, Triton X-100 0.1%, and the balance is 0.02mol/L Na ₂ B ₄ O ₇ ·10H ₂ O solution; the spraying method is: along the fiber pad The treatment liquid is sprayed in the center of the length direction, and after spraying, there are gaps between the edge lines on both sides of the treatment liquid on the fiber mat and the side edges on the corresponding side of the fiber mat.

The drying conditions are: 37-42°C, 10-70min.

Beneficial effects of the present invention:

1. The detection line antigen used in the test paper of the present invention uses a synthetic epitope polypeptide, and the artificial antigen coupled with the carrier protein has the advantages of easy access, stable structure, purity up to 99%, low cost, and rapid mass production. The use of artificial antigens solves the shortcomings of low expression of traditionally expressed proteins, difficulty in ensuring the natural spatial structure, difficulty in renaturation, difficulty in removing bacterial proteins, and affecting the specificity of test results. It also solves the problem of virus inactivation when using inactivated viruses. incomplete risk.

2. In the test strip mode of concatenated antibody detection, gold-labeled proteins can only choose proteins (such as SPA) or anti-antibodies that can bind to mammalian IgG. In the test paper system of the present invention, the control line is the secondary antibody IgG against the animal species to be detected, and the secondary antibody IgG can not only bind to the gold standard, but also bind to the IgG-gold standard that is not intercepted by the antigen protein of the detection line, ensuring Control line color stability.

3. The gold standard pad treatment solution of the present invention has a simple formula and does not contain sugar, which is more conducive to the water absorption of the gold standard pad and the release of gold standard protein. Among them, BSA and PVP-10 can seal the fiber cotton and NC membrane, and at the same time protect the stability of the gold-labeled protein; Na ₂ B ₄ O ₇ 10H ₂ O can provide an alkaline ion environment, which is more conducive to the antigen-antibody reaction and the gold-labeled protein. release; Triton X-100 for gold-labeled pad treatment can accelerate the release of gold-labeled protein, and can reduce the non-specificity of the product. The reason why sugar is not added to the treatment liquid of the present invention is that sugar is easy to crystallize during product storage, which will affect the release of gold-labeled protein and the moving speed of gold particles on the membrane, thereby affecting the shelf life and detection accuracy of the product.

4. The method disclosed in the present invention is different from the existing method of soaking the gold standard pad in the treatment liquid, but directly sprays it on the glass fiber cotton, and chooses the way of spraying to replace the soaking pretreatment of the gold standard pad, which can effectively improve the soaking of the gold standard pad. The edge effect produced by the standard pad; the spraying method will fix the treatment liquid locally, keep the rest of the gold standard pad in a fluffy state, increase the water absorption and adhesion of the gold standard pad, and have wider applicability; the spraying method The gold-labeled protein can be fixed within a certain range after spraying without spreading in a large area, which is more conducive to the stability and release of the gold-labeled protein, and greatly improves the detection sensitivity and stability of the colloidal gold immunochromatography test paper.

5. Easy and fast operation: the test strip of the present invention does not need to attach any other instruments and reagents during the test, just insert the test end into the serum to be tested for about 30 seconds, and then the test result can be judged in about 5 minutes.

6. Reduce investment and reduce detection cost: use the rapid detection test strip of the present invention, no need to be equipped with other instruments, equipment and reagents, save a lot of equipment, equipment and additional reagent costs; one test strip can detect the antibody of the immune population of foot-and-mouth disease at one time It can also detect foot-and-mouth disease infection or infected animals, and professionals and non-professionals can conduct real-time online detection anytime and anywhere, without paying expert diagnostic examination fees and related expenses. Therefore, the detection cost can be saved and the detection cost can be reduced.

7. Wide range of applications, easy to popularize and use: the rapid detection test strip of the present invention is easy to operate as a "one-step" or "fool-style", and is convenient to carry and store, and can meet the needs of personnel at different levels, including professional testing, customs quarantine , sanitation and epidemic prevention, quality monitoring, animal product processing, intensive breeding to individual breeding, etc., have broad market prospects and greater economic and social benefits.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1: FMD virus immune antibody evaluation and infection and immune differential diagnosis dual test strip specificity test results.

Figure 2: FMD virus immune antibody evaluation and sensitivity test results of dual test strips for differential diagnosis of infection and immunity.

Fig. 3: The effect of gold standard protein spraying on the gold standard pad treated in Example 1 and the gold standard pad without pretreatment.

Figure 4: Conditions of gold standard pads treated by the comparative method.

Fig. 5: The use situation of the gold standard pad processed by embodiment 2 and the comparative method.

Detailed ways

The specific implementation of the present invention will be described in further detail below in conjunction with the examples. Unless otherwise specified, "%" in the specific embodiments of the present invention is mass volume percentage (g/mL).

Example 1

Preparation of foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strips: First, according to the B-cell epitope on the foot-and-mouth disease virus VP1 and the B-cell epitope on the non-structural protein of the foot-and-mouth disease virus, artificially synthesize the foot-and-mouth disease virus structural protein VP1 on the table Epitope polypeptides and 3 epitope polypeptides on foot-and-mouth disease virus non-structural proteins 2B, 2C, and 3B are coupled to carrier proteins, respectively, for the preparation of detection line 1 (T1) and detection line 2 (T2) on cellulose membrane mats, and prepared simultaneously The secondary antibody IgG against the animal species to be tested is used to prepare the control line (C) on the cellulose membrane pad; the gold standard pad treatment solution and the gold standard pad pretreatment are prepared for spraying the gold standard protein; finally, the test strip is assembled.

1. Synthesis of foot-and-mouth disease virus structural protein and non-structural protein epitope polypeptide:

Synthesize the 142nd to 158th amino acid sequence on the foot-and-mouth disease virus VP1 (the amino acid sequence varies according to the detected foot-and-mouth disease strain), add a cysteine to its -NH ₄ end; Amino acid sequence 1106 (amino acid sequence: RTPEDLERAEK), amino acid sequence 1414-1425 on 2C (amino acid sequence: HEKVSSHPIFKQ), amino acid sequence 1602-1613 on 3B (amino acid sequence: GPYAGPMERQKP), respectively in -NH ₄ A cysteine was added to the end, and all peptide sequences were synthesized and purified by commercial companies.

2. Preparation of artificial antigens of foot-and-mouth disease virus structural protein and non-structural protein epitope polypeptide:

Using heterobifunctional reagent Sulfo-SMCC (MW: 436.37, Spacer Arm Length: Pierce) to activate -NH ₂ on the carrier protein BSA (or OVA), the specific operation steps are as follows:

(1) Coupling buffer (50mL): 0.15M NaCl, 0.1M PB buffer (pH 7.2), 1μM EDTA (ethylenediaminetetraacetic acid);

(2) BSA (bovine serum albumin) solution: weigh 8 mg of carrier protein BSA and dissolve in 1.0 mL of coupling buffer;

(3) Sulfo-SMCC solution: Weigh 2 mg of Sulfo-SMCC and add it to 100 μL of DMSO (dimethyl sulfoxide), pipetting repeatedly to make it fully dissolved;

(4) Mix the BSA solution and the Sulfo-SMCC solution, mix thoroughly, and react at room temperature for 1 hour or 37°C for 30 minutes, and mix from time to time;

(5) Dialyze the solution in step (4) with a coupling buffer at 4°C for 48 hours, and change the solution every 6 hours to remove excess coupling agent (Sulfo-SMCC) and DMSO;

(6) Adjust the concentration of the carrier protein BSA to 5 mg/mL with a coupling buffer, and this solution is the SMCC-activated carrier protein (SMCC-BSA), which is frozen at -20°C for future use.

The -NH ₂ on the carrier protein is activated and linked to the -SH of the N-terminal cysteine (Cys) of the polypeptide (Pep) to form an artificial binding antigen (BSA-Pep). Coupling steps: Weigh 4 mg of polypeptide and fully dissolve it in 0.01M PBS buffer (use DMF or DMSO to dissolve polypeptides with relatively low solubility); add 300 μL of 0.01M PB buffer (pH 7.2, containing 5mM EDTA) , prepare the peptide stock solution with a concentration of 10 mg/mL. For coupling, take 20 μL of the peptide stock solution, add it to an equal volume of 0.01M PB buffer (pH 7.2) containing 5 mM EDTA, then add 40 μL of SMCC-BSA solution, mix well, react at room temperature for 4 hours, and incubate overnight at 4°C. The above solution was dialyzed with normal saline at 4°C for 48 hours, and the solution was changed every 6 hours to remove excess coupling agent; the protein concentration was measured with an ultraviolet spectrophotometer.

3. Preparation of goat or rabbit anti-IgG secondary antibodies of the tested animal species:

Inject negative healthy sheep or rabbits 3 to 4 times subcutaneously and intramuscularly with 50 μg to 100 μg IgG/kg body weight, collect blood venously 20 days after the last immunization, and measure the serum antibody titer above 1:2000 by ELISA, collect blood from the heart or carotid artery Bloodletting and collection of hyperimmune serum. Take 1 volume of serum and 2 volumes of PBS buffer solution (pH 7.2) and mix well, add an equal volume of saturated ammonium sulfate solution and mix well, put it in a refrigerator at 4°C for 2 hours, centrifuge at 10,000 r/min at 4°C for 15 minutes, and discard the supernatant; Appropriate amount of PBS buffer (pH7.2) to dissolve the precipitate, add saturated ammonium sulfate solution to the final concentration of 33%, put it in the refrigerator at 4°C for 2h, centrifuge at 4°C and 10000r/min for 15min, discard the supernatant, and use Dissolve the precipitate with a small amount of PBS buffer (pH7.2), put it in a refrigerator at 4°C and dialyze with PBS buffer (pH7.2) overnight, change the medium 2-3 times, centrifuge at 4°C and 10000r/min for 15min, and collect the The protein concentration of the supernatant was determined by a UV spectrophotometer.

4. Preparation of gold-labeled protein:

Prepare gold sol by sodium citrate reduction method: add 2-4 mL of 0.5-2% trisodium citrate solution to 50-100 mL of boiling 0.01-0.05% mass fraction of chloroauric acid aqueous solution to obtain a colloid with a diameter of about 15 nm gold. Adjust the pH of the colloidal gold to 8.5-9.5 with 0.1mol/L K ₂ CO ₃ , and add the protein SPA to be labeled into the colloidal gold with a pH of 8.5-9.5 at a labeling ratio of 1:1000-1300. After labeling for 10 minutes, add Mass fraction 20% PEG-10000 to its final concentration of 0.05%, centrifuge at 4°C, 1500-3000r/min for 20min, remove unbound colloidal gold particles, centrifuge at 4°C, 15000r/min for 1h, discard the supernatant to obtain the colloid Gold-labeled protein.

5. Preparation of gold standard pad:

Gold standard pad pretreatment method: Spray the treatment solution on the fiber pad at an amount of 8 μL/cm by spraying equipment, and bake at 42°C for 50 minutes to obtain a pretreated gold standard pad. The gold standard pad treatment solution is composed of the following raw materials: BSA 5%, PVP-10 0.1%, Triton X-100 0.1%, and the balance is 0.02mol/L Na ₂ B ₄ O ₇ ·10H ₂ O solution. The spraying method is: spray the treatment liquid along the center of the length direction of the fiber mat, and after spraying, there are gaps between the edge lines on both sides of the treatment liquid on the fiber mat and the side edges of the corresponding side of the fiber mat. Then the prepared colloidal gold-labeled protein was evenly sprayed on the gold label pad, and baked at 42°C for 40min.

6. Assembly of test strips

Spray the artificial antigen of foot-and-mouth disease virus structural protein epitope polypeptide on the nitrocellulose membrane to form detection blot/line 1 (T1), spray the artificial antigen of foot-and-mouth disease virus non-structural protein epitope polypeptide on the nitrocellulose membrane to form To detect blot/line 2 (T2), spray goat or rabbit anti-IgG secondary antibody of the animal species to be tested on the nitrocellulose membrane to form a control blot/line (C); the sample pad, gold standard pad, cellulose The film pad and the absorbent pad are pasted on the bottom plate in turn, and cut to an appropriate size to obtain test paper.

A red sample marking line is printed on the surface layer corresponding to the junction of the sample pad and the gold standard pad, and the word max is printed. The warning line of the mark is 1.1-1.2cm away from the top side of the sample pad.

The principle of the rapid detection test strip of the present invention is detected:

When the test end of the rapid detection test strip of the present invention is inserted into the serum to be tested, the serum to be tested will drive the antibody of the serum to be tested and the gold standard protein in the glass wool of the gold standard protein to diffuse to the nitrocellulose membrane through the siphon, and finally penetrate into the handle end In the filter paper, the antibody can bind to the gold-labeled protein during the diffusion process, and the conjugate can then bind to the artificial antigen detection blot on the cellulose membrane, thus showing a brown-red detection blot (T1 and/or T2); while the control line The blot can be combined with a gold standard to form a brownish-red control blot (C). If there is no foot-and-mouth disease virus antibody and/or vaccine antibody in the serum to be tested, the test strip only shows one (one) brown-red control blot (C); if anti-foot-and-mouth disease virus antibody and/or vaccine antibody are contained in the serum to be tested, then First combine with its gold-labeled protein, and then combine with the artificial antigen detection blot T1 and/or T2 on the cellulose membrane, and a brown-red detection blot is displayed, which is a positive marker; if there is no brown-red blot on the cellulose membrane, then Indicates that the test strip has expired or was mishandled.

Test strip detection operation method:

(1) Preparation of detection sample liquid: aseptically take the blood of the tested animal, and separate the serum, and use physiological saline as a 1:200 times diluted serum to be tested; insert the test end of the rapid detection test strip of the present invention into the serum to be tested , the insertion depth does not exceed the marked line, take out the test strip after about 30s, place it horizontally for about 1-5min, and observe the result at the same time.

(2) Judgment of results: If only one (one) brown-red control blot (C) is displayed on the cellulose film layer of the test strip, it means that the test result is negative, indicating that no antibody to foot and mouth disease virus was detected in the tested serum And/or foot-and-mouth disease vaccine antibody, that is, the tested animal has no foot-and-mouth disease virus infection and/or foot-and-mouth disease vaccine immunity; if brown-red control imprint (C) and detection imprint 1 (T1) appear on the cellulose film layer on the test strip , indicating that the test result is positive, that is, the foot-and-mouth disease vaccine antibody is detected in the serum to be tested, that is, the animal under inspection has been immunized with the foot-and-mouth disease vaccine; if the detection blots T1 and T2 appear at the same time, it means that the antibody to foot-and-mouth disease virus infection is detected in the serum to be tested , that is, the animal under inspection has been infected or carries foot-and-mouth disease virus; if there is no brown-red imprint on the cellulose membrane, it indicates that the test strip has failed or has been mishandled.

Example 2

Foot-and-mouth disease virus immune antibody evaluation and the preparation method of infection and immune differential diagnosis dual test strips are as in Example 1, and the 142nd to 158th amino acid sequence NNVRGDLQVLAQKAERA on the detection line 1 is sprayed on the foot-and-mouth disease virus O/GX/09-7 strain VP1 (SEQ ID NO.1), adding a cysteine at its -NH4 end, coupling the artificial antigen prepared by carrier protein BSA or OVA; spraying the artificial antigen of non-structural protein of foot-and-mouth disease virus on the detection line 2, and the artificial antigen is selected at the same time The 1096th to 1106th amino acid (amino acid sequence: RTPEDLERAEK, SEQID NO.5) polypeptide on the nonstructural protein 2B of foot-and-mouth disease virus, and the 1414th to 1425th amino acid (amino acid sequence: HEKVSSHPIFKQ, SEQID NO.6) on the nonstructural protein 2C Polypeptide, amino acid 1602-1613 on non-structural protein 3B (amino acid sequence: GPYAGPMERQKP, SEQID NO.7) polypeptide, artificial antigen prepared by coupling carrier protein BSA or OVA, three artificial antigens in mass ratio 1:1: 1 mix.

Example 3

Foot-and-mouth disease virus immune antibody evaluation and the preparation method of infection and immune differential diagnosis dual test strips are as in Example 2, the difference is that the artificial antigen polypeptide on the detection line 1 is on the foot-and-mouth disease virus O/HN/CHA/93 strain VP1 The 142nd-158th amino acid sequence SNVRGDLQVLAQKAERA (SEQ ID NO.2).

Example 4

The preparation method of foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip is as embodiment 2, the difference is that the artificial antigen polypeptide on the detection line 1 is the 142nd on the foot-and-mouth disease virus O/TAW/97 strain VP1 The amino acid sequence at position 158 is NNVRGDLQVLAQKAERT (SEQ ID NO.3).

Example 5

The preparation method of foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip is as embodiment 2, the difference is that the artificial antigen polypeptide on the detection line 1 is the 142nd on the foot-and-mouth disease virus O/MYA/98 strain VP1 ～158 amino acid sequence TNVRGDLQVLAQKAARP (SEQ ID NO.4).

Verification example

1. Specific detection

The test paper of the present invention detects FMD infection serum, FMD immune serum, FMD negative serum, and swine fever virus, porcine blue ear disease virus, porcine circovirus, porcine pseudorabies virus, porcine B encephalitis virus positive serum, and the results show that the The specificity of the inventive test paper is 100%, and there is no cross-reaction with the foot-and-mouth disease negative serum and other virus positive serum (as shown in Figure 1).

2. Sensitivity detection

Use test paper of the present invention to detect the standard pig foot-and-mouth disease immune serum of doubling ratio dilution, the result shows that the sensitivity of test paper of the present invention to the standard positive serum of foot-and-mouth disease infection and immunity is not less than 1:3200 and 1:6400 (as shown in Figure 2) .

Comparative Experiment

1. Spray gold standard protein on the gold standard pad treated in embodiment 1 (spraying position is the same as the spraying position of treatment solution), gold standard protein becomes a line of aggregation and is fixed on the gold standard pad; while on the gold standard pad without pretreatment Gold-labeled protein was sprayed on the pad (the spraying position was the same as that of the treated gold-labeled pad), and the gold-labeled protein became dispersed and filled the entire gold-labeled pad, indicating that the gold-labeled pad treated by the present invention can fix the gold-labeled protein within a certain range , which is more conducive to the stability and release of the gold-labeled protein (Figure 3).

2. Taking the pretreatment methods of other gold standard pads as a contrast, compare with the pretreatment effect of the gold standard pad of the present invention. The pretreatment method of the comparison gold standard pad is to soak the fiber pad in the treatment solution for 15 minutes, and then take it out and dry it at 37°C to obtain the pretreated gold standard pad. The treatment solution is composed of the following raw materials: 3% BSA, 8% trehalose, 2% Tween-20, and the balance is 0.01mol/L phosphate buffer saline. It can be seen from the results (Fig. 4) that the distribution of the gold standard mat treated by the comparison method was uneven after drying, and the treated substance was precipitated and turned yellow at the edge, and the texture was relatively hard. Because the treatment solution contained Tween-20, it could not adhere to the support plate. The gold standard pad is prone to fall off during cutting, and this shortcoming can be improved by spraying.

3. Spray the SPA marked with colloidal gold on the gold standard pads pretreated in Example 2 and the comparative method, and make a test paper to detect the foot-and-mouth disease infected serum, and observe after 10min. In Example 2, the release rate of the gold-labeled protein on the gold-labeled pad was above 95%, and the gold-labeled SPA on the gold-labeled pad of the comparison method was not completely released, and the color development of the test strip was weaker than that of Example 2 (Figure 5).

The above description is only the best embodiment of the present invention, for those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Henan Academy of Agricultural Sciences

<120> A dual test strip for evaluation of immune antibody against foot-and-mouth disease virus and differential diagnosis of infection and immunity

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Foot and 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

<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

<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

<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

<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

<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

<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 antibody evaluation and infection and immune differential diagnosis dual test strip, it is characterized in that, described test strip comprises the detection line 1 that utilizes foot-and-mouth disease virus VP1 epitope polypeptide artificial antigen to print, and utilizes foot-and-mouth disease virus VP1 epitope polypeptide artificial antigen to print, and utilizes foot-and-mouth disease virus Detection line 2 of virus non-structural protein epitope peptide artificial antigen printing. 2. The test strip according to claim 1, characterized in that, the VP1 epitope polypeptide artificial antigen is the 142nd to 158th amino acid polypeptide on the VP1 of artificially synthesized foot-and-mouth disease virus, and the artificial antigen formed by coupling carrier protein; The 142-158th amino acid polypeptide on VP1 is the 142-158th amino acid sequence NNVRGDLQVLAQKAERA on VP1 of foot-and-mouth disease virus O/GX/09-7 strain; the 142-158th position on VP1 of foot-and-mouth disease virus O/HN/CHA/93 strain The amino acid sequence SNVRGDLQVLAQKAERA; the 142nd to 158th amino acid sequence NNVRGDLQVLAQKAERT on the foot-and-mouth disease virus O/TAW/97 strain VP1; at least one of the 142nd to 158th amino acid sequence TNVRGDLQVLAQKAARP on the foot-and-mouth disease virus O/MYA/98 strain VP1. 3. The test strip according to claim 2, characterized in that, preferably, foot-and-mouth disease virus O/GX/09-7 strain, O/HN/CHA/93 strain, O/TAW/97 strain, The 142nd to 158th amino acid polypeptides on the O/MYA/98 strain VP1 were mixed in a mass ratio of 1:1:1:1. 4. The test strip according to claim 1, characterized in that the nonstructural protein epitope polypeptide artificial antigen is artificially synthesized foot-and-mouth disease virus nonstructural protein 2B 1096～1106 amino acid RTPEDLERAEK, 2C 1414～1425 Amino acid HEKVSSHPIFKQ, at least one of the amino acids GPYAGPMERQKP at positions 1602-1613 on 3B, and a cysteine is added to its -NH ₄ end, and then coupled with a carrier protein to form an artificial antigen. 5. The test strip according to claim 4, characterized in that, preferably, the artificial antigen prepared from the 1096th to 1106th amino acid polypeptide on the foot-and-mouth disease virus nonstructural protein 2B, and the 1414th to 1425th amino acid polypeptide on the 2C Artificial antigen, the artificial antigen prepared from the 1602-1613 amino acid polypeptide on 3B, mixed according to the mass ratio of 1:1:1. 6. The test strip according to claim 1, further comprising a control line printed with a secondary antibody IgG against the animal species to be tested. 7. test strip according to claim 1, is characterized in that, described test strip also comprises sample pad, gold standard pad, absorption pad and base plate; Detection line and reference line are printed on nitrocellulose membrane pad. 8. The test strip according to claim 7, characterized in that, the gold standard pad is pretreated, the treatment solution is sprayed on the fiber pad, and dried to obtain the pretreated gold standard pad. 9. The test strip according to claim 7, characterized in that, the treatment liquid is made up of the following raw materials: BSA5%, PVP-100.1%, Triton X-100 0.1%, and a balance of 0.02mol/L _{Na2B 4} O ₇ ·10H ₂ O solution; the spraying method is: spray the treatment liquid along the center of the fiber mat in the length direction, and after spraying, there are gaps between the edge lines on both sides of the treatment liquid on the fiber mat and the side edges of the corresponding side of the fiber mat. 10. The test strip according to claim 8, characterized in that the drying conditions are: 37-42°C, 10-70min.