CN108931644B - Foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip - Google Patents
Foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip Download PDFInfo
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Abstract
The invention discloses a bigeminal test strip for evaluating immune antibodies of foot-and-mouth disease viruses and identifying and diagnosing infection and immunity, which comprises a detection line 1 printed by utilizing VP1 epitope polypeptide artificial antigens of the foot-and-mouth disease viruses and a detection line 2 printed by utilizing non-structural protein epitope polypeptide artificial antigens of the foot-and-mouth disease viruses. The artificial antigen prepared by the invention has the advantages of easy acquisition, stable structure, purity up to 99%, low cost, rapid mass production and the like, solves the defects of low expression level of traditional expression protein, difficult guarantee of natural spatial structure, difficult renaturation, difficult removal of mycoprotein, influence on specificity of detection results and the like, and also solves the risk of incomplete virus inactivation caused by using inactivated virus. The gold-labeled pad is pretreated, so that the gold-labeled pad is more favorable for water absorption and release of gold-labeled protein, and the problems that the gold-labeled protein is slowly and incompletely released, the detection accuracy, sensitivity and quality guarantee period of a test paper product are influenced and the like of the conventional test paper are effectively solved.
Description
Technical Field
The invention relates to a bigeminal test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis, belonging to the technical field of immune detection.
Background
Foot-and-mouth disease is a virulent infectious disease of artiodactyl caused by foot-and-mouth disease virus. Foot-and-mouth disease virus is heterogeneous, mutable, broad in host and extremely strong in infectivity, so that once the virus is attacked, the virus becomes epidemic and outbreak. More seriously, foot-and-mouth disease is a virulent infectious disease, and according to extremely strong infectivity, animals in the same group contacting with pathogeny and pathogenic animals need to be strictly isolated, blocked, prohibited to move, on the market of animal products and the like, so that the import and export trade of animal products in the pathogenic area and even in the pathogenic country is stopped, and huge economic loss is caused.
The main strategy for controlling the foot-and-mouth disease in developing countries is vaccine immunization, and the vaccine immunization effect of the foot-and-mouth disease immunization animal population, maternal antibodies and the establishment of an immunization program all need to rely on antibody detection. At present, serological methods for detecting the level of foot-and-mouth disease virus antibodies mainly comprise Liquid phase blocking ELISA (Liquid phase blocking ELISA, LPBE) and antibody detection test strips. The antigen used by the methods is mainly inactivated virus or expressed protein, and the inactivated virus has the problems of incomplete virus inactivation and difficult purification; the expressed protein has problems such as low expression level of the protein and difficulty in removal of the bacterial protein. The antibody detection test strip has the advantages of rapidness, simplicity, convenience and low cost, and the gold-labeled pad is an important component of the test strip and determines the activity and the release of gold-labeled protein, so that the effect of the colloidal gold immunochromatographic test strip is greatly influenced. At present, the preparation method of the gold-labeled pad is to directly spray or soak the colloidal gold-labeled protein/antibody on the cellucotton, or spray or soak the colloidal gold-labeled protein after the cellucotton is soaked by the treatment solution. However, in the using process, the problems that the gold-labeled protein is released slowly and incompletely due to improper treatment method of the gold-labeled pad and improper formula of the treatment solution, so that the detection accuracy, sensitivity, shelf life and the like of the test paper product are affected are often found.
Disclosure of Invention
In order to overcome the defects that the expression amount of expressed protein is low, the natural spatial structure is difficult to ensure, the renaturation is difficult, the specificity of a detection result is influenced by mycoprotein, the risk of incomplete virus inactivation exists in inactivated viruses and the like in the prior art, and the problems that the release of gold-labeled protein is slow and incomplete, the detection accuracy, the sensitivity, the quality guarantee period of a test paper product and the like in the prior art are influenced.
In order to achieve the purpose, the invention adopts the technical scheme that:
a bigeminal test strip for evaluating immune antibodies of foot-and-mouth disease viruses and identifying and diagnosing infection and immunity comprises a detection line 1 printed by using VP1 epitope polypeptide artificial antigens of the foot-and-mouth disease viruses and a detection line 2 printed by using non-structural protein epitope polypeptide artificial antigens of the foot-and-mouth disease viruses.
The VP1 epitope polypeptide artificial antigen is an artificial antigen formed by artificially synthesizing amino acid polypeptides at positions 142-158 of foot-and-mouth disease virus VP1 and coupling carrier proteins; the 142 th to 158 th amino acid polypeptides on the foot-and-mouth disease virus VP1 are amino acid sequences NNVRGDLQVLAQKAERA on the 142 th to 158 th positions on a foot-and-mouth disease virus O/GX/09-7 strain VP 1; an amino acid sequence SNVRGDLQVLAQKAERA at positions 142-158 on VP1 of a foot-and-mouth disease virus O/HN/CHA/93 strain; an amino acid sequence NNVRGDLQVLAQKAERT at positions 142-158 of a foot-and-mouth disease virus O/TAW/97 strain VP 1; at least one of amino acid sequences TNVRGDLQVLAQKAARP at positions 142-158 on VP1 of foot-and-mouth disease virus O/MYA/98 strain.
Preferably, the foot-and-mouth disease virus O/GX/09-7 strain, O/HN/CHA/93 strain, O/TAW/97 strain and O/MYA/98 strain VP1 have amino acid polypeptides at positions 142-158 in a mass ratio of 1:1: 1:1 and mixing.
The non-structural protein epitope polypeptide artificial antigen is at least one of artificially synthesized amino acids RTPEDLERAEK from 1096 to 1106, amino acids HEKVSSHPIFKQ from 1414 to 1425 and amino acids GPYAGPMERQKP from 1602 to 1613 on foot-and-mouth disease virus non-structural protein 2B, 2C and-NH thereof4Adding cysteine at the end, and coupling with carrier protein to form artificial antigen.
Preferably, the artificial antigen prepared from 1096-1106 th amino acid polypeptides on foot-and-mouth disease virus non-structural protein 2B, the artificial antigen prepared from 1414-1425 th amino acid polypeptides on 2C, and the artificial antigen prepared from 1602-1613 th amino acid polypeptides on 3B are mixed according to the mass ratio of 1:1:1 and mixing.
The test strip also includes control lines printed with a secondary antibody IgG directed against the species of animal to be tested.
The test strip comprises a sample pad, a gold label pad, an absorption pad and a bottom plate; the test and control lines were printed on nitrocellulose membrane pads.
And (3) pretreating the gold label pad, spraying the treatment liquid on the fiber pad, and drying to obtain the pretreated gold label pad.
The treatment fluid consists of the following raw materials: 5% of BSA, 100.1% of PVP, 1000.1% of Triton X and the balance of 0.02mol/L of Na2B4O7·10H2O solution; the spraying method comprises the following steps: and spraying treatment liquid along the center of the length direction of the fiber mat, wherein gaps are formed between edge lines on two sides of the treatment liquid on the fiber mat after spraying and the side edges of the corresponding side of the fiber mat.
The drying conditions were: 37-42 deg.C, 10-70 min.
The invention has the beneficial effects that:
1. the detection line antigen used by the test paper uses synthesized epitope polypeptide, and the artificial antigen coupled with carrier protein has the advantages of easy acquisition, stable structure, purity of 99%, low cost, rapid mass production and the like, and the use of the artificial antigen overcomes the defects of low expression level of the traditional expression protein, difficulty in ensuring the natural spatial structure, difficulty in renaturation, difficulty in removing mycoprotein, influence on the specificity of the detection result and the like, and also solves the risk of incomplete virus inactivation caused by using inactivated virus.
2. In the test paper mode of multi-joint antibody detection, the gold-labeled protein can only select protein (such as SPA) or anti-antibody capable of combining with mammal IgG. In the test paper system, the control line is a secondary antibody IgG resisting the animal species to be detected, the secondary antibody IgG can be combined with a gold marker and also can be combined with an IgG-gold marker which is not intercepted by the antigen protein of the detection line, and the color development stability of the control line is ensured.
3. The gold-labeled pad treatment fluid disclosed by the invention is simple in formula, does not contain sugar, and is more favorable for water absorption of the gold-labeled pad and release of gold-labeled protein. Wherein, BSA and PVP-10 can seal cellucotton and NC membrane, and protect the stability of gold-labeled protein; na (Na)2B4O7·10H2O can provide an alkaline ion environment, and is more beneficial to the antigen-antibody reaction and the release of gold-labeled protein; the treatment of Triton X-100 on the gold-labeled pad can accelerate the release of gold-labeled protein and reduce the non-specificity of the product. The reason why the treatment solution of the invention does not contain sugar is as follows: sugar is easy to crystallize in the product storage process, and can affect the release of gold-labeled protein and the moving speed of gold particles on a film, thereby affecting the shelf life and the detection accuracy of the product.
4. The method disclosed by the invention is different from the existing method for soaking the gold mark pad in the treatment solution, but is directly sprayed on the glass fiber cotton, and the spraying mode is selected to replace the soaking pretreatment of the gold mark pad, so that the edge effect generated by soaking the gold mark pad can be effectively improved; the spraying mode can fix the treatment liquid locally, so that the rest part of the gold label pad can keep a fluffy state, the water absorption and the adhesion of the gold label pad are increased, and the applicability is wider; the spraying mode can ensure that the gold-labeled protein is fixed in a certain range without large-area diffusion after being sprayed, is more favorable for the stability and release of the gold-labeled protein, and greatly improves the detection sensitivity and stability of the colloidal gold immunochromatographic test paper.
5. The operation is simple, convenient and quick: the test strip of the invention is used without any additional instrument and reagent in the detection process, and the detection result can be judged only by inserting the test end of the test strip into the serum to be detected for about 30s and then about 5 min.
6. The investment and the detection cost are reduced: the rapid detection test strip does not need to be additionally provided with other instruments, equipment and reagents, so that the cost of a large number of instruments, equipment and additional reagents is saved; one test paper can detect the antibody level of the immune group of the foot-and-mouth disease at one time, can also detect the foot-and-mouth disease infected or toxic animals, and can carry out real-time online detection by professional and non-professional persons at any time and any place without paying the diagnosis and examination cost of experts and the related cost thereof. Therefore, the detection cost can be saved, and the detection cost can be reduced.
7. Wide application range and convenient popularization and use: the test strip for rapid detection is simple to operate in a one-step mode or a fool mode, is convenient to carry and store, can meet the requirements of personnel at different levels, comprises professional assay, customs quarantine, health and epidemic prevention, quality monitoring, livestock product processing, intensive culture to individual culture and the like, and has wide market prospect and great economic and social benefits.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1: and (3) evaluating immune antibodies of the foot-and-mouth disease virus and carrying out specific test results of the duplex test strip for infection and immune differential diagnosis.
FIG. 2: and (3) evaluating immune antibodies of the foot-and-mouth disease virus and carrying out sensitivity test on the duplex test strip for infection and immune differential diagnosis.
FIG. 3: example 1 effect of spraying gold-labeled protein on treated gold-labeled pad and non-pretreated gold-labeled pad.
FIG. 4: and comparing the gold-labeled pad condition treated by the method.
FIG. 5: example 2, use of gold pad treated by comparative method.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail. In the embodiments of the present invention, "%" is mass-volume percent (g/mL), unless otherwise specified.
Example 1
Preparing a bigeminal test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis: firstly, artificially synthesizing epitope polypeptides on a foot-and-mouth disease virus structural protein VP1 and 3 epitope polypeptides on foot-and-mouth disease virus non-structural proteins 2B, 2C and 3B according to B cell epitopes on a foot-and-mouth disease virus VP1 and B cell epitopes on a foot-and-mouth disease virus non-structural protein, respectively coupling carrier proteins, and preparing a detection line 1(T1) and a detection line 2(T2) on a cellulose membrane pad, and simultaneously preparing a secondary antibody IgG (immunoglobulin G) for resisting animal species to be detected and a control line (C) on the cellulose membrane pad; preparing a gold-labeled pad treatment solution and pretreating a gold-labeled pad for spraying gold-labeled protein; and finally assembling the test strip.
1. Synthesizing foot-and-mouth disease virus structural protein and non-structural protein epitope polypeptide:
synthesizing amino acid sequences of 142 th to 158 th positions (the amino acid sequences are different according to the detected foot-and-mouth disease strains) on the VP1 of the foot-and-mouth disease virus, and carrying out-NH reaction on the amino acid sequences4Adding a cysteine into the end; synthesizing the 1096 th to 1106 th amino acids (the amino acid sequence is RTPEDLERAEK) on the non-structural protein 2B of the foot-and-mouth disease virus, 1414 th to 1425 th amino acids (the amino acid sequence is HEKVSSHPIFKQ) on the 2C, 1602 th to 1613 th amino acid sequences (the amino acid sequence is GPYAGPMERQKP) on the 3B, respectively4One cysteine was added at the end and all polypeptide sequences were synthesized and purified by commercial companies.
2. Preparing foot-and-mouth disease virus structural protein and non-structural protein epitope polypeptide artificial antigen:
using the heterobifunctional reagent sulfoco-SMCC (MW: 436.37, Spacer Arm Length:
pierce) activates-NH on Carrier protein BSA (or OVA)2The specific operation steps are as follows：
(1) Coupling buffer (50 mL): 0.15M NaCl, 0.1M PB buffer (pH7.2), 1. mu.M EDTA (ethylenediaminetetraacetic acid);
(2) BSA (bovine serum albumin) solution: weighing 8mg of carrier protein BSA and dissolving in 1.0mL of coupling buffer solution;
(3) Sulfo-SMCC solution: weighing 2mg of Sulfo-SMCC, adding the weighed Sulfo-SMCC into 100 mu L of DMSO (dimethyl sulfoxide), and repeatedly beating to fully dissolve the Sulfo-SMCC;
(4) mixing the BSA solution and the Sulfo-SMCC solution, fully and uniformly mixing, reacting for 1h or 37 ℃ for 30min at room temperature, and mixing uniformly from time to time;
(5) dialyzing the solution obtained in the step (4) with a coupling buffer solution at 4 ℃ for 48h, and changing the solution every 6h to remove the excessive coupling agent (Sulfo-SMCC) and DMSO;
(6) adjusting the concentration of carrier protein BSA to 5mg/mL by using a coupling buffer solution, wherein the solution is the SMCC activated carrier protein (SMCC-BSA), and freezing and storing at 20 ℃ for later use.
-NH on a carrier protein2After activation, the protein is connected with-SH of cysteine (Cys) at the N terminal of the polypeptide (Pep) to form artificial combined antigen (BSA-Pep). The coupling steps are as follows: weighing 4mg of polypeptide, and dissolving in 0.01M PBS buffer (DMF or DMSO is used for the polypeptide with low solubility); a polypeptide stock solution was prepared at a concentration of 10mg/mL by adding 300. mu.L of 0.01M PB buffer (pH7.2, containing 5mM EDTA). In the coupling, 20. mu.L of the polypeptide stock solution was added to an equal volume of 0.01M PB buffer (pH7.2) containing 5mM EDTA, and then 40. mu.L of SMCC-BSA solution was added thereto and mixed well, followed by reaction at room temperature for 4 hours and incubation at 4 ℃ overnight. Dialyzing the solution with normal saline at 4 deg.C for 48h, and changing the solution every 6h to remove excessive coupling agent; the protein concentration was measured by UV spectrophotometer.
3. Preparation of secondary antibody of goat or rabbit anti-IgG of the animal species to be tested:
injecting 50-100 mu g IgG/kg body weight into negative healthy sheep or rabbit by subcutaneous and intramuscular injection for 3-4 times, performing intravenous blood sampling 20 days after the last immunization, and determining the serum antibody titer by ELISA to be 1: and (5) collecting hyperimmune serum of the heart or carotid artery blood collection above 2000. Adding 2 volumes of PBS buffer solution (pH7.2) into 1 volume of serum, mixing, adding equal volume of saturated ammonium sulfate solution, mixing, placing in a refrigerator at 4 deg.C for 2h, centrifuging at 4 deg.C and 10000r/min for 15min, and removing supernatant; dissolving the precipitate with a proper amount of PBS buffer solution (pH7.2), adding saturated ammonium sulfate solution until the final concentration is 33%, placing the precipitate in a 4 ℃ refrigerator for 2h, centrifuging the precipitate for 15min at 4 ℃ under the condition of 10000r/min, removing the supernatant, dissolving the precipitate with a small amount of PBS buffer solution (pH7.2), placing the precipitate in a 4 ℃ refrigerator, dialyzing the precipitate with the PBS buffer solution (pH7.2) overnight, changing the solution for 2-3 times, centrifuging the precipitate for 15min at 4 ℃ under the condition of 10000r/min, collecting the supernatant, and measuring the protein concentration of the supernatant with an ultraviolet spectrophotometer.
4. Preparing gold-labeled protein:
preparing gold sol by a sodium citrate reduction method: namely, 2-4 mL of trisodium citrate solution with the mass fraction of 0.5-2% is added into 50-100 mL of boiling chloroauric acid aqueous solution with the mass fraction of 0.01-0.05% to obtain colloidal gold with the diameter of about 15 nm. With a K of 0.1mol/L2CO3Adjusting the pH value of the colloidal gold to 8.5-9.5, and mixing the gold powder with the weight ratio of 1: adding the protein SPA to be labeled into colloidal gold with the pH value of 8.5-9.5 at the labeling ratio of 1000-1300, labeling for 10min, adding 20% by mass of PEG-10000 until the final concentration is 0.05%, centrifuging for 20min at 4 ℃ and 1500-3000 r/min, removing unbound colloidal gold particles, centrifuging for 1h at 4 ℃ and 15000r/min, and discarding the supernatant to obtain the colloidal gold labeled protein.
5. Preparing a gold label pad:
the gold-labeled pad pretreatment method comprises the following steps: and (3) spraying the treatment liquid on the fiber pad by using a spraying instrument in an amount of 8 mu L/cm, and drying at 42 ℃ for 50min to obtain the pretreated gold-labeled pad. The gold-labeled pad treatment solution consists of the following raw materials: BSA 5%, PVP-100.1%, Triton X-1000.1%, and Na 0.02mol/L2B4O7·10H2And (4) O solution. The spraying method comprises the following steps: and spraying treatment liquid along the center of the length direction of the fiber mat, wherein gaps are formed between edge lines on two sides of the treatment liquid on the fiber mat after spraying and the side edges of the corresponding side of the fiber mat. Then the prepared colloidal gold labeled protein is evenly sprayed on a gold labeled pad and dried for 40min at 42 ℃.
6. Assembly of test strips
Spraying the foot-and-mouth disease virus structural protein epitope polypeptide artificial antigen on a nitrocellulose membrane to form a detection blot/line 1(T1), spraying the foot-and-mouth disease virus non-structural protein epitope polypeptide artificial antigen on the nitrocellulose membrane to form a detection blot/line 2(T2), and spraying a secondary antibody of goat or rabbit anti-detected animal species IgG on the nitrocellulose membrane to form a control blot/line (C); and (3) sequentially sticking the sample pad, the gold label pad, the cellulose membrane pad and the absorption pad on a bottom plate, and cutting into a proper size to obtain the test paper.
And red sample mark lines are printed on the surface layer corresponding to the junction of the sample pad and the gold mark pad, and max characters are printed on the surface layer, wherein the mark warning lines are 1.1-1.2cm away from one side of the top end of the sample pad.
The principle of the rapid detection test strip for implementing detection is as follows:
after the test end of the rapid detection test strip is inserted into the serum to be detected, the serum to be detected drives an antibody of the serum to be detected and gold-labeled protein in gold-labeled protein glass wool to diffuse to a nitrocellulose membrane through siphoning and finally permeate into the handle end filter paper, the antibody can be combined with the gold-labeled protein in the diffusion process, and the combination is further combined with an artificial antigen detection print on the cellulose membrane, so that a brownish red detection print (T1 and/or T2) is displayed; while the control line blot can bind to the gold label to form a red-brown control blot (C). If the serum to be detected does not contain the foot-and-mouth disease virus antibody and/or the vaccine antibody, the test strip only displays one (one) reddish brown control blot (C); if the serum to be detected contains an anti-foot-and-mouth disease virus antibody and/or a vaccine antibody, the serum is firstly combined with the gold-labeled protein thereof and then combined with the artificial antigen detection blot T1 and/or T2 on the cellulose membrane to display a brownish red detection blot which is a positive marker; if no red-brown blot on the cellulose membrane is revealed, this indicates that the test strip has failed or has been mishandled.
The detection operation method of the test strip comprises the following steps:
(1) preparation of a detection sample solution: blood of the test animal was aseptically taken, and serum was separated, and the ratio of physiological saline solution was set as 1: diluting serum by 200 times to be tested; the test end of the rapid detection test strip is inserted into the serum to be detected, the insertion depth does not exceed the mark line, the test strip is taken out after about 30s, the test strip is horizontally placed for about 1-5 min, and the result is observed at the same time.
(2) And (5) judging a result: if only one (one) brownish red control blot (C) is displayed on the cellulose membrane layer of the detection test strip, the detection result is negative, which indicates that the foot-and-mouth disease virus antibody and/or the foot-and-mouth disease vaccine antibody are not detected in the detected serum, namely that the detected animal is free from foot-and-mouth disease virus infection and/or foot-and-mouth disease vaccine immunity; if a brownish red control blot (C) and a detection blot 1(T1) appear on the cellulose membrane layer on the detection test strip, the detection result shows that the detection result is positive, namely, the aftosa vaccine antibody is detected in the serum to be detected, namely, the detected animal is immunized by the aftosa vaccine; if the detection blots T1 and T2 appear simultaneously, the detection of the foot-and-mouth disease virus infection antibody in the serum to be detected indicates that the detected animal is infected with or carries the foot-and-mouth disease virus; if no red-brown blot on the cellulose membrane strip is revealed, the test strip is failed or mishandled.
Example 2
A preparation method of a dual test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis is as in example 1, the foot-and-mouth disease virus O/GX/09-7 strain VP1 is sprayed with amino acid sequence NNVRGDLQVLAQKAERA (SEQ ID NO.1) at positions 142-158 on a detection line 1, cysteine is added at the-NH 4 end of the test strip, and the test strip is coupled with artificial antigen prepared from carrier protein BSA or OVA; the foot-and-mouth disease virus non-structural protein artificial antigen is sprayed on the detection line 2, the artificial antigen simultaneously selects 1096-1106 th amino acid (amino acid sequence is RTPEDLERAEK, SEQ ID NO.5) polypeptide on the foot-and-mouth disease virus non-structural protein 2B, 1414-1425 th amino acid (amino acid sequence is HEKVSSHPIFKQ, SEQ ID NO.6) polypeptide on the non-structural protein 2C, 1602-1613 th amino acid (amino acid sequence is GPYAGPMERQKP, SEQ ID NO.7) polypeptide on the non-structural protein 3B, and is coupled with an artificial antigen prepared from carrier protein BSA or OVA, and the three artificial antigens are mixed according to the mass ratio of 1:1: 1.
Example 3
The preparation method of the dual test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis is as in example 2, and the difference is that the artificial antigen polypeptide on the detection line 1 is the amino acid sequence SNVRGDLQVLAQKAERA (SEQ ID NO.2) at the 142 th to 158 th positions of the foot-and-mouth disease virus O/HN/CHA/93 strain VP 1.
Example 4
The preparation method of the combined test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis is as in example 2, and the difference is that the artificial antigen polypeptide on the detection line 1 is the amino acid sequence NNVRGDLQVLAQKAERT (SEQ ID NO.3) at the 142 th to 158 th positions on the foot-and-mouth disease virus O/TAW/97 strain VP 1.
Example 5
The preparation method of the combined test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis is as in example 2, and the difference is that the artificial antigen polypeptide on the detection line 1 is the amino acid sequence TNVRGDLQVLAQKAARP (SEQ ID NO.4) at the 142 th to 158 th positions on the foot-and-mouth disease virus O/MYA/98 strain VP 1.
Verification example
1. Specificity detection
The test paper provided by the invention is used for detecting foot-and-mouth disease infection serum, foot-and-mouth disease immune serum, foot-and-mouth disease negative serum, swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine pseudorabies virus and porcine encephalitis B virus positive serum, and the result shows that the specificity of the test paper provided by the invention is 100%, and the test paper has no cross reaction with the foot-and-mouth disease negative serum and other virus positive serum (as shown in figure 1).
2. Sensitivity detection
The test paper of the invention is used for detecting the standard swine foot-and-mouth disease immune serum diluted by multiple proportions, and the result shows that the sensitivity of the test paper of the invention to the foot-and-mouth disease infection and the immune standard positive serum is respectively not lower than 1: 3200 and 1: 6400 (see fig. 2).
Comparative experiment
1. Spraying gold-labeled protein on the gold-labeled pad treated in the example 1 (the spraying position is the same as that of the treatment liquid), and fixing the gold-labeled protein on the gold-labeled pad in an aggregated line; and the gold-labeled protein is sprayed on the non-pretreated gold-labeled pad (the spraying position is the same as that of the treated gold-labeled pad), and the gold-labeled protein is dispersed to fully impregnate the whole gold-labeled pad, which shows that the gold-labeled pad treated by the method can fix the gold-labeled protein within a certain range, and is more favorable for the stability and release of the gold-labeled protein (figure 3).
2. The pretreatment effect of the gold mark pad of the invention is compared with that of other gold mark pads. The pretreatment method of the comparative gold-labeled pad comprises the steps of soaking the fiber pad in the treatment solution for 15min, taking out the fiber pad, and drying the fiber pad at 37 ℃ to obtain the pretreated gold-labeled pad. The treatment fluid consists of the following raw materials: 3% of BSA, 8% of trehalose, 202% of Tween and the balance of 0.01mol/L phosphate buffer solution. As a result (fig. 4), the gold-labeled pad treated by the comparative method was not uniformly distributed after drying, and the gold-labeled pad had a hard texture and a yellow precipitate at the edge, and was easily peeled off during cutting because the treatment solution contained Tween-20 and the treatment solution could not adhere to the support plate.
3. SPA labeled with colloidal gold is sprayed on the gold-labeled pad pretreated in example 2 and the comparison method, and test paper is prepared to detect foot-and-mouth disease infected serum, and the test paper is observed after 10 min. In example 2, the release rate of the gold-labeled protein on the gold-labeled pad was 95% or more, the gold-labeled SPA on the gold-labeled pad in the comparative method was not completely released, and the color development of the test strip was weaker than that in example 2 (FIG. 5).
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> agricultural science institute of Henan province
<120> a bigeminal test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis
<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 (6)
1. A bigeminal test strip for foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis is characterized in that the test strip comprises a sample pad, a gold-labeled pad, an absorption pad, a bottom plate, a detection line and a contrast line, wherein the detection line and the contrast line are printed on a nitrocellulose membrane pad; the detection line comprises a detection line 1 printed by using the foot-and-mouth disease virus VP1 epitope polypeptide artificial antigen and a detection line 2 printed by using the foot-and-mouth disease virus non-structural protein epitope polypeptide artificial antigen;
the non-structural protein epitope polypeptide artificial antigen is artificially synthesized by amino acids RTPEDLERAEK at 1096-1106, amino acids HEKVSSHPIFKQ at 1414-1425 and amino acids GPYAGPMERQKP at 1602-1613 on foot-and-mouth disease virus non-structural protein 2B, 2C and-NH thereof4Adding cysteine at the end, and coupling with an artificial antigen formed by carrier protein;
adopting an artificial antigen prepared from 1096-1106 th amino acid polypeptides on a foot-and-mouth disease virus non-structural protein 2B, an artificial antigen prepared from 1414-1425 th amino acid polypeptides on a 2C, and an artificial antigen prepared from 1602-1613 th amino acid polypeptides on a 3B, wherein the mass ratio of the artificial antigen to the 3B is 1:1:1, mixing;
pretreating the gold label pad, spraying a treatment solution on the fiber pad, and drying to obtain a pretreated gold label pad;
the treatment fluid consists of the following raw materials: BSA 5%, PVP-100.1%, Triton X-1000.1%, and Na 0.02mol/L2B4O7·10H2And (4) O solution.
2. The test strip of claim 1, wherein the VP1 epitope polypeptide artificial antigen is an artificial antigen formed by artificially synthesizing amino acid polypeptides at positions 142-158 of VP1 of foot-and-mouth disease virus and coupling with a carrier protein; the 142 th to 158 th amino acid polypeptides on the foot-and-mouth disease virus VP1 are amino acid sequences NNVRGDLQVLAQKAERA on the 142 th to 158 th positions on a foot-and-mouth disease virus O/GX/09-7 strain VP 1; an amino acid sequence SNVRGDLQVLAQKAERA at positions 142-158 on VP1 of a foot-and-mouth disease virus O/HN/CHA/93 strain; an amino acid sequence NNVRGDLQVLAQKAERT at positions 142-158 of a foot-and-mouth disease virus O/TAW/97 strain VP 1; at least one of amino acid sequences TNVRGDLQVLAQKAARP at positions 142-158 on VP1 of foot-and-mouth disease virus O/MYA/98 strain.
3. The test strip of claim 2, wherein the amino acid polypeptides at positions 142-158 of the foot-and-mouth disease virus O/GX/09-7 strain, O/HN/CHA/93 strain, O/TAW/97 strain and O/MYA/98 strain VP1 are expressed in a mass ratio of 1:1: 1:1 and mixing.
4. The strip of claim 1, further comprising control lines printed with a secondary antibody IgG directed against the species of animal to be tested.
5. The test strip of claim 1, wherein the spraying method comprises: and spraying treatment liquid along the center of the length direction of the fiber mat, wherein gaps are formed between edge lines on two sides of the treatment liquid on the fiber mat after spraying and the side edges of the corresponding side of the fiber mat.
6. The test strip of claim 1, wherein the drying conditions are: 37-42 deg.C, 10-70 min.
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| FR3111432A1 (en) * | 2020-06-16 | 2021-12-17 | Ng Biotech | System for rapid analysis of a biological sample, to discriminate serological antibodies specific to an infectious agent present in said biological sample |
| CN112698028B (en) * | 2020-12-22 | 2023-03-03 | 海卫特(广州)医疗科技有限公司 | Immunochromatographic gasket and preparation method and application thereof |
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