A foot-and-mouth disease virus antibody microparticle manipulation flash test kit

Abstract

The application relates to a foot-and-mouth disease virus antibody microparticle manipulation flash test kit and belongs to the technical field of biological detection. 2+ The application uses the coordination of His tags and Ni to fix recombinant VP2 proteins on the surface of a microparticle manipulation chip, improves the problems of random protein orientation and epitope burying in a traditional physical adsorption method. A chemiluminescence immunoassay method is established by taking VP2 proteins as coated antigens and taking HRP-labeled anti-FMDV monoclonal antibodies as competitive antibodies. The application can detect A-type and O-type foot-and-mouth disease virus antibodies, the detection time is only 5 minutes, the A-type / O-type antibody detection titer reaches 1:355, the specificity is 100%, the within-batch variation coefficient is 0.14%-4.96%, and the between-batch variation coefficient is 0.20%-1.23%.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to a foot-and-mouth disease virus antibody particle manipulation flash assay kit. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious disease caused by the foot-and-mouth disease virus (FMDV), classified as a Class A animal disease in my country. FMDV has seven serotypes: O, A, Asia I, C, SAT1, SAT2, and SAT3. Currently, my country primarily uses vaccination against FMD serotypes O and A. It is a major and highly contagious disease that is a key focus of prevention and control in the livestock industry. FMD is characterized by rapid transmission, diverse transmission routes, and strong cross-regional spread. Once introduced into susceptible animal populations, it can trigger regional outbreaks, causing significant economic losses to livestock production and seriously affecting the safety of livestock product circulation and trade. Therefore, rapid and accurate FMDV serological antibody detection technology is crucial for effective FMD control and is of great significance for conducting epidemiological monitoring, evaluating vaccine efficacy, and ensuring the safety of cross-regional trade in live animals.

[0003] Current serological testing technologies for FMDV all have varying degrees of technical shortcomings, making it difficult to meet the actual needs of comprehensive FMD prevention and control. For example, the virus neutralization test (VNT) requires the use of live virus and must be performed in a BSL-3 biosafety laboratory, which is not feasible for grassroots testing institutions; moreover, the testing cycle is as long as several days, which cannot meet the needs of rapid response after an outbreak; liquid-phase blocking ELISA (LPBE), as the standard detection method recommended by WOAH, takes 2-3 hours to detect, has poor specificity, and the aggregation of non-specific proteins in serum can easily interfere with the reaction system, leading to false positive results; solid-phase competitive immunoadsorption assay (SPC) has insufficient sensitivity and is prone to missing low-level antibody samples, making it unsuitable for early antibody monitoring after vaccination. These technical limitations restrict the accuracy and efficiency of FMD immune monitoring, epidemiological screening, and emergency response to outbreaks.

[0004] Developing a detection kit that is stable in antigen binding, highly active, and cost-effective is of great value for achieving rapid and sensitive detection of foot-and-mouth disease virus antibodies. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a particle manipulation flash assay kit and its detection method that is rapid, sensitive, highly specific, and capable of simultaneously detecting antibodies against type A and type O foot-and-mouth disease virus.

[0006] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a foot-and-mouth disease virus antibody particle manipulation flash assay kit, comprising: (a) A microparticle manipulation chip with recombinant foot-and-mouth disease virus VP2 protein immobilized on its surface; (b) Enzyme-labeled monoclonal antibody against foot-and-mouth disease virus; (c) Chemiluminescent substrate solution; (d) Sample pretreatment solution.

[0007] As used in this text, “Microparticle Manipulation Control” (MCC) refers to a technique that uses electrodynamic effects such as alternating current heating to generate a micro-electric field, actively controlling the movement of microparticles in a solution, accelerating the binding reaction between antigens and antibodies, and thus significantly shortening the detection time.

[0008] VP2 protein is one of the structural proteins of the foot-and-mouth disease virus capsid, containing multiple neutralizing antigenic epitopes. It is the main immunogenic protein that induces the production of neutralizing antibodies. Studies have shown that VP2 protein exhibits high conservation among different serotypes, with significantly higher amino acid invariance than VP1 protein, and multiple conserved antigenic epitopes exposed on its surface exist in the N-terminal region. It can be used to establish serological detection methods for foot-and-mouth disease.

[0009] Preferably, the surface of the particle manipulation chip is sequentially modified from the inside out with a polydopamine coating, Nα,Nα-bis(carboxymethyl)-L-lysine, and Ni 2+ The chelating layer of the recombinant VP2 protein carries a His tag, which binds to Ni via the His tag. 2+ The coordination effect fixes it to the chip surface.

[0010] Preferably, the sample pretreatment solution contains at least one of casein, Tween-20, and ProClin 300. This pretreatment solution is used to dilute the serum to be tested, which can effectively block non-specific binding sites, eliminate interference, and improve detection specificity. More preferably, the sample pretreatment solution contains 0.5-2% (w / v) casein, 0.1-0.5% (v / v) Tween-20, and 0.01-0.05% (v / v) ProClin 300.

[0011] Preferably, the enzyme-labeled anti-foot-and-mouth disease virus monoclonal antibody is a horseradish peroxidase-labeled foot-and-mouth disease virus monoclonal antibody, prepared according to the instructions of the HRP conjugation kit (Abcam), and stored at -20°C. It includes horseradish peroxidase-labeled type A FMDV monoclonal antibody (BC2B7) or horseradish peroxidase-labeled type O FMDV monoclonal antibody (4A51B10).

[0012] Preferably, the chemiluminescent substrate solution is a commercially available luminol chemiluminescent substrate kit (such as Roche), and is prepared according to the instructions and stored at 4°C in the dark.

[0013] Preferably, the kit further comprises one or more auxiliary reagents selected from the following: concentrated washing solution, positive control serum, and negative control serum.

[0014] Furthermore, the method for fabricating the particle manipulation chip includes the following steps: (1) Immerse the chip in a dopamine solution to form a polydopamine coating; (2) Immerse the polydopamine-coated chip in an Nα,Nα-bis(carboxymethyl)-L-lysine solution; (3) Immerse the chip in NiCl2 solution; (4) Immerse the chip in a His-tagged recombinant VP2 protein solution to fix the VP2 protein onto the chip surface; (5) Wash, seal and dry.

[0015] Preferably, the concentration of the dopamine solution in step (1) is 2-3 mg / mL, the pH is 8.0-8.5, and the time is 3-6 hours.

[0016] Preferably, the concentration of the Nα,Nα-bis(carboxymethyl)-L-lysine solution in step (2) is 1-3 mg / mL, and the time is 1-2 hours.

[0017] Preferably, the concentration of the NiCl2 solution in step (3) is 5-20 mM and the time is 30-60 minutes.

[0018] Preferably, the concentration of the His-tagged recombinant VP2 protein solution in step (4) is 2-4 μg / mL, and the incubation time is 30-60 minutes.

[0019] Secondly, the present invention provides a method for using the foot-and-mouth disease virus antibody particle manipulation flash assay kit described in the first aspect, comprising the following steps: (a) Dilute the serum to be tested with sample pretreatment solution; (b) The diluted serum to be tested and the enzyme-labeled anti-foot-mouth disease virus monoclonal antibody were added to the microparticle control chip at the same time and incubated under microparticle control conditions. (c) After washing, add chemiluminescent substrate solution, measure the luminescence value after reaction, and calculate the inhibition rate.

[0020] Preferably, the microparticle manipulation conditions are: accelerating voltage 6V, accelerating frequency 20K, and incubation time 90 seconds.

[0021] Preferably, the inhibition rate is calculated as PI = [1 - (sample luminescence value - blank control) / (negative control luminescence value - blank control)] × 100%. The PI value reflects the degree of inhibition by which the foot-and-mouth disease virus antibody in the serum being tested inhibits the binding of the enzyme-labeled antibody to the solid-phase antigen.

[0022] This invention is based on the principle of chemiluminescent immunoassay (CLIA), combined with microparticle manipulation (MMC) technology and a competitive detection mode. Specifically: the recombinant foot-and-mouth disease virus (FMDV) VP2 protein is immobilized on the surface of a microparticle manipulation chip; during detection, anti-FMDV antibodies from the test serum and horseradish peroxidase-labeled anti-FMDV monoclonal antibodies are simultaneously added to the chip, competing to bind to the VP2 protein on the chip surface; after washing, a chemiluminescent substrate solution is added, and the luminescence value is negatively correlated with the antibody content in the test serum; the result is determined by calculating the inhibition rate. Microparticle manipulation technology accelerates the antigen-antibody reaction through a micro-electric field, shortening the traditional detection time to less than 5 minutes.

[0023] Thirdly, the present invention provides the application of the foot-and-mouth disease virus antibody particle manipulation flash assay kit as described in the first aspect in the detection of foot-and-mouth disease virus antibodies.

[0024] The applications include, but are not limited to, the following scenarios: monitoring and screening for foot-and-mouth disease outbreaks in farms, slaughterhouses, and live animal markets; monitoring antibody levels and evaluating the effectiveness of immunization in animal populations vaccinated against foot-and-mouth disease; conducting serological epidemiological surveys of foot-and-mouth disease in different regions and under different farming models to understand the virus infection status and epidemic patterns; and conducting rapid antibody testing on animals to be transported across regions and during import and export quarantine to reduce the risk of cross-regional spread of the epidemic.

[0025] The beneficial effects of this invention are as follows: (1) This invention utilizes His tags and Ni 2+ The coordination effect of the protein immobilizes the recombinant VP2 protein on the chip surface, improving upon the problems of random protein orientation, epitope embedding, and easy detachment in traditional physical adsorption methods. This ensures full exposure of the antigenic epitopes of the VP2 protein, effectively maintaining protein activity and improving batch-to-batch repeatability and detection stability. This invention requires only 2-4 μg / mL of VP2 protein for efficient immobilization, with the protein amount used being only 1 / 5-1 / 3 of that required by traditional physical adsorption methods, thus reducing reagent costs.

[0026] (2) This invention combines particle manipulation technology with chemiluminescence immunoassay, and accelerates antigen-antibody reaction through alternating current electrothermal effect. The incubation time is only 60-120 seconds, and the total detection time can be controlled within 5 minutes, which is faster than traditional ELISA method and virus neutralization test.

[0027] (3) This invention utilizes the high conservation of VP2 protein among different serotypes to achieve simultaneous detection of A and O type foot-and-mouth disease virus antibodies without the need for typing detection, with a specificity of 100%, providing an efficient and reliable technical means for epidemic monitoring and immune assessment. Attached Figure Description

[0028] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0029] Figure 1 The results are the sensitivity measurement results of this invention; Figure 2 The results of serum O-type antibody detection using a commercial O-type foot-and-mouth disease virus LPBE kit; Figure 3 The results of serum A-type antibody detection using a commercial foot-and-mouth disease virus type A LPBE kit; Figure 4 These are the results of the cross-reactivity test of the present invention. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided.

[0031] Example 1: Fabrication of a Particle Manipulation Chip (1) Immerse the chip in anhydrous ethanol, sonicate for 15 minutes, rinse three times with ultrapure water, and dry with nitrogen. Prepare a 2 mg / mL dopamine hydrochloride solution, immerse the pretreated chip in the dopamine solution, adjust the pH to 8.5, and keep at room temperature in the dark for 4 hours. Remove the chip and wash it three times with ultrapure water.

[0032] (2) Prepare a 2 mg / mL solution of Nα,Nα-bis(carboxymethyl)-L-lysine (dissolved in PBS, pH 7.4). Immerse the polydopamine-coated chip in this solution at room temperature for 2 hours. Remove the chip and wash it three times with PBS.

[0033] (3) Prepare a 10 mM NiCl2·6H2O solution (dissolved in pure water). Immerse the chip in the NiCl2 solution at room temperature for 30 minutes. Remove the chip and wash it three times with PBS.

[0034] (4) Dilute the recombinant VP2 protein (Wuhan Aibisai, VK164012) to 2 μg / mL with PBS. Immerse the chip in the VP2 solution at room temperature for 1 hour. Remove the chip and wash it 3 times with PBS.

[0035] (5) Prepare a PBS blocking solution containing 3% casein. Immerse the chip in the blocking solution at room temperature for 1 hour. Remove the chip, wash it 3 times with PBS, dry it with nitrogen, and store it in a sealed container at 4°C for later use.

[0036] Example 2: Composition of the Foot-and-Mouth Disease Virus Antibody Particle Manipulation Flash Detection Kit The kit contains the following components: (1) Two microparticle manipulation chips with His-tagged recombinant VP2 protein fixed on their surfaces, sealed and stored at 4°C; (2) Two vials (12 mL) of HRP-labeled anti-foot-mouth disease virus monoclonal antibody were stored at -20℃; (3) Chemiluminescent substrate solution: Reagent A (containing luminol buffer) and reagent B (containing buffer containing stable form H2O2). When using, mix solution A and solution B at a ratio of 1:100 and store at 4°C in the dark. (4) Two bottles of sample pretreatment solution (12 mL / bottle) containing 1% (w / v) casein, 0.2% (v / v) Tween-20 and 0.02% (v / v) ProClin 300 in 0.01 M PBS buffer (pH 7.4), stored at 4℃; (5) One tube (1 mL) of positive control serum, stored at -20℃; (6) One tube (1 mL) of negative control serum, stored at -20℃; (7) One bottle (50 mL) of concentrated washing solution, stored at 4℃.

[0037] The sample pretreatment solution was prepared as follows: 1 g of casein was weighed and dissolved in 100 mL of PBS buffer (0.01 M, pH 7.4). 0.2 mL of Tween-20 and 20 μL of ProClin 300 were added. After stirring to dissolve, the solution was filtered through a 0.22 μm filter membrane for sterilization and stored at 4°C.

[0038] Concentrated washing buffer: 0.2 M PBS buffer (pH 7.2-7.4) containing 0.5% (v / v) Tween-20, diluted 20 times with distilled water before use.

[0039] Example 3: Detection method for foot-and-mouth disease virus antibodies (1) Remove the kit from the refrigerated environment and allow it to equilibrate to room temperature for 30 minutes; (2) Dilute the concentrated washing solution 20 times with distilled water; (3) Dilute the serum to be tested with sample pretreatment solution at a ratio of 1:10; take 40 μL of diluted serum and add it to the reaction well of the microparticle control chip, and add 40 μL of 4A51B10 monoclonal antibody (0.1 μg / mL). (4) Set up positive control wells (with positive control serum), negative control wells (with negative control serum), and blank control wells (without serum); (5) Place the chip in a microparticle manipulation incubator, set the accelerating voltage to 6V and the accelerating frequency to 20K, and incubate at room temperature for 90 seconds; (6) Discard the reaction solution, add washing solution to each well, soak for 15 seconds, discard the washing solution, repeat washing 4 times, and pat dry. (7) Add 40 μL of chemiluminescent substrate solution to each well and react at room temperature in the dark for 2 minutes; measure the luminescence value at a wavelength of 435 nm using a chemiluminescence detector; calculate the inhibition rate PI according to the formula and determine the result.

[0040] Calculate the inhibition rate (PI) using the following formula: PI = 1 - (Sample luminescence value - Blank control luminescence value) / (Negative control luminescence value - Blank control luminescence value) To determine the cutoff, this study measured the PI values ​​of the 104 known negative samples. The mean PI value and standard deviation (SD) of these negative samples were 14.47% and 10.51%, respectively. The cutoff was defined as the mean PI value of all negative samples plus three times the standard deviation (mean + 3SD). The calculated cutoff PI value was 46%. The criteria were defined as follows: PI > 46% was considered FMDV antibody positive; PI ≤ 46% was considered FMDV antibody negative.

[0041] At this threshold, the diagnostic sensitivity (Dsn) of this kit FMDV-VP2-cMMC is 100% (76 out of 76 known positive samples were correctly identified) and the diagnostic specificity (Dsp) is 100% (114 out of 114 known negative samples were correctly identified).

[0042] To further validate the performance of this method, the FMDV O / A type liquid phase blocking ELISA (LPBE) kit was used as a reference standard to perform parallel testing on the 190 samples. For FMDV O type positive serum (n=54, including 38 O / A bivalent positive serums and 16 O type positive serums), the relative sensitivity of FMDV-VP2-cMMC was 100% (both methods detected 54 positive samples); for FMDV A type positive serum (n=60, including 38 O / A bivalent positive serums and 22 A type positive serums), the relative sensitivity of FMDV-VP2-cMMC was 105.3% (both methods detected 57 positive samples; and FMDV-VP2-cMMC detected 3 more positive samples that were missed by FMDV A type LPBE). Based on all FMDV type O and A positive samples detected (n=76), the overall relative sensitivity of FMDV-VP2-cMMC was 104.1% (both methods detected 73 positive samples; and FMDV-VP2-cMMC detected 3 more positive samples that were missed by FMDV type A LPBE); the overall relative specificity was 101% (both methods detected 112 negative samples; and FMDV-VP2-cMMC correctly identified 2 samples that were misclassified as false positives by FMDV / type A LPBE).

[0043] Table 1 Specificity determination

[0044] Table 2 Sensitivity Measurement

[0045] Sensitivity: Foot-and-mouth disease virus (FMD) serotype O / A bivalent positive sera were serially diluted twofold (from 1:8 to 1:1024) and detected using the kit of this invention. The results were paralleled with commercially available FMD virus type O and A liquid phase blocking ELISA (LPBE) kits. The endpoint titer was defined as the highest serum dilution that yielded a positive result. The results showed that the kit of this invention detected a serum FMD virus antibody titer of 1:355 (…). Figure 1 ); while the commercial O-type foot-and-mouth disease virus LPBE kit detected a serum O-type antibody titer of 1:355 ( ); Figure 2 The commercial foot-and-mouth disease virus type A LPBE kit showed a serum type A antibody titer of 1:90 (). Figure 3 ).

[0046] Specificity (cross-reactivity): The kit of this invention was used to detect positive sera for type A FMDV, type O FMDV, classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca virus type A (SVA). The results showed that ( Figure 4 Only FMDV positive sera of types A and O were considered positive, while all others were negative, with a specificity of 100%.

[0047] Repeatability: Intra-batch repeatability: For the same batch of chips, 3 positive serum samples and 1 negative serum sample were tested, with each sample tested 3 times. The coefficient of variation was 0.14%-4.96%. Inter-batch repeatability: For the same batch of chips, 3 different batches of chips were tested, with the coefficient of variation being 0.20%-1.23%.

[0048] Table 3 Repeatability Tests

[0049] Sample validation: The positive detection rate of 40 field serum samples was 95%, which was higher than the 92.5% of type O and type A FMDV LPBE.

[0050] Table 4 Sample Detection Results

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A foot-and-mouth disease virus antibody particle manipulation flash assay kit, characterized in that, include: (a) A microparticle manipulation chip with recombinant foot-and-mouth disease virus VP2 protein immobilized on its surface; (b) Enzyme-labeled monoclonal antibody against foot-and-mouth disease virus; (c) Chemiluminescent substrate solution; (d) Sample pretreatment solution.

2. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 1, characterized in that, The surface of the particle manipulation chip is sequentially modified from the inside out with a polydopamine coating, as well as Nα,Nα-bis(carboxymethyl)-L-lysine and Ni. 2+ The chelating layer contains a His tag on the recombinant VP2 protein, which binds to Ni via the His tag. 2+ The coordination effect fixes it to the chip surface.

3. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 1, characterized in that, The sample pretreatment solution contains at least one of casein, Tween-20, and ProClin 300.

4. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 1, characterized in that, The enzyme-labeled anti-foot-and-mouth disease virus monoclonal antibody is a horseradish peroxidase-labeled foot-and-mouth disease virus monoclonal antibody.

5. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 1, characterized in that, The method for preparing the particle manipulation chip includes the following steps: (1) Immerse the chip in a dopamine solution to form a polydopamine coating; (2) Immerse the polydopamine-coated chip in an Nα,Nα-bis(carboxymethyl)-L-lysine solution; (3) Immerse the chip in NiCl2 solution; (4) Immerse the chip in a His-tagged recombinant VP2 protein solution to fix the VP2 protein onto the chip surface; (5) Wash, seal and dry.

6. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 5, characterized in that, The concentration of the dopamine solution in step (1) is 2-3 mg / mL, the pH is 8.0-8.5, and the time is 3-6 hours.

7. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 5, characterized in that, The concentration of the Nα,Nα-bis(carboxymethyl)-L-lysine solution in step (2) is 1-3 mg / mL, and the time is 1-2 hours.

8. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 5, characterized in that, The concentration of the NiCl2 solution in step (3) is 5-20 mM, and the time is 30-60 minutes.

9. The foot-and-mouth disease virus antibody particle manipulation flash assay kit according to claim 5, characterized in that, The concentration of the His-tagged recombinant VP2 protein solution in step (4) is 2-4 μg / mL, and the incubation time is 30-60 minutes.

10. The application of the foot-and-mouth disease virus antibody particle manipulation flash assay kit according to any one of claims 1-9 in the detection of foot-and-mouth disease virus antibodies.

Images