West nile virus IgM antibody detection card as well as preparation method and application thereof

Abstract

The invention discloses a west nile virus IgM antibody detection card as well as a preparation method and application thereof. The west nile virus IgM antibody detection card comprises a bottom plate, absorbent paper, a reaction pad, a combination pad and a sample pad, the absorbent paper, the reaction pad, the combination pad and the sample pad are sequentially arranged on the bottom plate from bottom to top; a detection line and a quality control line which are spaced from each other are arranged on the reaction pad, the detection line is positioned at one end close to the combination pad, and the quality control line is positioned at one end close to the absorbent paper; an anti-human IgM antibody is fixed on the detection line, and a quality control antibody is fixed on the quality control line; the anti-human IgM antibody and the quality control antibody are both diluted by using a coating diluent and then lineation is performed; and the surface of the conjugate pad is coated with a Nicovirus antigen-colloidal gold conjugate and an antibody-colloidal gold conjugate of an anti-quality control antibody. The detection card provided by the invention can accurately and sensitively detect the West Nile virus antibody.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical detection, and particularly relates to a West Nile virus IgM antibody detection card as well as a preparation method and application thereof. BACKGROUND

[0002] The West Nile virus (WNV) belongs to the flavivirus genus of the Flaviviridae family, has a capsid structure, and has a single-stranded positive-strand RNA genome. The virus has homology with the Japanese encephalitis virus, St. Louis encephalitis virus, and dengue virus, and mainly causes infectious diseases of the nervous system, and can cause encephalitis symptoms and even death in severe cases. In clinical diagnosis, the detection of West Nile virus IgM antibodies has important value for early diagnosis and recent infection confirmation.

[0003] Currently, in the field of West Nile virus detection technology, the problem of cross-reaction is prominent. Due to the antigen similarity between the West Nile virus and other members of the Flaviviridae family (such as dengue, Zika, and Japanese encephalitis viruses), serological detection (such as ELISA) is prone to false positives, and needs to be combined with neutralization tests or nucleic acid detection for secondary confirmation. Secondly, it is difficult to balance the sensitivity and specificity of West Nile virus detection. Although traditional PCR has high sensitivity, low viral load samples (such as asymptomatic infected persons) are prone to missed detection. Although the rapid method such as colloidal gold test strip is convenient (15 minutes for results), it has insufficient specificity, and the coincidence rate with ELISA is only 86.5%. Although digital PCR can achieve single molecule detection, the equipment is expensive, and popularization is limited. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a West Nile virus IgM antibody detection card, which can quickly and accurately detect West Nile virus IgM antibodies.

[0005] The present application also proposes a preparation method of the West Nile virus IgM antibody detection card.

[0006] The present application also proposes a kit.

[0007] The present application also proposes an application.

[0008] According to a first aspect of the present application, a West Nile virus IgM antibody detection card is proposed, which comprises a bottom plate, an absorbent paper, a reaction pad, a binding pad and a sample pad.

[0009] The absorbent paper, the reaction pad, the binding pad and the sample pad are sequentially arranged on the bottom plate from bottom to top.

[0010] The reaction pad is provided with detection lines and quality control lines spaced apart from each other, the detection lines are located at one end close to the binding pad, and the quality control lines are located at one end close to the water absorption paper; the detection lines are fixed with anti-human IgM antibodies, and the quality control lines are fixed with quality control antibodies; the anti-human IgM antibodies and the quality control antibodies are diluted with a coating diluent and then line-marked;

[0011] The surface of the binding pad is coated with a Nile virus antigen-gold colloid combination and an antibody-gold colloid combination of anti-quality control antibodies;

[0012] The surface of the sample pad is coated with a blocking agent.

[0013] In some embodiments of the present application, the blocking agent includes mouse IgG antibodies, anti-HAMA blocking agents, and bovine serum albumin.

[0014] In some embodiments of the present application, the quality control antibodies include goat anti-chicken IgY antibodies.

[0015] In some embodiments of the present application, the antibodies of the anti-quality control antibodies include chicken IgY antibodies.

[0016] In some embodiments of the present application, the coating diluent includes a PBS buffer containing 0.005-0.02 g / mL sucrose.

[0017] According to a second aspect of the present application, a preparation method of the West Nile virus IgM antibody detection card of the first aspect of the present application is provided, and the preparation method includes the following steps:

[0018] S1: coating a labeled gold colloid solution containing a Nile virus antigen-gold colloid combination and an antibody-gold colloid combination of anti-quality control antibodies on the surface of a glass fiber membrane to obtain a binding pad;

[0019] S2: immersing the glass fiber membrane in a sample pad treatment solution containing a blocking agent to obtain a sample pad;

[0020] S3: line-marking and fixing anti-human IgM antibodies and quality control antibodies diluted with a coating diluent on the surface of a nitrocellulose membrane to form detection lines and quality control lines, and obtaining a reaction pad;

[0021] S4: sequentially arranging a water absorption pad, a reaction pad, a binding pad, and a sample pad from bottom to top on the bottom plate to obtain the West Nile virus IgM antibody detection card.

[0022] In some embodiments of the present application, the preparation method of the Nile virus antigen-gold colloid combination in step S1 includes the following steps:

[0023] The colloidal gold sol is mixed with the West Nile virus antigen, and after incubation, blocking and purification treatment are performed to obtain the West Nile virus antigen-colloidal gold combination; wherein the mixing ratio of the colloidal gold sol and the West Nile virus antigen is 1:(5-20) mL / μg, and the mass fraction of chloroauric acid in the colloidal gold sol is 0.01%-0.05%.

[0024] In some embodiments of the present application, the preparation method of the antibody-colloidal gold combination of the quality control antibody in step S1 comprises the following steps:

[0025] The antibody of the quality control antibody is mixed with the West Nile virus antigen, and after incubation, blocking and purification treatment are performed to obtain the antibody-colloidal gold combination of the quality control antibody; wherein the mixing ratio of the colloidal gold sol and the antibody of the quality control antibody is 1:(5-20) mL / μg, and the mass fraction of chloroauric acid in the colloidal gold sol is 0.01%-0.05%.

[0026] In some embodiments of the present application, the blocking is performed using 1%-5% casein solution.

[0027] In some embodiments of the present application, the mass ratio of the West Nile virus antigen-colloidal gold combination and the antibody-colloidal gold combination of the quality control antibody is (1-3):1.

[0028] In some embodiments of the present application, after the coating in step S1, the combination pad is vacuum dried at 35-40°C for 12-18h.

[0029] In some embodiments of the present application, the sample pad treatment solution in step S2 comprises the following components at the following final concentrations: 5-30 mg / mL sucrose, 2-10 mg / mL mouse IgG antibody, 0.5-3 mg / mL anti-HAMA blocking agent, 0.5-3 mg / mL Proclin 300, 0.1-2 mg / mL Tween-20 and 5-30 mg / mL BSA, and the solvent is Tris buffer.

[0030] In some embodiments of the present application, the immersion time in step S2 is 0.5-2h.

[0031] In some embodiments of the present application, after the immersion in step S2, the sample pad is baked at 35-40°C for 8-12h.

[0032] In some embodiments of the present application, the coating diluent in step S3 comprises PBS buffer containing 5-20 mg / mL sucrose.

[0033] In some embodiments of the present application, after the fixing in step S3, the reaction pad is dried at 40-60 DEG C for 48-56 hours.

[0034] In some embodiments of the present application, the preparation method of the colloidal gold sol comprises:

[0035] After heating the chloroauric acid to boiling, the trisodium citrate solution is quickly injected, and the solution color changes from bright yellow to nearly colorless; continue to heat and observe the solution change from transparent to black, and finally present a purple red phase transition process, and the room temperature is cooled to obtain.

[0036] According to a third aspect of the present application, a kit for detecting West Nile virus antibody is provided, which comprises the West Nile virus IgM antibody detection card according to the first aspect of the present application or the West Nile virus IgM antibody detection card prepared by the preparation method according to the second aspect of the present application; and a sample diluent.

[0037] In some embodiments of the present application, the sample diluent comprises Tris buffer (pH 9.0) containing 0.5% Tween-20.

[0038] According to a fourth aspect of the present application, the West Nile virus IgM antibody detection card according to the first aspect of the present application or the West Nile virus IgM antibody detection card prepared by the preparation method according to the second aspect of the present application is applied to prepare a product for detecting West Nile virus IgM antibody.

[0039] The present application has at least the following beneficial effects:

[0040] The kit for detecting West Nile virus antibody provided by the present application meets the rapid detection demand of the virus, and through specific antigen-antibody reaction, the visualization of West Nile virus IgM antibody can be realized directly according to the color development result of the detection line without professional equipment assistance. Moreover, the reagents in the kit provided by the present application have high stability, the detection sensitivity of the present application is higher than that of the enzyme-linked immunoassay kit, reaching 98%; at the same time, false negative or false positive problems are less likely to occur. This instant detection (POCT) method does not require complex sample processing or additional color development reagents, and is particularly suitable for on-site rapid screening and early infection identification, and provides effective technical support for timely clinical intervention. BRIEF DESCRIPTION OF DRAWINGS

[0041] The present application will be further described below in combination with the drawings and examples, in which:

[0042] Figure 1 FIG. 1 is a structural schematic diagram of the West Nile virus IgM antibody detection card in Example 1 of the present application;

[0043] Figure 2Schematic diagram for judging the color development result in Example 2 of the present application;

[0044] Figure 3 Appearance diagram of the coating diluent of four different formulations in the test example of the present application when freshly prepared;

[0045] Figure 4 Appearance diagram of the coating diluent of four different formulations in the test example of the present application after standing at room temperature for 1 month.

[0046] Reference signs:

[0047] Sample pad 1;

[0048] Bonding pad 2;

[0049] Reaction pad 3, detection line 31, and quality control line 32;

[0050] Absorbent paper 4;

[0051] Base plate 5. DETAILED DESCRIPTION

[0052] The concept and technical effects of the present application will be described below in conjunction with examples for a clear and complete understanding of the purpose, features and effects of the present application. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0053] The following examples facilitate a better understanding of the present application, but do not limit the present application.

[0054] In the following examples, the experimental methods are all conventional methods unless otherwise specified.

[0055] In the following examples, the experimental materials used are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0056] In the following examples, the percentage of substances refers to mass fraction unless otherwise specified.

[0057] Example 1

[0058] The present example provides a kit for detecting West Nile virus antibody, which comprises a West Nile virus IgM antibody detection card and a sample diluent. The sample diluent is Tris buffer (pH 9.0) containing 0.5% Tween-20. The West Nile virus IgM antibody detection card comprises a PVC base plate, absorbent paper, a reaction pad, a bonding pad and a sample pad, and the specific preparation method is as follows:

[0059] 1. Preparation of colloidal gold sol:

[0060] (1) Preparation of colloidal gold precursor: Take 3 mL of 0.1 g / mL HAuCl4 solution and dilute to 1 L with purified water, and place it in an electric heating jacket. Heat to boiling at a stirring rate of 300 r / min;

[0061] (2) Reaction: After maintaining vigorous boiling for 60 seconds, quickly inject 3 mL of 0.1 g / mL trisodium citrate solution. The solution color changes from bright yellow to nearly colorless. Continue heating and observe the solution changing from transparent to black, and finally showing a purple-red phase change process. When a stable purple-red color appears, reduce the stirring rate to 200 r / min and terminate heating. Cool to 25±3°C at room temperature to obtain a highly dispersed colloidal gold sol.

[0062] 2. Immunolabeling agent construction:

[0063] (1) Preparation of West Nile virus recombinant antigen-colloidal gold conjugate solution:

[0064] ① pH adjustment: Take 1 mL of colloidal gold sol prepared in step 1 and adjust the system pH to 8.5 with 0.2 mol / L K2CO3;

[0065] ② Antigen coupling: Add 10 μg (8-15 μg can be used) of West Nile virus recombinant antigen (Maiyue Biological, MR870202), and incubate at room temperature for 18 min (15-20 min can be used) to complete directional coupling;

[0066] ③ Site blocking: Add 20 μL of 0.03 g / mL casein solution and continue to incubate for 20 min to block unbound sites;

[0067] ④ Purification and preservation: Centrifuge at 12000 rpm and 4°C for 25 min, then take the precipitate and resuspend it in 100 μL of PBS buffer (pH 7.4) containing 0.01 g / mL BSA, 0.1 g / mL sucrose, and 0.5 mg / mL Proclin to obtain the West Nile virus recombinant antigen-colloidal gold conjugate solution.

[0068] (2) Preparation of chicken IgY antibody-colloidal gold conjugate solution:

[0069] ① pH adjustment: Take 1 mL of colloidal gold sol prepared in step 1 and adjust the system pH to 8.5 with 0.2 mol / L K2CO3;

[0070] ② Antigen coupling: Add 18 μg (15-20 μg can be used) of chicken IgY antibody (Feifeng Biological, chicken IgY-3#),

[0071] incubate at room temperature for 18 min (15-20 min can be used) to complete directional coupling;

[0072] (3) Site blocking: 20 μL of 0.03 g / mL casein solution was added to continue incubation for 20 min to block unbound sites;

[0073] (4) Purification and storage: after centrifugation at 12000 rpm and 4°C for 25 min, the precipitate was resuspended in 100 μL of PBS buffer (pH 7.4) containing 0.01 g / mL BSA, 0.1 g / mL sucrose and 0.5 mg / mL Proclin to obtain a chicken IgY antibody-gold conjugate solution.

[0074] 3. Preparation of functional components:

[0075] (1) Formation of conjugate pad:

[0076] The West Nile virus recombinant antigen-gold conjugate solution, chicken IgY antibody-gold conjugate solution and gold labeling resuspension solution prepared in step 2 were mixed in a volume ratio of 2:1:1 to obtain a labeled gold solution. The labeled gold solution was uniformly sprayed on a glass fiber membrane with a width of 8 mm at a spraying amount of 8 μL / cm using a gold spraying instrument, and was vacuum dried at 37°C for 15 h (12-18 h) to obtain a conjugate pad. The formulation of the above gold labeling resuspension solution is as follows: 0.05 mol / L Tris buffer solution (pH 9.0) containing 0.01 g / mL BSA, 0.15 g / mL sucrose, 1 mg / mL ProClin 300 and 1 mg / mL Tween-20.

[0077] (2) Sample pad treatment:

[0078] The glass fiber membrane with a width of 18 mm was immersed in a sample pad treatment solution (0.01 g / mL sucrose, 4 mg / mL mouse IgG antibody (original valley biological), 1 mg / mL anti-HAMA blocking agent (Feifeng biological, HIER-E-010), 1 mg / mL Proclin 300, 0.5 mg / mL Tween-20 and 0.01 g / mL BSA in a pH 7.4 Tris buffer system) for 1 h and then dried at 37°C for 10 h to obtain a sample pad.

[0079] (3) Reaction pad construction:

[0080] A nitrocellulose membrane (chromatographic rate 130 s / 4 cm) was fixed on a three-section structure PVC substrate (size: upper 15 mm x middle 25 mm x lower 20 mm), and goat anti-chicken IgY antibody (Feifeng Biological, goat anti-chicken IgY-3#) and anti-human IgM antibody (Feifeng Biological, anti-u-3#) were diluted to 1 mg / mL working solution using coating diluent, respectively, and then a pneumatic dot membrane system was used to dot the detection line (anti-human IgM antibody) and the control line (goat anti-chicken IgY antibody), dried at 50°C for 50 h (48-56 h) to form a double-line recognition system, with the control line being 25 mm from the top edge of the substrate and the detection line being 29 mm from the bottom edge, and the distance between the two lines being 6 mm ± 0.5 mm. Among them, the coating diluent was 20 mmol / L PBS buffer solution (pH 7.4) containing 0.01 g / mL sucrose by mass fraction.

[0081] 4. Chromatographic assembly:

[0082] 1-2 mm lamination was performed in the order of water absorption pad (width 17 mm)-reaction pad-combination pad-sample pad, and was cut into 3.5 mm wide test strips to obtain the West Nile virus IgM antibody detection card, and a structural diagram thereof is shown in Figure 1 .

[0083] Figure 1 The West Nile virus IgM antibody detection card shown in the structural diagram comprises a sample pad 1, a combination pad 2, a reaction pad 3, a water absorption paper 4, and a bottom plate 5; the sample pad 1, the combination pad 2, the reaction pad 3, and the water absorption paper 4 are sequentially arranged on the bottom plate 5 from left to right; the combination pad 2 is connected with the sample pad 1 and the reaction pad 3 at both ends, respectively, and the end of the reaction pad 3 away from the combination pad 2 is connected with the water absorption paper 4; the reaction pad 3 is provided with a detection line 31 and a control line 32 spaced from each other, the detection line 31 is located at one end close to the combination pad 2, and the control line 32 is located at one end close to the water absorption paper 4; the surface of the combination pad 2 is coated with a Nile virus antigen-colloidal gold conjugate and an antibody-colloidal gold conjugate of an anti-control antibody, the detection line 31 is fixed with an anti-human IgM antibody, and the control line 32 is fixed with a goat anti-chicken IgY antibody.

[0084] Example 2

[0085] The present embodiment provides a West Nile virus IgM antibody detection method based on the kit for detecting West Nile virus antibodies prepared in Example 1, comprising the following steps:

[0086] The sample to be tested was gradient diluted (2, 4, 8 times dilution) using Tris buffer solution (pH 9.0) containing 5 mg / mL Tween-20, 50 μL of the diluted sample was added to the sample pad, and then reacted for 15-30 min, and visual judgment was performed; a result determination diagram is shown in Figure 2 .

[0087] This detection system improves the detection sensitivity by 2 to 3 levels compared to traditional methods through the control of nanomaterial morphology and optimization of process parameters, with an intra-batch coefficient of variation of <8% and room temperature storage stability of up to 18 months.

[0088] Test case

[0089] This experiment investigated the effects of different coating diluent formulations and different sample pad treatment formulations on the detection results, and tested the detection performance of the kit provided in Example 1 for detecting West Nile virus antibodies using clinical samples. The specific experimental methods and results are as follows:

[0090] 1. The effect of different coating diluent formulations on detection results

[0091] In this experiment, 20 mmol / L PBS buffer (pH 7.4) was used as the base solution. Four different coating diluents were prepared according to the formula shown in Table 1. After sterilization by 0.22 μm filter membrane, the resulting coating diluents should meet the following technical indicators: transmittance > 95% (λ = 600 nm), conductivity ≤ 200 μS / cm, and no visible suspended matter or crystal precipitation.

[0092] Then, the performance of the four coating diluents was tested based on the West Nile virus IgM antibody detection card system provided in Example 1. All reagents and preparation methods, except for the coating diluents, were consistent with those in Example 1. Colorimetric ability and stability tests were performed based on clinical samples, and the results are shown in Table 2. Figure 3 and Figure 4 As shown.

[0093] Table 1 Formulations of four coating diluents

[0094]

[0095]

[0096] Table 2. Effects of different coating diluents on colorimetric contrast.

[0097] Sample Coating liquid 1 Coating liquid 2 Coating liquid 3 Coating liquid 4 Sample No. 1 +++ ++ +++ +++ Sample No. 2 +++ +++ ++ + Sample No. 3 +++ +++ ++ + Sample No. 4 +++ +++ +++ +++ Sample No. 5 +++ +++ +++ +++ Sample No. 6 +++ +++ ++ +++ Sample No. 7 +++ ++ ++ ++ Sample No. 8 +++ +++ +++ +++ Sample No. 9 +++ +++ +++ +++ Sample No. 10 +++ ++ ++ ++

[0098] Figure 3 The fresh appearance of four different coating diluents upon completion of preparation. Figure 4 The appearance after standing at room temperature for 1 month.

[0099] Combined with Table 2, Figure 3 and Figure 4 The results showed that coating solution 1 had the most stable color development effect and could maintain a colorless and clear state without crystal precipitation even after long-term storage. Therefore, based on multi-dimensional performance evaluation and cost-benefit analysis, coating solution 1 was finally selected as the better coating diluent formulation.

[0100] 2. The influence of different sample pad treatment liquid formulations on detection results

[0101] In this test example, 0.01 mol / L Tris buffer (pH 7.4) was used as the buffer system, and four different sample pad treatment liquids were prepared according to the formulations shown in Table 3. Then, the performance of the above-mentioned four sample pad treatment liquids was tested based on the West Nile virus IgM antibody detection card system provided in Example 1, and the reagents and preparation methods other than the sample pad treatment liquid were consistent with Example 1.

[0102] Table 3 Formulations of four sample pad treatment liquids

[0103]

[0104] 1) Coincidence rate test:

[0105] The positive and negative coincidence rates of the test were tested using samples with known results, and 25 positive and 25 negative samples were selected. The blank control group was the group that omitted the sample pad treatment liquid. The results are shown in Table 4.

[0106] Table 4 Coincidence rate test

[0107] Group Number of positive samples detected Number of negative samples detected Accuracy Treatment liquid 1 25 25 100％ Treatment liquid 2 24 25 98％ Treatment liquid 3 24 25 98％ Treatment liquid 4 23 25 96％ Blank control 25 23 96％

[0108] As can be seen from Table 4, the blank control group had a false positive result due to not using the sample pad treatment liquid for treatment. The addition of an appropriate dose of blocking agent (mouse IgG antibody and anti-HAMA blocking agent) can effectively solve the false positive problem. None of treatment liquids 1-4 had a false positive problem. Furthermore, treatment liquid 1 also did not have a false negative, i.e., a missed detection, and treatment liquids 2-4 all had false negative results.

[0109] 2) Sensitivity test:

[0110] Three detection limit reference products were tested. The three reference products were all weakly positive. If the detection result was positive, the sensitivity was good. Detection limit reference products L1, L2, and L3 were samples with suspicious results when tested for the first time using the ELISA West Nile virus kit, and the ISR value was positive after repeated testing.

[0111] Table 5 Sensitivity test

[0112] Sample L1 L2 L3 Treatment liquid 1 + + + Treatment liquid 2 + + - Treatment liquid 3 + + - Treatment liquid 4 + + -

[0113] 3) Reproducibility test:

[0114] The same sample was tested five times, and the differences in the five test results were observed. If the five test results were the same, the reproducibility was good.

[0115] Table 6 Repetitive test

[0116] Times 1 2 3 4 5 Treatment liquid 1 +++ +++ +++ +++ +++ Treatment liquid 2 +++ +++ ++ +++ +++ Treatment liquid 3 +++ +++ +++ +++ +++ Treatment liquid 4 +++ +++ +++ ++ +++

[0117] Based on multi-dimension performance evaluation and cost-benefit analysis, the sample pad treatment liquid 1 is finally selected as the better sample pad treatment liquid formula.

[0118] 3. Clinical performance evaluation:

[0119] Using the kit for detecting the antibody of the Nile virus provided in Example 1 and the enzyme-linked immunoassay kit (purchased from Guangzhou Jianlun Biological Technology Co., Ltd.) to detect the serum samples of 50 patients infected with the West Nile virus and 50 normal persons respectively, and the detection results are shown in Table 6.

[0120] Table 6 Clinical sample test

[0121]

[0122]

[0123] As shown in Table 6, the kit for detecting the antibody of the West Nile virus provided in Example 1 has excellent specificity in detecting the serum sample, and has higher detection sensitivity of 98% compared with the enzyme-linked immunoassay kit.

[0124] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A West Nile virus IgM antibody detection card, characterized in that, The West Nile virus IgM antibody detection card includes a base plate, absorbent paper, reaction pad, conjugate pad, and sample pad; The absorbent paper, reaction pad, conjugate pad, and sample pad are arranged sequentially from bottom to top on the base plate; The reaction pad is provided with a detection line and a control line spaced apart from each other. The detection line is located at one end near the conjugate pad, and the control line is located at one end near the absorbent paper. Anti-human IgM antibody is fixed on the detection line, and control antibody is fixed on the control line. Both the anti-human IgM antibody and the control antibody are diluted with coating diluent before being drawn. The surface of the conjugate pad is coated with Nile virus antigen-colloidal gold conjugate and antibody-colloidal gold conjugate against quality control antibody; The sample pad surface is coated with a blocking agent.

2. The West Nile virus IgM antibody detection card according to claim 1, characterized in that, The blocking agents include mouse IgG antibodies, anti-HAMA blocking agents, and bovine serum albumin.

3. The West Nile virus IgM antibody detection card according to claim 1, characterized in that, The quality control antibody includes goat anti-chicken IgY antibody; Preferably, the antibody against the quality control antibody includes chicken IgY antibody.

4. The West Nile virus IgM antibody detection card according to claim 1, characterized in that, The coating diluent includes PBS buffer containing 0.005–0.02 g / mL sucrose.

5. The method for preparing the West Nile virus IgM antibody detection card according to any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: S1: A labeled colloidal gold solution containing Nile virus antigen-colloidal gold conjugate and anti-quality control antibody is coated onto the surface of a glass fiber membrane to obtain a conjugate pad; S2: Impregnate the glass fiber membrane with a sample pad treatment solution containing a blocking agent to obtain a sample pad; S3: Anti-human IgM antibody and quality control antibody diluted with coating diluent are streaked and fixed on the surface of nitrocellulose membrane to form detection line and quality control line, respectively, to obtain reaction pad; S4: The absorbent pad, reaction pad, conjugate pad, and sample pad are sequentially placed on the base plate from bottom to top to obtain the West Nile virus IgM antibody detection card.

6. The preparation method according to claim 5, characterized in that, The preparation method of the Nile virus antigen-colloidal gold conjugate in step S1 includes the following steps: The colloidal gold sol was mixed with West Nile virus antigen and incubated. After incubation, the mixture was blocked and purified to obtain the Nile virus antigen-colloidal gold conjugate. The mixing ratio of the colloidal gold sol to the West Nile virus antigen was 1:(5-20)mL / μg, and the mass fraction of chloroauric acid in the colloidal gold sol was 0.01%-0.05%. Preferably, the preparation method of the antibody-colloidal gold conjugate against the quality control antibody in step S1 includes the following steps: The antibody against the quality control antibody was mixed with West Nile virus antigen and incubated. After incubation, the mixture was blocked and purified to obtain the antibody-colloidal gold conjugate against the quality control antibody. The mixing ratio of the colloidal gold sol to the antibody against the quality control antibody was 1:(5-20)mL / μg, and the mass fraction of chloroauric acid in the colloidal gold sol was 0.01%-0.05%. Preferably, the mass ratio of the Nile virus antigen-colloidal gold conjugate to the antibody-colloidal gold conjugate of the quality control antibody is (1-3):

1.

7. The preparation method according to claim 5, characterized in that, The sample pad treatment solution described in step S2 comprises the following components at final concentrations: 5–30 mg / mL sucrose, 2–10 mg / mL mouse IgG antibody, 0.5–3 mg / mL anti-HAMA blocker, 0.5–3 mg / mL Proclin 300, 0.1–2 mg / mL Tween-20, and 5–30 mg / mL BSA, in Tris buffer.

8. The preparation method according to claim 5, characterized in that, The coating diluent described in step S3 includes a PBS buffer containing 5–20 mg / mL sucrose.

9. A kit for detecting West Nile virus antibodies, characterized in that, The kit includes a West Nile virus IgM antibody detection card according to any one of claims 1 to 4 or a West Nile virus IgM antibody detection card prepared by the preparation method according to any one of claims 5 to 8; and a sample diluent.

10. The use of the West Nile virus IgM antibody detection card according to any one of claims 1 to 4 or the West Nile virus IgM antibody detection card prepared by the preparation method according to any one of claims 5 to 8 in the preparation of products for detecting West Nile virus IgM antibodies.

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