Detection test strip and detection card for detecting coxsackie virus group B IgM antibody as well as preparation method and application of detection test strip and detection card

Abstract

The invention discloses a detection test strip and a detection card for detecting a coxsackie virus group B IgM antibody as well as a preparation method and application of the detection test strip and the detection card. The detection test strip comprises a bottom plate as well as a nitrocellulose membrane, absorbent paper, a conjugate pad and a sample pad which are sequentially overlapped and fixed on the bottom plate, the nitrocellulose membrane is coated with a detection line and a quality control line, the detection line is coated with an antigen of an anti-coxsackie virus group B IgM antibody, and the quality control line is coated with an IgG polyclonal antibody; the conjugate pad comprises an antigen conjugate of a colloidal gold-anti-coxsackie virus group B IgM (Immunoglobulin M) antibody; the particle size of the colloidal gold is larger than or equal to 100 nm. The detection test strip prepared by the invention is high in sensitivity, good in repeatability and strong in specificity, and can be used for detecting the coxsackie virus group B IgM antibody. The device is convenient to use, easy to operate, short in consumed time and convenient to carry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a detection test strip for detecting coxsackievirus B group IgM antibody, a detection card and a preparation method and application thereof. BACKGROUND

[0002] Coxsackievirus B group belongs to small RNA virus and belongs to enterovirus family, and its infection can cause various clinical manifestations, from mild respiratory infection to severe myocarditis, meningitis and the like.

[0003] At present, the coxsackievirus B group virus diagnosis for clinical and scientific research mainly includes etiological diagnosis, molecular biology diagnosis and immunological diagnosis, etc. The etiological diagnosis mainly separates and identifies the pathogen in clinical urine, blood and the like through the method of cell culture, and has high specificity, large technical operation difficulty, complicated operation, long separation period, is mainly applied to scientific research and teaching aspects, is not suitable for large-scale, rapid and accurate clinical detection demand, has certain condition restriction, and is time-consuming. Molecular biology mainly detects pathogen nucleic acid, and this method has strict requirements for the collection of samples, such as time, position and the like, and has high requirements for experimental technology, and false positive results are easy to produce. The immunological diagnosis mainly detects the antibody in serum, including colloidal gold method and enzyme-linked immunosorbent assay. The colloidal gold method generally carries out experimental operation manually, and finally carries out qualitative determination by naked eye, and this technology has low sensitivity, is easy to appear false positive, and is greatly influenced by human subjective factors. The sensitivity of enzyme-linked immunosorbent assay is improved compared with colloidal gold method, but is still low, and the linear range is narrow, and the reaction time is also long, and the clinical application cannot be well met. SUMMARY

[0004] The present application aims at at least solving one of the technical problems existing in the prior art. To this end, the present application provides a detection test strip for detecting coxsackievirus B group IgM antibody, which can specifically detect coxsackievirus B group IgM antibody.

[0005] The present application also provides a preparation method of the above-mentioned detection test strip.

[0006] The present application also provides the application of the above-mentioned detection test strip.

[0007] The present application also provides a detection card for detecting coxsackievirus B group IgM antibody.

[0008] According to the first aspect of the present application, a detection test strip for detecting coxsackievirus B group IgM antibody is provided, which comprises a bottom plate and a nitrocellulose membrane, a water absorption paper, a combination pad and a sample pad which are sequentially lapped and fixed on the bottom plate.

[0009] The nitrocellulose membrane is coated with a detection line and a quality control line, the detection line is coated with an antigen of anti-cosxackie virus group B IgM antibody, and the quality control line is coated with an IgG polyclonal antibody;

[0010] The binding pad contains a colloidal gold-anti-cosxackie virus group B IgM antibody antigen conjugate.

[0011] The particle size of the colloidal gold is greater than or equal to 100 nm.

[0012] The present application adopts colloidal gold with a length of 100 nm and above, and the two ends also have a larger curvature, which can avoid the problem of labeling efficiency caused by steric hindrance when the antibody is combined, and effectively improve the detection sensitivity and the detection background is cleaner.

[0013] According to some embodiments of the present application, the bottom plate comprises at least one of a polyvinyl chloride (PVC) plate, a polyethylene plate, and a glass sheet.

[0014] According to some embodiments of the present application, the preparation method of the nitrocellulose membrane comprises the following steps:

[0015] Prepare a coating liquid and a detection mixture of the antigen of anti-cosxackie virus group B IgM antibody, draw a line on the nitrocellulose membrane to form a detection line; prepare a quality control mixture of the coating liquid and the IgG polyclonal antibody, draw a line on the nitrocellulose membrane to form a quality control line, and dry it to obtain it;

[0016] The coating liquid is a buffer solution comprising sucrose and dextran.

[0017] According to some embodiments of the present application, the IgG polyclonal antibody comprises a goat anti-mouse IgG polyclonal antibody.

[0018] According to some embodiments of the present application, the concentration of sucrose in the buffer solution is 1-2 (w / v) %. It can be 1 (v / v) %, 1.5 (v / v) %, 1.8 (v / v) %, 2 (v / v) %.

[0019] According to some embodiments of the present application, the concentration of dextran in the buffer solution is 1-2 (w / v) %. It can be 1 (v / v) %, 1.5 (v / v) %, 1.8 (v / v) %, 2 (v / v) %.

[0020] According to some embodiments of the present application, the buffer solution comprises at least one of a PBS buffer solution and a PB buffer solution.

[0021] According to some embodiments of the present application, the concentration of the PBS buffer solution is 0.01-0.05 M. For example: it can be 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M.

[0022] According to some embodiments of the application, the pH of the buffer solution is 7.0-8.0. For example, the pH can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0023] According to some embodiments of the application, the sample pad comprises at least one of a glass fiber membrane, a high molecular polymer membrane.

[0024] According to some embodiments of the application, the method for preparing the sample pad comprises the following steps:

[0025] The sample pad treatment solution is allowed to soak the sample pad, and the sample pad is dried to obtain the sample pad.

[0026] The sample pad treatment solution is a buffer solution comprising 1-2 (w / v)% sucrose, 1-2 (w / v)% trehalose, 0.4-0.8 (w / v)% blocker 003, 0.1-0.2 (w / v)% blocker 004, 0.05-1 (w / v)% Tween-20, and 1-2 (w / v)% BSA.

[0027] According to some embodiments of the application, the sample pad treatment solution is a buffer solution comprising sucrose at a concentration of 0.01-0.02 mg / mL, trehalose at a concentration of 0.01-0.02 mg / mL, blocker 003 at a concentration of 0.004-0.008 mg / mL, blocker 004 at a concentration of 0.001-0.002 mg / mL, bovine serum albumin at a concentration of 0.01-0.02 mg / mL, and 0.05-1 mL Tween-20.

[0028] According to some embodiments of the application, the blocker 003 comprises mouse IgG antibodies.

[0029] According to some embodiments of the application, the blocker 004 comprises an anti-HAMA blocker.

[0030] According to some embodiments of the application, the sample pad treatment solution comprises sucrose at a concentration of 0.01 mg / mL, trehalose at a concentration of 0.01 mg / mL, blocker 003 at a concentration of 0.004 mg / mL, blocker 004 at a concentration of 0.002 mg / mL, bovine serum albumin at a concentration of 0.01 mg / mL, and 0.5 mL Tween-20.

[0031] According to some embodiments of the application, the sample pad treatment solution further comprises a preservative.

[0032] According to some embodiments of the present application, the preservative comprises at least one of ProClin 300, ProClean 150.

[0033] According to some embodiments of the present application, the concentration of the preservative is 0.1-0.2 (v / v)%. For example, it can be 0.1 (v / v)%, 0.15 (v / v)%, 0.18 (v / v)%, 0.2 (v / v)%.

[0034] According to some embodiments of the present application, the amount of the preservative added is 0.05-0.2 mL.

[0035] According to some embodiments of the present application, the buffer comprises at least one of PBS buffer, PB buffer.

[0036] According to some embodiments of the present application, the concentration of the PBS buffer is 0.01-0.05 M. For example, it can be 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M.

[0037] According to some embodiments of the present application, the pH of the buffer is 7.0-8.0. For example, the pH can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0038] According to some embodiments of the present application, the temperature of the drying in the preparation method of the sample pad is 35-40℃. For example, the temperature of the drying is 35℃, 36℃, 37℃, 38℃, 39℃, 40℃.

[0039] According to some embodiments of the present application, the time of the drying in the preparation method of the sample pad is 8-12 h. For example, the time of the drying is 8 h, 9 h, 10 h, 11 h, 12 h.

[0040] According to some embodiments of the present application, the preparation method of the colloidal gold comprises the following steps:

[0041] After the HAuCl4 solution is heated to boiling, a reducing agent is added for reaction;

[0042] The mass ratio of the HAuCl4 to the reducing agent is 1:(1-5).

[0043] According to some embodiments of the present application, the reducing agent comprises at least one of trisodium citrate, citric acid.

[0044] According to some embodiments of the present application, the concentration of HAuCl4 in the HAuCl4 solution is 0.01-0.04 wt%. For example, it can be 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%.

[0045] According to some embodiments of the present application, the method further comprises the step of mixing the HAuCl4 solution with 950-1100 mL of deionized water.

[0046] According to some embodiments of the present application, the method for preparing the colloidal gold specifically comprises the following steps:

[0047] After mixing the HAuCl4 solution with 950-1100 mL of deionized water, heating to boiling at a rotation speed of 300 r / min-500 r / min, adding a reducing agent, waiting for the solution to become transparent purple red, reducing the rotation speed to 100 r / min-300 r / min, and stopping heating. The method for preparing the colloidal gold provided by the present application has good stability of the prepared colloidal gold solution, which can be stored for a long time.

[0048] According to some embodiments of the present application, after adding the reducing agent, the color change further comprises the solution changing from the original bright yellow to nearly colorless, continuing to heat to boiling, and observing that the solution changes from transparent to black and then from black to purple red transparent.

[0049] According to some embodiments of the present application, the method for preparing the colloidal gold-anti-Coxsackievirus group B IgM antibody antigen conjugate comprises the following steps: mixing the colloidal gold solution with the anti-Coxsackievirus group B IgM antibody antigen, incubating for 15-30 min, and adding a blocking solution to obtain the colloidal gold-anti-Coxsackievirus group B IgM antibody antigen conjugate.

[0050] In some embodiments of the present application, the mass-volume ratio of the anti-Coxsackievirus group B IgM antibody to the colloidal gold solution is 40-80 μg: 1 mL.

[0051] In some embodiments of the present application, the blocking solution comprises a BSA solution.

[0052] In some embodiments of the present application, the concentration of the BSA solution is 8%-12%.

[0053] In some embodiments of the present application, the concentration of the BSA solution is 9%-11%.

[0054] In some embodiments of the present application, the method further comprises the step of centrifuging the colloidal gold-anti-Coxsackievirus group B IgM antibody antigen conjugate obtained after blocking.

[0055] In some embodiments of the present application, the centrifuging condition is centrifuging at 3-5℃, 9,000-12,000 r / min for 30 min.

[0056] In some embodiments of the present application, the centrifugation condition is 10,000 r / min for 30 min at 4℃.

[0057] According to some embodiments of the present application, the method for preparing the conjugate pad comprises the following steps:

[0058] The antigen conjugate of colloidal gold-anti-Coxsackie virus B group IgM antibody is sprayed on the conjugate pad, and dried to obtain.

[0059] According to some embodiments of the present application, the conjugate pad comprises at least one of glass fiber membrane and high molecular polymer membrane.

[0060] According to some embodiments of the present application, the width of the conjugate pad is 7-9 mm. For example, the width can be 7 mm, 8 mm or 9 mm.

[0061] According to some embodiments of the present application, the spraying amount of the antigen conjugate of colloidal gold-anti-Coxsackie virus B group IgM antibody is 7-9 μL / cm. For example, the spraying amount can be 7 μL / cm, 8 μL / cm or 9 μL / cm.

[0062] According to some embodiments of the present application, the drying temperature in the method for preparing the conjugate pad is 35-40℃. For example, the drying temperature can be 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

[0063] According to some embodiments of the present application, the drying time in the method for preparing the conjugate pad is 2-4 h. For example, the drying time can be 2 h, 3 h or 4 h.

[0064] The test strip according to the embodiments of the present application has at least the following beneficial effects:

[0065] The test strip prepared by the present application has high sensitivity, good repeatability and strong specificity, and can be used for detecting Coxsackie virus B group IgM antibody. The test strip is convenient to use, simple to operate, time-saving and convenient to carry.

[0066] The method for preparing the test strip according to the first aspect of the second aspect embodiments of the present application comprises the following steps:

[0067] The nitrocellulose membrane, the absorbent paper, the conjugate pad and the sample pad are sequentially attached to the bottom plate.

[0068] The test strip according to the first aspect of the third aspect embodiments of the present application is applied to the preparation of a product for detecting Coxsackie virus B group IgM antibody.

[0069] According to a fourth aspect of the present application, a detection card for detecting Coxsackie virus B group IgM antibody is provided, which comprises the detection test strip as described in the first aspect of the present application.

[0070] Other features and advantages of the present application will be set forth in the description that follows, and in part will be apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 Flow chart for preparing the detection card for detecting Coxsackie virus B group IgM antibody;

[0072] Figure 2 Structure diagram of the detection card for detecting Coxsackie virus B group IgM antibody; wherein, 1 is a sample pad, 2 is a sample pad, 3 is a reaction pad, 4 is a sample pad, 5 is a sample pad, 31 is a detection line, and 32 is a quality control line.

[0073] Figure 3 Schematic diagram for judging positive, negative and invalid results of the detection card for detecting Coxsackie virus B group IgM antibody according to the present application. DETAILED DESCRIPTION

[0074] The concept and the technical effects of the present application will be described in detail below in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0075] In the description of the present application, if the first, second, etc. are described, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0076] When a numerical range is disclosed herein, the above range is considered to be continuous, and includes the minimum value and the maximum value of the range, and every value between the minimum value and the maximum value. Further, when the range refers to an integer, every integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0077] "and / or" is used to indicate that one or both of the described conditions can occur, for example, A and / or B includes (A and B) and (A or B).

[0078] Unless otherwise specified, the examples were carried out under conventional conditions or according to the manufacturer's instructions. Unless otherwise specified, the reagents or instruments used were conventional products available commercially.

[0079] Unless otherwise specified, "room temperature" in the present application means (25±5) °C.

[0080] Example 1

[0081] This example provides a detection card for detecting coxsackie virus group B IgM antibody, the preparation process is as shown in Figure 1 Specifically, the method is as follows:

[0082] 1. Preparation of colloidal gold solution:

[0083] Mix 4.0 mL of 10% HAuCl4 solution with 1000 mL of deionized water, heat to boiling under stirring at 400 r / min using an electric heating jacket, and then boil for 1 min. Immediately add 4 mL of 10% trisodium citrate solution, and the solution changes from bright yellow to nearly colorless. Continue heating to boiling, and observe that the solution changes from transparent to black. When the solution changes from black to purple red transparent, reduce the stirring speed to 200 r / min (maintain for 10-30 min, 20 min in this example), and stop heating. Cool and stand at room temperature for 1.5 h (1-2 h), to obtain a colloidal gold solution with a particle size of 100 nm.

[0084] 2. Antibody labeling of colloidal gold

[0085] (1) Take 1 mL of the colloidal gold solution in a 1.5 mL centrifuge tube, and add 0.2 mol / L K2CO3 aqueous solution to adjust the pH to 9.0. Mix well at room temperature for 10 min.

[0086] (2) Add 1.51 μL of anti-coxsackie virus group B IgM antibody antigen (the labeling amount is 40-80 μg, 75 μg in this example, supplied by Guangzhou Hekun Biotechnology Co., Ltd.), and incubate at room temperature for 20 min (15-30 min).

[0087] (3) Add 20 μL of 10% BSA solution, and block the excess binding sites on the colloidal gold at room temperature for 30 min.

[0088] (4) Centrifuge at 10,000 r / min at 4 °C for 30 min, discard the supernatant, and add 100 μL of gold label reconstitution solution. Gently blow to reconstitute the colloidal gold-antigen complex, and store at 4 °C for standby use.

[0089] 3. Preparation of the conjugate pad

[0090] The colloidal gold-antigen complex reconstitution solution was evenly sprayed on the 8mm wide glass fiber membrane using a gold spraying instrument, and the spraying amount was 8μL / cm. Then it was placed in a 37℃ oven for 2h to dry.

[0091] 4. Preparation of sample pad

[0092] The 18mm wide glass fiber membrane was soaked in the sample treatment solution (1% sucrose, 1% trehalose, 0.4% blocker 003 (mouse IgG antibody, purchased from Genway Biologics), 0.2% blocker 004 (anti-HAMA blocker, purchased from Fipeng Biology), 0.1% Proclin 300, 0.05% Tween-20 and 1% BSA (specific preparation method as follows: according to 100mL preparation, containing: sucrose 1mg, trehalose 1mg, 0.4mg blocker 003, 0.2mg blocker 004, 0.1mL Proclin 300, bovine serum albumin 1mg, 0.5mL Tween-20, 0.01M pH7.4 PBS 100mL)) for 1h, and then dried in a 37℃ oven for 10h to prepare the sample pad.

[0093] 5. Coating of nitrocellulose membrane

[0094] (1) Preparation of coating solution, which is 0.01M PBS solution pH7.4 containing 1% sucrose and 1% dextran by mass fraction;

[0095] (2) Using a PVC base plate with adhesive (upper 15mm + middle 25mm + lower 20mm), tear off the middle 25mm wide adhesive strip, and paste a 25cm wide nitrocellulose membrane on it (flow rate 130s);

[0096] (3) Dilute the goat anti-mouse IgG polyclonal antibody (item number 11031, supplier Dynabeads TM ) to a concentration of 1mg / mL with the coating solution to obtain the quality control line solution. Dilute the antigen of anti-coxsackie virus group B IgM antibody (item number 20230711, supplier Guangzhou Hekun Biological Technology Co., Ltd.) to a concentration of 1mg / mL with the coating solution to obtain the detection line solution;

[0097] (4) Draw the diluted coating solution on the nitrocellulose membrane with a line marker at 1μL / cm, with the quality control line 6mm away from the upper edge of the 25mm on the PVC base plate, and the detection line 6mm away from the lower edge of the 29mm on the PVC base plate;

[0098] (5) Dry the drawn base plate in a 37℃ oven for 48-56h to obtain the reaction pad.

[0099] 6. Assembly of colloidal gold immunochromatography test strip

[0100] (1) Tear off the 15mm adhesive tape on the top of the PVC base plate with the nitrocellulose membrane coated with the antibody, and stick the 17mm wide absorbent paper to the top of the base plate, so that it is aligned with the top edge of the nitrocellulose membrane and covers the top edge of the nitrocellulose membrane by 1-2mm;

[0101] (2) Tear off the 20mm adhesive tape on the bottom of the base plate, and stick the dried conjugate pad to cover the bottom edge of the nitrocellulose membrane by 1-2mm;

[0102] (3) Stick the sample pad to the bottom edge of the base plate and cover part of the conjugate pad;

[0103] (4) Finally, cut into 3mm wide test strips on the strip cutter.

[0104] 7. Obtaining of the detection card

[0105] The colloidal gold immunochromatographic test strip obtained above is placed in a shell to obtain a detection card, and a structural diagram of the detection card is shown in Figure 2 .

[0106] The use method of the detection card is as follows:

[0107] (1) Sample processing

[0108] Collect the whole blood or serum sample according to the conventional method, and ensure aseptic operation during sample collection and storage.

[0109] The whole blood sample can be anticoagulated by a conventional amount of heparin (9.8-28IU / mL), sodium citrate (3.8%), and ethylenediaminetetraacetic acid (4.55mmoL / mL±0.85mmoL / mL).

[0110] The whole blood sample cannot be frozen, and can be stored at 2-8℃ for 3 days. The sample is best detected on the day of collection, and the diluted sample should not be stored. The serum sample should not be repeatedly frozen and thawed to prevent the antibody titer from decreasing. The number of freezing and thawing times should not be more than 6 times. The serum sample can be stored at 2-8℃ for 7 days, at -20℃ for 2 years, and at -70℃ for 5 years. The sample should be mixed after thawing before detection. The turbid or precipitated sample should be centrifuged or filtered before detection.

[0111] (2) Sample detection

[0112] Drop 60μL of the sample to be tested (such as serum or whole blood) into the sample addition hole of the detection card. Observe and record the experimental results within 5-30min after sample addition.

[0113] The result judgment standard is as follows (as shown in Figure 3 ):

[0114] (1) Positive: If purple bands appear on both the quality control line and the detection line, it indicates that the sample contains Coxsackie virus B group IgM antibody.

[0115] (2) Negative: If only a purple band appears on the quality control line and no purple band appears on the detection line, it indicates that the sample does not contain Coxsackie virus B group IgM antibody or its content is below the detectable range.

[0116] (3) Invalid: The quality control line does not appear a purple band. This indicates that the operation process is incorrect or the test card is damaged, and should be retested.

[0117] Example 2

[0118] This example provides a detection card for detecting Coxsackie virus B group IgM antibody, the difference between its preparation method and that of Example 1 is only in the preparation method of colloidal gold solution, specifically, the preparation method of colloidal gold solution is as follows:

[0119] (1) Take 100 mL of deionized water and add 1 mL of 1% HAuCl4 solution to a round-bottom flask, heat to 93°C with stirring at 400 r / min using an electric heating jacket;

[0120] (2) Immediately add 0.14 mL of 1% trisodium citrate solution, the solution changes from bright yellow to nearly colorless;

[0121] (3) Continue to heat to boiling, immediately add 0.250 mL of 1 mM Na2HPO4 solution, wait for 5 s, immediately add 0.3 mL of 1% trisodium citrate solution, continue to heat to keep the mixed reaction liquid boiling;

[0122] (4) After the solution changes from gray blue to brown, reduce the stirring speed to 200 r / min and continue to heat for 15 min;

[0123] (5) Stand at room temperature for 16 h to obtain a colloidal gold solution with a particle size of 90 nm.

[0124] Example 3

[0125] This example provides a detection card for detecting Coxsackie virus B group IgM antibody, the difference between its preparation method and that of Example 1 is only in the preparation method of colloidal gold solution, specifically, the preparation method of colloidal gold solution is as follows:

[0126] (1) Take 100 mL of deionized water and add 4 mL of 1% HAuCl4 solution to a round-bottom flask, heat to boiling with stirring at 400 r / min using an electric heating jacket;

[0127] (2) Immediately add 4 mL of 1% trisodium citrate solution, the solution changes from bright yellow to nearly colorless;

[0128] (3) Continue heating to boiling, observed the solution from transparent to black;

[0129] (4) After the solution from black to purple red transparent color, reduce the stirring speed to 200 r / min stop heating;

[0130] (5) Room temperature for 16 h to get 82 nm colloidal gold solution.

[0131] Example 4

[0132] This example provides a detection card for detecting coxsackie virus group B IgM antibody, the preparation method is the same as example 1, the difference is only in the preparation method of colloidal gold solution, specifically, the preparation method of colloidal gold solution is as follows:

[0133] (1) Take 100 mL of deionized water into a round bottom flask, add 1 mL of 1% HAuCl4 solution, use electric heating sleeve 400 r / min stirring under heating to 93℃;

[0134] (2) Immediately add 0.15 mL of 1% trisodium citrate solution, the solution from originally bright yellow to nearly colorless;

[0135] (3) Continue heating to boiling, immediately add 0.250 mL of 1 mM Na2HPO4 solution, wait for 5 s immediately add 0.4 mL of 1% trisodium citrate solution, continue heating to keep the mixed reaction liquid boiling;

[0136] (4) After the solution from dark purple to red, reduce the stirring speed to 200 r / min continue heating for 15 min;

[0137] (5) Room temperature for 16 h to get 70 nm colloidal gold solution.

[0138] Example 5

[0139] This example screens the preparation conditions of test paper strip, except for the change of screening factors, other conditions are the same as example 1.

[0140] 1. Screening factor: coating liquid.

[0141] The formula of each coating liquid to be screened is shown in table 1.

[0142] Table 1

[0143] Name Sucrose (g) Glucan (g) 0.01M PBS buffer (pH 7.4) Coating liquid 1 2.00 1.00 100 mL Coating liquid 2 1.00 2.00 100 mL Coating liquid 3 1.00 1.00 100 mL

[0144] The goat anti-mouse IgG polyclonal antibody and the anti-cosavirus group B IgM antibody were diluted with different coating liquids as shown in Table 1, and nitrocellulose membranes were coated. The test cards were prepared according to the method of Example 1. The reference product of 800 IU / mL was diluted to 10 IU / mL using fetal bovine serum, and then diluted to 1 IU / mL, 0.5 IU / mL, 0.25 IU / mL, 0.125 IU / mL, and 0 IU / mL (the diluent did not contain the reference product). The prepared test cards were used for detection. The sample addition amount of each sample well was 60 uL. Timing was started after sample addition, and false positives, complete release of the gold label pad, uniformity of chromatography, and reverse white conditions at different chromatography times were recorded.

[0145] Table 2: Test results of three different coating liquids

[0146] Coating liquid 1 Coating liquid 2 Coating liquid 3 Sample concentration 1 IU / mL + + + Sample concentration 0.5 IU / mL + + + Sample concentration 0.25 IU / mL + + + Sample concentration 0.125 IU / mL + + + Sample concentration 0 IU / mL - - - False positive No No No Gold mark pad release Complete Complete Complete Whether the chromatography is uniform Chromatography uniform Chromatography uniform Chromatography uniform Chromatography 5 minutes reverse white case Red Red Red Chromatography 8 minutes reverse white case Red Red Red Chromatography 10 minutes reverse white case Red Red White Chromatography 13 minutes reverse white case White White White Chromatography 15 minutes reverse white case White White White Chromatography 20 minutes reverse white case White White White Chromatography 25 minutes reverse white case White White White Chromatography 30 minutes reverse white case White White White

[0147] The results are shown in Table 2. As can be seen from the table, considering the sensitivity, specificity, chromatography reverse white conditions, and cost factors, the 0.01M PBS solution containing 1% sucrose and 1% dextran was selected as the final formula of the coating liquid.

[0148] 2. Screening factor: sample pad treatment liquid

[0149] The formula of each sample pad treatment liquid to be screened is shown in Table 3.

[0150] Table 3

[0151]

[0152] The sample pads were prepared using different sample pad treatment liquids as shown in Table 3, and the test cards were prepared. The reference product of 800 IU / mL was diluted to 10 IU / mL using fetal bovine serum, and then diluted to 1 IU / mL, 0.5 IU / mL, 0.25 IU / mL, 0.125 IU / mL, and 0 IU / mL (the diluent did not contain the reference product). The prepared test cards were used for detection. The sample addition amount of each sample well was 60 uL. Timing was started after sample addition, and false positives, complete release of the gold label pad, uniformity of chromatography, and reverse white conditions at different chromatography times were recorded.

[0153] Table 4

[0154]

[0155]

[0156] The results are shown in Table 4. Considering the sensitivity, specificity, anti-interference and cost factors, the sample pad treatment liquid 4 is finally selected as the sample pad treatment liquid formula (prepared according to 100 mL: sucrose 1 mg, trehalose 1 mg, 0.4 mg blocker 003, 0.2 mg blocker 004, 0.1 mL Proclin 300, 1 mg bovine serum albumin, 0.5 mL Tween-20, 0.01 M pH 7.4 PBS 100 mL) as the sample pad treatment liquid formula.

[0157] Comparative Example 1

[0158] This example provides a detection card for detecting coxsackie virus group B IgM antibody, and the preparation method is the same as that of Example 1, except that the colloidal gold solution is replaced by colloidal gold ball solution. Specifically, the preparation method of the colloidal gold ball solution is as follows:

[0159] Mix 4.0 mL of 10% mass fraction HAuCl4 solution with 1000 mL of deionized water, heat to boiling under stirring at 400 r / min using an electric heating jacket, and then fully boil for 1 min; immediately add 4 mL of 10% trisodium citrate solution, and the solution changes from bright yellow to nearly colorless. Continue heating to boiling, and observe that the solution changes from transparent to black. Reduce the stirring speed to 200 r / min and stop heating. Cool and stand at room temperature to obtain the colloidal gold ball solution.

[0160] Detection Example

[0161] 1. Screening of colloidal gold solution

[0162] Dilute 800 IU / mL of the reference into 10 IU / mL using fetal bovine serum, and then dilute 10 IU / mL into 1 IU / mL, 0.5 IU / mL, 0.25 IU / mL, 0.125 IU / mL and 0 IU / mL (the diluent does not contain the reference). Use the detection cards prepared according to Examples 1-4 and Comparative Example 1 to perform detection, and the sample amount of each sample hole is 60 μL. Start timing after sample addition, and record the false positive of each group, the release of the gold label pad, the uniformity of the chromatography and the reverse white condition at different chromatography times.

[0163] Table 5

[0164]

[0165]

[0166] The results are shown in Table 5. It can be seen from the table that the detection card prepared according to Example 1 has obviously higher sensitivity than that of Examples 2-4 and Comparative Example 1.

[0167] 2. Detection of clinical samples

[0168] The detection card provided in Example 1 and the commercially available enzyme-linked immunoassay kit for detecting coxsackie virus group B IgM antibody were used to detect serum samples from 50 coxsackie virus infected patients and 50 healthy volunteers (not infected with coxsackie virus).

[0169] Table 6

[0170]

[0171]

[0172] The results are shown in Table 6. As can be seen from the table, the detection card provided in Example 1 has excellent specificity and sensitivity, and is superior to the commercially available enzyme-linked immunoassay kit.

[0173] The above embodiments of the present application have been described in detail, 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 spirit of the present application.

Claims

1. A test strip for detecting coxsackie virus group B IgM antibody, characterized by, The bottom plate and the nitrocellulose membrane, the water absorption paper, the binding pad and the sample pad are sequentially and fixedly connected to the bottom plate; The nitrocellulose membrane is coated with a detection line and a quality control line, the detection line is coated with an antigen of anti-Coxsackievirus B group IgM antibody, and the quality control line is coated with an IgG polyclonal antibody; The binding pad contains a colloidal gold-anti-Coxsackievirus B group IgM antibody antigen conjugate; The particle size of the colloidal gold is ≥100 nm.

2. The test strip of claim 1, wherein, The preparation method of the nitrocellulose membrane comprises the following steps: Preparation of a coating liquid and a detection mixture of the antigen of anti-Coxsackievirus B group IgM antibody, the detection mixture is drawn on the nitrocellulose membrane to form a detection line; preparation of a coating liquid and a quality control mixture of an IgG polyclonal antibody, the quality control mixture is drawn on the nitrocellulose membrane to form a quality control line, and drying is obtained; The coating liquid is a buffer solution containing sucrose and dextran; Preferably, the concentration of sucrose in the buffer solution is 1-2 (w / v) %; Preferably, the concentration of dextran in the buffer solution is 1-2 (w / v) %.

3. The test strip of claim 1, wherein, The sample pad comprises at least one of a glass fiber membrane and a high molecular polymer membrane.

4. The test strip of claim 1, wherein, The preparation method of the sample pad comprises the following steps: The sample pad treatment liquid is used to soak the sample pad, and drying is obtained; The sample pad treatment liquid is a buffer solution containing 1-2 (w / v) % sucrose, 1-2 (w / v) % trehalose, 0.4-0.8 (w / v) % blocking agent 003, 0.1-0.2 (w / v) % blocking agent 004, 0.05-1 (w / v) % Tween-20 and 1-2 (w / v) % BSA.

5. The test strip of claim 1, wherein, The preparation method of the colloidal gold comprises the following steps: After the HAuCl4 solution is heated to boiling, a reducing agent is added for reaction; The mass ratio of the HAuCl4 to the reducing agent is 1:(1-5).

6. The test strip of claim 1, wherein, The preparation method of the colloidal gold-anti-Coxsackievirus B group IgM antibody antigen conjugate comprises the following steps: the colloidal gold solution is mixed with the antigen of anti-Coxsackievirus B group IgM antibody, incubated for 15-30 min, and then a blocking liquid is added for blocking to obtain the colloidal gold-anti-Coxsackievirus B group IgM antibody antigen conjugate.

7. The test strip of claim 1, wherein, The preparation method of the binding pad comprises the following steps: The colloidal gold-anti-Coxsackievirus B group IgM antibody antigen conjugate is sprayed on the binding pad, and drying is obtained; Preferably, the binding pad comprises at least one of a glass fiber membrane and a high molecular polymer membrane.

8. A method for the production of a test strip according to any one of claims 1 to 7, characterized in that The following steps are included: The nitrocellulose membrane, the water absorption paper, the binding pad and the sample pad are sequentially and fixedly connected to the bottom plate.

9. The use of the test strip according to any one of claims 1 to 7 in the preparation of a product for detecting Coxsackievirus B group IgM antibody.

10. A test card for detecting coxsackie virus group B IgM antibody, characterized by, The test strip according to any one of claims 1 to 7 is contained.

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