Detection card, kit and preparation method for quantitatively detecting IgG subtype antibodies

Abstract

This application relates to the field of gene detection, and specifically discloses a test strip, a kit and a preparation method for quantitatively detecting IgG subtype antibodies. The test strip includes a sample pad, a conjugate pad, an NC membrane and an absorbent pad; the conjugate pad is sprayed with conjugate antibodies, and the conjugate antibodies include: anti-human IgG antibody-fluorescent microsphere complexes of different subtypes, and quality control antibody A-fluorescent microsphere complexes; on the NC membrane, a test line and a quality control line are provided, there are at least two test lines, which are respectively coated with anti-human IgG antibodies of different subtypes, and the quality control line is coated with quality control antibody B, and quality control antibody A and quality control antibody B can specifically bind. Such a test strip and kit are simple to operate, do not require reliance on large-scale instruments, have low detection costs, have low professional requirements for testers, and have good sensitivity and specificity, and can achieve quantitative and qualitative detection of IgG subtypes.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and more specifically, it relates to a test card, a kit and a preparation method for quantitatively detecting IgG subtype antibodies. Background Art

[0002] IgG is the main antibody in the secondary immune response and has four subclasses, namely IgG1, IgG2, IgG3, and IgG4, the contents of which decrease in sequence in the blood of normal humans. It can activate complement and play roles such as lysing bacteria and cells. Clinically, the determination of serum IgG subclasses is of great value for studying immunodeficiency diseases and hypersensitivity diseases.

[0003] The physiological characteristics and functions of the 4 subclasses of IgG are different. For example, IgG1, IgG2, and IgG3 forming complexes with antigens can activate complement through the classical pathway, among which IgG3 has the strongest ability to activate complement, while IgG4 polymerizes and can activate complement through the alternative pathway. The functions of each subclass in the immune response are also not the same. For example, IgG1 and IgG3 have a response to bacterial and viral antigen proteins, while IgG2 plays a major immune response role against the capsular polysaccharide antigens of Streptococcus pneumoniae, Group A Streptococcus, and Haemophilus influenzae, and IgG4 is related to chronic allergic stimulation. Therefore, the determination of IgG subclasses is of great significance for infection recurrence, immunodeficiency, and autoimmune diseases, etc.

[0004] However, currently, there are few products for detecting IgG subtype antibodies, and mainly detect IgG in serum. The main detection method used is chemiluminescence, which requires the use of supporting instruments and consumables. The instrument is large, the operation is cumbersome, the professional degree requirement for the detection personnel is high, and the detection cycle is long, which is not conducive to the rapid quantitative monitoring of IgG subtype antibodies. Summary of the Invention

[0005] In order to be able to rapidly qualitatively and quantitatively monitor IgG subtype antibodies, the present application provides a test card, a kit and a preparation method for quantitatively detecting IgG subtype antibodies.

[0006] The present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a test card for quantitatively detecting IgG subtype antibodies, and the test card includes a sample pad, a conjugate pad, an NC membrane, and a water absorption pad;

[0008] The conjugate pad is sprayed with conjugate antibodies, and the conjugate antibodies include: anti-human IgG antibody-fluorescent microsphere complexes of different subtypes, and quality control antibody A-fluorescent microsphere complexes;

[0009] The NC membrane is provided with a detection line and a quality control line. There are at least two detection lines, which are respectively coated with anti-human IgG antibodies of different subtypes. The quality control line is coated with quality control antibody B, and quality control antibody A can specifically bind to quality control antibody B.

[0010] Further, the anti-human IgG antibodies of different subtypes include at least two of anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody and anti-IgG4 antibody.

[0011] Further, the quality control antibody A is a rabbit anti-mouse antibody or a rabbit anti-DNP-BSA; the quality control antibody B is a goat anti-rabbit antibody or DNP-BSA.

[0012] In a second aspect, the present application provides a detection card for quantitatively detecting IgG subtype antibodies as described above, which includes: preparing anti-human IgG antibody-fluorescent microsphere complexes of different subtypes and quality control antibody A-fluorescent microsphere complexes, mixing the anti-human IgG antibody-fluorescent microsphere complexes of different subtypes and the quality control antibody A-fluorescent microsphere complexes with a protective solution to obtain a conjugate pad treatment solution, and spraying the conjugate pad treatment solution on glass fiber to obtain a conjugate pad;

[0013] Preparing working solutions of anti-human IgG antibodies of different subtypes, respectively drawing the working solutions to the detection area of the NC membrane, and drying to obtain at least two detection lines;

[0014] Drawing a working solution containing quality control antibody B to the quality control area of the NC membrane, and drying to obtain a quality control line C;

[0015] Assembling and cutting the sample pad, conjugate pad, NC membrane and absorbent pad to obtain a detection card.

[0016] Further, the preparation method of the anti-human IgG antibody-fluorescent microsphere complex is as follows:

[0017] Disperse the activated fluorescent microspheres in the first buffer solution, add anti-human IgG antibodies of different subtypes under vortex conditions, and continue to vortex for 8-12 h, and centrifuge to obtain fluorescent microspheres labeled with anti-human IgG antibodies of different subtypes;

[0018] Redisperse the fluorescent microspheres labeled with anti-human IgG antibodies in the second buffer solution, add a BSA solution for blocking for 1-3 h to obtain the anti-human IgG antibody-fluorescent microsphere complex.

[0019] Further, the anti-human IgG antibodies of different subtypes are anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody and anti-IgG4 antibody with a mass ratio of 1:0.8-1.2:0.8-1.2:0.8-1.2.

[0020] Further, the method for activating the fluorescent microspheres is as follows:

[0021] The fluorescent microspheres are ultrasonically dispersed in MES buffer, and then mixed with an EDC-NHS conjugate at a concentration of 3-7 mg / mL, and vortexed for 15-30 min for activation.

[0022] Further, the protective solution includes trehalose and polyvinylpyrrolidone.

[0023] In a third aspect, the present application provides a kit for quantitatively detecting IgG subtype antibodies, which includes the above-mentioned test card and loading buffer.

[0024] Further, the loading buffer contains 0.1-0.3 wt% of Tween-20 and 0.05-0.15 wt% of preservative.

[0025] In summary, the present application has the following beneficial effects:

[0026] The test card provided by the present application includes a sample pad, a conjugate pad, an NC membrane and an absorbent pad, and is mainly used for quantitatively detecting the subtypes of IgG antibodies. During the preparation of this test card, different subtypes of anti-human IgG antibodies and quality control antibodies labeled with fluorescent microspheres are sprayed on the conjugate pad, corresponding IgG antibodies are coated on the test line, and corresponding quality control antibodies are coated on the quality control line. When the sample to be tested contains IgG subtype antibodies, an immune complex condensate of fluorescent microspheres-anti-human IgG antibody-IgG subtype antibody-anti-human IgG antibody will be formed on the test card, and thus will be intercepted at the corresponding test line (T line) position of the NC membrane. If the sample to be tested does not contain IgG subtype antibodies, the above immune complex condensate cannot be formed and cannot be intercepted on the NC membrane. At the same time, since the amount of the above immune complex condensate is proportional to the amount of IgG antibodies in the sample to be tested, the fluorescence intensity of the fluorescent microspheres in the immune complex condensate can be measured by a dry fluorescence analyzer. By establishing a standard curve with the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line and the known concentration, the IgG subtype antibodies in the sample to be tested can be quantitatively analyzed by measuring the fluorescence intensity.

[0027] At the same time, regardless of whether there are IgG antibodies in the sample to be tested, the quality control antibody B will bind to the quality control antibody A labeled with fluorescent microspheres on the quality control line to form an immune complex condensate of fluorescent microspheres-quality control antibody A-quality control antibody B, so that the fluorescence intensity of the C line remains unchanged. Thus, false negatives can be reduced and the detection sensitivity can be improved.

[0028] Therefore, compared with the existing problems in the detection of IgG subtypes, such as relying on supporting instruments and consumables, large and cumbersome instruments, high professional requirements for testers, and long detection cycles, the test card and kit provided in this application are easy to operate, do not rely on large instruments, have low detection costs, low professional requirements for testers, and have good sensitivity and specificity, and can achieve quantitative and qualitative detection of IgG subtypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the linear curve of the test card provided in Example 1 of this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0031] The technical solution of the present invention is as follows:

[0032] The test card provided in this application for quantitatively detecting IgG subtype antibodies includes a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. Specifically:

[0033] (1) Sample pad: It is one of polyester film or glass fiber, mainly used for loading samples and adsorbing samples, so that the analytes in the samples can be further chromatographically developed along the direction of the conjugate pad.

[0034] (2) Conjugate pad: The conjugate pad overlaps with the sample pad and is also one of polyester film or glass fiber. It is mainly used to bind to the IgG subtype antibodies to be detected in the test samples, and is coated with two binding antibodies: anti-human IgG antibody-fluorescent microsphere complex and quality control antibody A-fluorescent microsphere complex.

[0035] Among them, the anti-human IgG antibody includes at least two of anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody; preferably, the anti-human IgG antibody is anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody. The quality control antibody A is rabbit anti-mouse antibody or rabbit anti-DNP-BSA, and the quality control antibody B is goat anti-rabbit antibody or DNP-BSA; the quality control antibody A and the quality control antibody B can specifically bind and are set in pairs, such as rabbit anti-mouse antibody and goat anti-rabbit antibody, or rabbit anti-DNP-BSA and DNP-BSA.

[0036] Furthermore, the preparation method of the anti-human IgG antibody-fluorescent microsphere complex is as follows:

[0037] Step a: Disperse the activated fluorescent microspheres in the first buffer solution, add anti-human IgG antibodies of different subtypes under vortexing conditions, and continue vortexing for 8 - 12 h, then centrifuge to obtain fluorescent microspheres labeled with anti-human IgG antibodies of different subtypes;

[0038] Among them, the first buffer solution is an aqueous solution of HEPES with a pH of 7.5 - 8.5 and a concentration of 0.02 - 0.07 M.

[0039] The dispersed fluorescent microspheres are added with anti-human IgG mixed antibodies under vortexing. The mass ratio of anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody is 1:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2. Preferably, the mass ratio is 1:1:1:1.

[0040] Preferably, the method for activating fluorescent microspheres is: ultrasonically disperse the fluorescent microspheres in MES buffer solution (pH 5.5 - 6.5, 0.05 - 0.15 M), then mix with an EDC - NHS conjugate with a concentration of 3 - 7 mg / mL, and vortex for 15 - 30 min for activation.

[0041] Step b: Redisperse the fluorescent microspheres labeled with anti-human IgG antibodies in the second buffer solution, add a BSA solution for blocking for 1 - 3 h to obtain the anti-human IgG antibody - fluorescent microsphere complex.

[0042] Adding BSA for blocking treatment to reduce non-specific binding in the reagent and improve the accuracy of detection.

[0043] (3) NC membrane: Overlapping with the conjugate pad, on which a detection line and a control line are provided. There are at least two detection lines, which are respectively coated with anti-human IgG antibodies of different subtypes, and the control line is coated with a control antibody B.

[0044] (4) Absorbent pad.

[0045] The raw materials, models, and usage purposes adopted in the embodiments of the present application are shown in Table 1:

[0046] Table 1. Sources of raw materials and models

[0047]

[0048]

[0049] The following details the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for explaining and understanding the present invention, and are not used to limit the present invention.

[0050] Preparation Example

[0051] Preparation Example 1

[0052] This preparation example provides an anti-human IgG antibody-fluorescent microsphere complex, and its preparation method includes the following steps:

[0053] 1. Take 100 μl of fluorescent microspheres, add them to 500 μl of 0.1 M pH 6.0 MES buffer solution, then add 200 μl of pure water, vortex and mix evenly, and ultrasonicate for 2 min with an ultrasonic cell disruptor;

[0054] 2. Prepare an EDC-NHS aqueous solution with the final concentrations of both EDC and NHS being 5 mg / mL;

[0055] 3. While vortexing, add 100 μl of the above EDC-NHS aqueous solution dropwise to the fluorescent microspheres, and activate at room temperature for 20 min on a rotary mixer;

[0056] 4. Centrifuge the activated fluorescent microspheres at 10,000 rpm for 15 min at a temperature below 10 °C;

[0057] 5. Disperse the precipitate in 900 μl of 0.05 M pH 8.0 HEPES aqueous solution, and ultrasonicate for 2 min with an ultrasonic cell disruptor;

[0058] 6. While vortexing, add 0.5 mg of anti-human IgG antibody (where the mass ratio of anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody is 1:1:1:1) dropwise to the activated and dispersed fluorescent microspheres, and label overnight at room temperature on a rotary mixer;

[0059] 7. After the labeling is completed, take out the reacted fluorescent microspheres, centrifuge at 10,000 rpm for 15 min at a temperature below 10 °C, disperse the precipitate in 200 μl of 0.05 M pH 9.0 Tris-HCl buffer solution, and ultrasonicate for 2 min with an ultrasonic cell disruptor;

[0060] 8. Add 10 μl of 10% BSA aqueous solution to block for 2 h, and then place it at 2 - 8 °C for standby.

[0061] Preparation Example 2

[0062] This preparation example provides a quality control antibody A-fluorescent microsphere complex, where the quality control antibody A is a rabbit anti-mouse antibody. The preparation method of this complex is basically the same as that of Preparation Example 1, except that:

[0063] Step 6: While vortexing, add 0.5 mg of rabbit anti-mouse antibody dropwise to the activated and dispersed fluorescent microspheres, and label overnight at room temperature on a rotary mixer.

[0064] Preparation Example 3

[0065] This preparation example provides an anti-human IgG antibody-fluorescent microsphere complex. The preparation method of this complex is basically the same as that of Preparation Example 1, except that:

[0066] Step 6: 0.5 mg of anti-human IgG antibody (including anti-IgG1 antibody and anti-IgG3 antibody, with a mass ratio of 1:1 between the two) was added dropwise to the above-activated and dispersed fluorescent microspheres under vortexing, and labeled overnight at room temperature on a rotary mixer.

[0067] Preparation Example 4

[0068] This preparation example provides a quality control antibody A-fluorescent microsphere complex, where the quality control antibody A is rabbit anti-DNP-BSA. The preparation method of this complex is basically the same as that of Preparation Example 1, except that:

[0069] Step 6. 0.5 mg of rabbit anti-DNP-BSA was added dropwise to the above-activated and dispersed fluorescent microspheres under vortexing, and labeled overnight at room temperature on a rotary mixer.

[0070] Example

[0071] Example 1

[0072] This example provides a test card for quantitatively detecting IgG subtype antibodies. Its preparation method includes the following steps:

[0073] (1) Preparation of the conjugate pad:

[0074] 125 μl of 0.4 M pH 8.5 Tris-HCl buffer, 100 μl of 20% trehalose aqueous solution, 100 μl of 10% polyvinylpyrrolidone-K30 (PVP-K30) aqueous solution, 20 μl of anti-human IgG antibody-fluorescent microsphere complex (prepared in Preparation Example 1), and 20 μl of rabbit anti-mouse antibody-fluorescent microsphere complex (prepared in Preparation Example 2) were added, and finally pure water was added to make up to 950 μl to form the conjugate pad treatment solution; the above conjugate pad treatment solution was sprayed on SB08 glass fiber, dried at 45 °C for 2 h, and placed in an aluminum foil bag with a desiccant for later use.

[0075] (2) Preparation of the NC membrane:

[0076] The stock solutions of anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4 antibodies were diluted to 0.3 mg / ml with 1×PBS, and the goat anti-rabbit antibody was diluted to 0.8 mg / ml for use as the working solution;

[0077] Coat the above working fluid on the NC membrane respectively through a three-dimensional gold spraying and scribing instrument to obtain 4 test lines, namely T1, T2, T3 and T4; and 1 quality control line C which is goat anti-rabbit. The concentrations of the test line and the quality control line are 1 μl / cm. Put the scribed NC membrane into an oven, dry it at 50 °C for 14 h, and place it in an aluminum foil bag with a desiccant for later use.

[0078] (3) Preparation of the test card:

[0079] Take 1 rubber plate, stick the NC membrane, the absorbent pad and the conjugate pad on it respectively to form a large plate;

[0080] Put the large plate on a high-speed chopping machine and cut it into small strips with a width of 4 mm;

[0081] Put the small strips into the bottom cover of a special card shell, cover the upper cover, and open a sample adding hole at the corresponding position of the sample pad to obtain the test card; place the test card in an aluminum foil bag with a desiccant for later use.

[0082] For the convenience of detection, this embodiment also provides a test kit, which includes a sample loading buffer and the above test card, and the preparation of the sample loading diluent is as follows:

[0083] Measure 99.7 mL of 1×PBS, add 200 μl of Tween-20 and 100 μl of preservative to it, and stir well to form the sample loading buffer; the sample loading buffer is dispensed into 10 mL dropping bottles, 6 mL per bottle, and placed at room temperature for later use.

[0084] Example 2

[0085] This embodiment provides a test card for quantitatively detecting IgG subtype antibodies, and its preparation method is basically the same as that of Example 1, except that:

[0086] (1) Preparation of the conjugate pad:

[0087] Add 125 μl of 0.4 M pH 8.5 Tris-HCl buffer solution, 100 μl of 20% trehalose aqueous solution, 100 μl of 10% polyvinylpyrrolidone-K30 (PVP-K30) aqueous solution, 20 μl of anti-human IgG antibody-fluorescent microsphere complex (prepared in Preparation Example 3) and 20 μl of rabbit anti-mouse antibody-fluorescent microsphere complex (prepared in Preparation Example 2), and finally add pure water to make up to 950 μl to form the conjugate pad treatment solution; spray the above conjugate pad treatment solution on SB08 glass fiber, dry it at 45 °C for 2 h, and place it in an aluminum foil bag with a desiccant for later use.

[0088] (2) Preparation of the NC membrane:

[0089] Dilute the stock solutions of anti-IgG1 and anti-IgG3 antibodies to 0.3 mg / ml with 1×PBS, and dilute the goat anti-rabbit antibody to 0.8 mg / ml for use as working solutions.

[0090] Coat the above working solutions onto the NC membrane using a three-dimensional gold spraying and membrane scribing instrument to obtain 2 test lines, namely T1 and T3; and 1 control line C which is goat anti-rabbit. The concentration of the test lines and the control line is 1 μl / cm. Place the scribed NC membrane in an oven and dry it at 50°C for 14 h, then place it in an aluminum foil bag with a desiccant for later use.

[0091] Example 3

[0092] This example provides a test card for quantitatively detecting IgG subtype antibodies. Its preparation method is basically the same as that of Example 1, except that:

[0093] (1) Prepare the conjugate pad:

[0094] Add 125 μl of 0.4 M pH 8.5 Tris-HCl buffer, 100 μl of 20% trehalose aqueous solution, 100 μl of 10% polyvinylpyrrolidone-K30 (PVP-K30) aqueous solution, 20 μl of anti-human IgG antibody-fluorescent microsphere complex (prepared in Preparation Example 1), and 20 μl of rabbit anti-DNP-BSA-fluorescent microsphere complex (prepared in Preparation Example 4), and finally supplement pure water to 950 μl to form the conjugate pad treatment solution; spray the above conjugate pad treatment solution onto SB08 glass fiber, dry it at 45°C for 2 h, and place it in an aluminum foil bag with a desiccant for later use.

[0095] (2) Prepare the NC membrane:

[0096] Dilute the stock solutions of anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4 antibodies to 0.3 mg / ml with 1×PBS, and dilute DNP-BSA to 0.8 mg / ml for use as working solutions.

[0097] Coat the above working solutions onto the NC membrane using a three-dimensional gold spraying and membrane scribing instrument to obtain 4 test lines, namely T1, T2, T3, and T4; and 1 control line C which is DNP-BSA. The concentration of the test lines and the control line is 1 μl / cm. Place the scribed NC membrane in an oven and dry it at 50°C for 14 h, then place it in an aluminum foil bag with a desiccant for later use.

[0098] Performance detection test

[0099] I. Linear range and clinical sample detection:

[0100] Using the test card prepared in Example 1, standard samples with concentrations ranging from 20 - 25000 ng / mL and the serum samples to be tested (from a scientific research cooperation unit) were tested as follows:

[0101] (1) Take a test card, add 10 μl of the standard sample or the serum sample to be tested to the sample addition hole of the sample pad, and wait for 2 min;

[0102] (2) Add 4 drops of the loading buffer to the buffer hole of the sample pad of the test card and wait for 10 min;

[0103] (3) After 10 min, read the fluorescence peak value with a fluorescence instrument to obtain the test result.

[0104] Each sample was measured in parallel 3 times, and the mean value (y) of the measurement results was calculated respectively. Taking the standard sample concentration (x) as the independent variable and the mean value (y) of the measurement results as the dependent variable, the result average value and dilution ratio were linearly fitted by the least square method, and the correlation coefficient (r) of the linear regression was calculated. The results are as Figure 1 shown.

[0105] It can be seen from Figure 1 that the linear equation is: y = 0.0026x + 0.0308, and in the concentration range of 50 ng / mL - 2000 ng / mL, the linear correlation coefficient r ≥ 0.9500.

[0106] Using the same method to test 5 serum samples to be tested, and substituting the test results into the linear equation to obtain the content of each IgG subtype antibody in the serum samples to be tested, as shown in Table 2:

[0107] Table 2. Measurement results of samples to be tested

[0108] serum sample IgG1 IgG2 IgG3 IgG4 sample to be tested 1 200 ng / mL 140 ng / mL 65 ng / mL 25 ng / mL sample to be tested 2 160 ng / mL 55 ng / mL 26 ng / mL 23 ng / mL sample to be tested 3 260 ng / mL 150 ng / mL 87 ng / mL 65 ng / mL sample to be tested 4 110 ng / mL 150 ng / mL 86 ng / mL 150 ng / mL sample to be tested 5 158 ng / mL 53 ng / mL 56 ng / mL 78 ng / mL

[0109] II. Repeatability test:

[0110] A sample within the linear range was repeatedly measured 10 times, and the mean value (M) and standard deviation (SD) of the measured values were calculated. The coefficient of variation (CV) was calculated according to formula (1):

[0111] CV = SD / M × 100%.................................(1)

[0112] Where:

[0113] CV - coefficient of variation;

[0114] SD - standard deviation of the 10 measurement results;

[0115] M - average value of the 10 measurement results.

[0116] The results are shown in Table 3 as follows:

[0117] Table 3. Results of Repeatability Determination

[0118]

[0119] It is calculated that the coefficient of variation CV is 0.84 - 3.15, indicating that the repeatability of this test strip is good. In addition, since below 20 ng / ML is negative, the numerical results in the range of 0 - 15 in the determination results do not differ much, mainly because the content of its antibody itself is very small, and the numerical difference mainly comes from the error of the instrument itself.

[0120] III. Precision Detection:

[0121] Samples with two different concentrations were measured respectively, and each was measured 3 times repeatedly. The measurement results were recorded as (X), and the coefficient of variation (CV) was calculated respectively according to formula (2).

[0122] Coefficient of variation (CV) = SD / M × 100%...............................(2)

[0123] In the formula:

[0124] CV - coefficient of variation;

[0125] SD - standard deviation of 10 measurement results;

[0126] M - average value of 10 measurement results.

[0127] The results are shown in Table 4 and Table 5 as follows:

[0128] Table 4. Precision Determination Results (200 ng / mL)

[0129]

[0130]

[0131] Table 5. Precision Determination Results (800 ng / mL)

[0132]

[0133] It can be calculated from Table 4 and Table 5 that:

[0134] For the test sample with a concentration of 200 ng / mL, the coefficient of variation CV is 0.05 - 0.09;

[0135] For the test sample with a concentration of 800 ng / mL, the coefficient of variation CV is 0.04 - 0.05;

[0136] It can be seen that for the test card provided in Embodiment 1 of the present application, the relative deviation of the test result does not exceed ±15%, and the precision is good.

[0137] IV. Sensitivity detection:

[0138] Samples with gradually decreasing theoretical addition amounts were respectively detected, measured three times repeatedly, and the average value was taken to calculate the sensitivity:

[0139] The test results are shown in Table 6:

[0140] Table 6. Sensitivity detection results

[0141]

[0142] As can be seen from Table 6, for the test card provided in Embodiment 1 of the present application, the lowest detection limit is 50 ng / mL.

[0143] V. Stability detection:

[0144] Samples with two different concentrations (200 ng / ml and 800 ng / ml) stored at 4°C and 37°C were measured, and the measurement was carried out continuously for one week. The results are shown in Table 7 and Table 8:

[0145] Table 7. Stability measurement results (200 ng / ml)

[0146]

[0147]

[0148] Table 8. Stability measurement results (800 ng / ml)

[0149]

[0150] As can be seen from Table 7 and Table 8, for the two samples within the detection range, the test card provided in Embodiment 1 of the present application has good stability in continuous measurement for one week.

[0151] This specific embodiment is only an explanation of the present application, and it does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A test card for quantitatively detecting IgG subtype antibodies, characterized in that, The detection card includes a sample pad, a conjugate pad, an NC membrane, and a wicking pad; The conjugate pad is sprayed with conjugate antibodies, and the conjugate antibodies include: anti-human IgG antibody-fluorescent microsphere complexes of different subtypes, and quality control antibody A-fluorescent microsphere complexes; The NC membrane is provided with a test line and a quality control line. There are at least two test lines, which are respectively coated with anti-human IgG antibodies of different subtypes. The quality control line is coated with quality control antibody B, and quality control antibody A and quality control antibody B can specifically bind; The preparation method of the conjugate pad includes: (1) Disperse the activated fluorescent microspheres in the first buffer solution, and simultaneously add anti-human IgG antibodies of different subtypes under vortex conditions, and continue to vortex for 8-12 h, and centrifuge to obtain fluorescent microspheres labeled with anti-human IgG antibodies of different subtypes; (2) Redisperse the fluorescent microspheres labeled with anti-human IgG antibodies of different subtypes in the second buffer solution, add a BSA solution for blocking for 1-3 h to obtain the anti-human IgG antibody-fluorescent microsphere complex; (3) Mix the anti-human IgG antibody-fluorescent microsphere complexes of different subtypes, the quality control antibody A-fluorescent microsphere complexes with a protective solution to obtain a conjugate pad treatment solution, and spray the conjugate pad treatment solution on glass fiber to prepare a conjugate pad; Among them, in step (1), simultaneously adding the anti-human IgG antibodies of different subtypes includes at least two of anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody; when simultaneously detecting four anti-human IgG antibody subtypes, the added anti-IgG1 antibody, anti-IgG2 antibody, anti-IgG3 antibody, and anti-IgG4 antibody have a mass ratio of 1:0.8-1.2:0.8-1.2:0.8-1.2; The protective solution includes trehalose and polyvinylpyrrolidone.

2. The test strip for quantitatively detecting IgG subtype antibodies according to claim 1, characterized in that, The quality control antibody A is a rabbit anti-mouse antibody or a rabbit anti-DNP-BSA; the quality control antibody B is a goat anti-rabbit antibody or DNP-BSA.

3. A method for preparing a test card for quantitatively detecting IgG subtype antibodies according to claim 1 or 2, characterized in that, It includes: Prepare anti-human IgG antibody-fluorescent microsphere complexes of different subtypes and quality control antibody A-fluorescent microsphere complexes, mix the anti-human IgG antibody-fluorescent microsphere complexes of different subtypes, the quality control antibody A-fluorescent microsphere complexes with a protective solution to obtain a conjugate pad treatment solution, and spray the conjugate pad treatment solution on glass fiber to prepare a conjugate pad; Prepare working solutions of anti-human IgG antibodies of different subtypes, respectively draw the working solutions to the test area of the NC membrane, and after drying, obtain at least two test lines; Draw the working solution containing quality control antibody B to the quality control area of the NC membrane, and after drying, obtain a quality control line C; Assemble and cut the sample pad, conjugate pad, NC membrane, and wicking pad to obtain a detection card.

4. The preparation method of the detection card for quantitatively detecting IgG subtype antibodies according to claim 3, characterized in that The method for activating the fluorescent microspheres is: Ultrasonically disperse the fluorescent microspheres in MES buffer solution, then mix with an EDC-NHS conjugate with a concentration of 3-7 mg / mL, and vortex for 15-30 min for activation.

5. A kit for quantitatively detecting IgG subtype antibodies, characterized in that, It includes the detection card as described in claim 1 or 2 and a sample loading buffer solution.

6. The kit for quantitatively detecting IgG subtype antibodies according to claim 5, wherein, The sample loading buffer solution contains 0.1 - 0.3 wt% of Tween-20 and 0.05 - 0.15 wt% of a preservative.

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