A test strip and a test kit for detecting IgG / IgM antibodies

Abstract

The present invention provides a test strip and a test kit for detecting IgG / IgM antibodies, comprising a labeled pad, a sample pad, a test pad and a water-absorbing pad which are adhered to a bottom card and arranged in an overlapping manner from upstream to downstream; the test pad is coated with an IgG test line and an IgM test line, and the IgG test line is located upstream of the IgM test line; during detection, the sample reaches the test line of the test pad prior to the label. The test strip of the present invention has a simple structure, is convenient to use, has strong anti-interference ability and high detection sensitivity. The test kit of the present invention can improve the accuracy of early diagnosis of diseases such as infectious diseases, enable early diagnosis and early treatment, thereby reducing the spread of infectious diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and particularly relates to a test strip and a test kit for detecting IgG / IgM antibodies. Background Art

[0002] When pathogenic microorganisms such as bacteria and viruses invade the body, B lymphocytes will transform into plasma cells and produce antibodies that specifically bind to the corresponding antigens, forming immunoglobulins, including immunoglobulins such as IgM and IgG.

[0003] Immunoglobulin M (abbreviated IgM) is the earliest antibody to appear in the human body after being infected. It plays a role in clearing viruses and anti-infection in the early stage of infection, and has functions such as lysing bacteria, activating complement, immune regulation and agglutination.

[0004] Abnormal IgM values often indicate the possibility of infection. In the early stage of infection or the active stage of certain autoimmune diseases, such as rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, etc., the IgM level in serum may increase. The abnormality of IgM is also related to daily living habits. Bad living habits such as long-term alcoholism or overeating may lead to abnormal IgM values. IgM is also used in prenatal examinations to clarify whether the fetus has intrauterine infection. In some chronic liver diseases such as hepatitis A, hepatitis B, and hepatitis C, the increase in IgM may indicate that the virus is in the active stage.

[0005] IgM can be used as an early diagnostic indicator for infectious diseases, and its positivity usually indicates that the body is in the acute infection period. The detection of IgM helps in the auxiliary diagnosis of various diseases, including autoimmune diseases, digestive system diseases, blood system diseases, respiratory system diseases, infectious diseases, and urinary system diseases, etc.

[0006] Immunoglobulin G (abbreviated IgG) is the main component of immunoglobulins in serum, accounting for about 75% of the total content of immunoglobulins in serum. It is the most important anti-pathogenic microorganism antibody in body fluids and is also the main antibody in the secondary immune response.

[0007] Dengue fever is an acute arbovirus infectious disease caused by the dengue virus, which is mainly transmitted by Aedes aegypti and Aedes albopictus. The detection methods of dengue fever mainly include antigen detection, viral nucleic acid detection and antibody detection. These methods can help doctors diagnose whether a patient is infected with the dengue virus.

[0008] Antibody detection is achieved by detecting specific antibodies IgG and IgM in the patient's body. When an infected person is infected with the dengue virus, the body's immune system produces two types of antibodies, IgG and IgM, to fight the virus. In patients with primary infection, IgM antibodies can be detected 3-5 days after the onset of the disease, and IgG antibodies can be detected 1 week after the onset of the disease. The detection of these antibodies helps diagnose dengue fever and can be used as a reference in secondary infections.

[0009] As Figure 1 shown, the test strip is a conventional product for detecting dengue antibodies by the lateral flow (LF) method. Figure 1 The test strip shown includes a buffer pad 1, a conjugate pad 2, a test pad 4 (nitrocellulose membrane), and an absorbent pad 8 that are adhered to the base card 5 and arranged in sequence. The blood sample is directly added to the conjugate pad and reacts with the dengue recombinant antigen-gold conjugate on the conjugate pad to form a dengue recombinant antigen-gold-Dengue antibody complex. Buffer is added to the buffer pad to move the dengue recombinant antigen-gold-Dengue antibody complex along the test pad and bind to the anti-human IgM antibody or / and anti-human IgG antibody pre-coated on the test line of the test pad, resulting in color development.

[0010] For products that simultaneously detect Dengue IgG antibodies and Dengue IgM antibodies on a single test strip, since the content of IgM in the blood is relatively small, when using the test strip structure and method of conventional products for detection, the sensitivity of IgM will be interfered by IgG and be relatively low. Summary of the Invention

[0011] To overcome the defects of the prior art, the present invention provides a test strip for detecting IgG / IgM antibodies, including a conjugate pad, a sample pad, a test pad, and an absorbent pad that are adhered to the base card and overlapped in sequence from upstream to downstream; the test pad includes a first test line and a second test line, the first test line is coated with an anti-IgM antibody, the second test line is coated with an anti-IgG antibody, the first test line is located downstream of the second test line, and the conjugate pad includes a label that binds to the antigen.

[0012] Further, it also includes a buffer pad for receiving buffer, and the buffer pad is located upstream of the conjugate pad.

[0013] Further, the material of the buffer pad is hydrophobic.

[0014] Further, the buffer pad is made of a glass fiber or polyester film material with strong hydrophobicity.

[0015] Further, the release rate of the markers on the marker pad is slower than the release rate of the sample on the sample pad, that is, the time for the markers on the marker pad to reach the detection pad is later than the time for the added sample to reach the detection pad.

[0016] Further, the IgG / IgM antibody is a dengue IgG / IgM antibody.

[0017] Further, the marker pad includes a marker complex in which dengue antigen is combined with a marker.

[0018] More specifically, the dengue antigen is: type I, type II, type III, and type IV dengue recombinant antigens. The marker is selected from colloidal gold, latex, etc. More specifically, the marker pad is coated with type I, type II, type III, and type IV dengue recombinant antigen-colloidal gold complexes that are separately labeled.

[0019] Further, the detection pad of the test strip further includes a quality control line, and the quality control line is coated with streptavidin; the marker pad is coated with a biotin-marker complex. The marker is selected from colloidal gold, latex, etc. More specifically, the marker pad is coated with a biotin-colloidal gold complex.

[0020] In the specific scheme of the dengue IgG / IgM antibody test strip of the present invention, a sample pad is added downstream of the marker pad, and the sample is directly added to the sample pad and then flows along the detection pad. At this time, the sample does not come into contact with the dengue recombinant antigen-colloidal gold complex. IgG and IgM in the sample are successively bound to the anti-human IgG antibody and anti-human IgM antibody pre-coated on the detection pad and are fixed on the detection pad. Then, with the addition of the buffer solution, the dengue recombinant antigen-colloidal gold complex fixed on the marker pad dissolves and begins to flow on the detection pad. The dengue recombinant antigen-colloidal gold complex successively binds to the Dengue-IgG antibody and Dengue-IgM antibody fixed on the detection pad, causing the corresponding positions on the detection pad to develop color, achieving the purpose of testing.

[0021] The present invention also provides a test kit for detecting IgG / IgM antibodies, and the kit includes the test strip and buffer solution of the present invention.

[0022] Further, the buffer solution includes 0.01M PBS buffer solution, wherein the PBS buffer solution contains 0.2% to 1% Tween20, 0.1% NaN 3 。

[0023] In the specific scheme of the dengue IgG / IgM antibody detection kit of the present invention, its usage steps include: (1) directly adding the sample onto the sample pad; (2) the sample flows from the sample pad to the detection pad, and the sample chromatographs (flows) along the detection pad, and IgG and IgM in the sample are successively fixed on the detection pad by binding to the anti-human IgG antibody and anti-human IgM antibody pre-coated on the detection pad; (3) adding buffer solution onto the buffer pad and flowing it to the labeling pad, and the dengue recombinant antigen-gold colloid complex fixed on the labeling pad is dissolved; (4) the dengue recombinant antigen-gold colloid complex flows along with the buffer solution to the detection pad and continues to flow. On the detection pad, the dengue recombinant antigen-gold colloid complex successively binds to the Dengue-IgG antibody and Dengue-IgM antibody that have been fixed on the detection pad by the anti-human IgG antibody and anti-human IgM antibody, causing the corresponding positions on the detection pad to develop color.

[0024] The present invention also provides a detection method for detecting IgG / IgM antibodies. The method includes: providing a test strip for detecting IgG / IgM antibodies, the test strip includes a labeling pad, a sample pad, a detection pad, and a water absorption pad that are adhered to the base card and are arranged in a lap joint manner from upstream to downstream in sequence; the detection pad includes a first detection line and a second detection line, the first detection line is coated with an anti-IgM antibody, the second detection line is coated with an anti-IgG antibody, the first detection line is located downstream of the second detection line, and the labeling pad includes a label combined with an antigen; adding a sample to be detected to the sample pad of the test strip; adding buffer solution to the test strip to release the label on the labeling pad and flow it to the detection pad; the time when the label on the labeling pad reaches the detection pad is later than the time when the added sample reaches the detection pad.

[0025] Furthermore, it also includes a buffer pad for receiving buffer solution, and the buffer pad is located upstream of the labeling pad.

[0026] Furthermore, the material of the buffer pad is hydrophobic.

[0027] Furthermore, the material of the buffer pad is preferably a glass fiber or polyester film material with strong hydrophobicity.

[0028] Furthermore, the release rate of the label on the labeling pad is slower than the release rate of the sample on the sample pad, that is, the time when the label on the labeling pad reaches the detection pad is later than the time when the added sample reaches the detection pad. Even further, after adding the sample to be detected to the sample pad of the test strip and waiting for a preset time, then add buffer solution to the test strip.

[0029] Furthermore, the preset time is 1 to 2 minutes.

[0030] Furthermore, the IgG / IgM antibody is a dengue IgG / IgM antibody.

[0031] Furthermore, the marking pad includes a marking complex in which a dengue antigen is combined with a marker.

[0032] More specifically, the dengue antigen is: type I, type II, type III, and type IV dengue recombinant antigens. The marker is selected from colloidal gold, latex, etc. More specifically, the marking pad is coated with type I, type II, type III, and type IV dengue recombinant antigen - colloidal gold complexes separately marked.

[0033] Furthermore, the test pad of the test strip also includes a quality control line, and the quality control line is coated with streptavidin; the marking pad is coated with a complex of biotin - marker. The marker is selected from colloidal gold, latex, etc. More specifically, the marking pad is coated with a biotin - colloidal gold complex.

[0034] Since the products for routinely detecting IgG / IgM antibodies first bind the sample to the marker marked with antigen on the marking pad, and then the binding flow enters the test pad for chromatography and reacts with the substances on the test line of the test pad. For example, after the dengue antibody in the sample binds to the dengue recombinant antigen - colloidal gold complex on the marking pad, it enters the nitrocellulose membrane of the test pad for chromatography and binds to the anti - IgM antibody on the first test line and the anti - IgG antibody on the second test line of the test pad, thereby showing the test result. This will lead to a decrease in the sensitivity of IgM.

[0035] However, for the test strip for detecting IgG / IgM antibodies, the corresponding test kit and the detection method of the present invention are such that before the marker on the marking pad is released, the sample is allowed sufficient time to flow and chromatograph independently on the test strip, and the sample reaches the test line before the marker. For example, a sample pad is added downstream of the marking pad of the test strip, the sample is added to the sample pad, and the sample enters the nitrocellulose membrane of the test pad for chromatography before binding to the marker, and a binding reaction occurs with the substances on the test line of the test pad. Subsequently, the marker on the marking pad flows into the test pad for chromatography driven by the buffer solution and reacts with the analytes such as IgG and IgM in the sample, showing the test result.

[0036] The detection test strip structure and detection method of the present invention effectively weaken the interference effect of interfering substances in the sample before the sample contacts the labeling agent. For example, the interference effect of a large amount of IgG in the sample on relatively less IgM is effectively weakened. The present invention separately and independently sets a sample pad and a labeling pad, which also allows for more selectivity in sample pad treatment without having to overly consider the stability of related substances in the labeling agent, such as active substances like colloidal gold antigen. Moreover, the present invention can also delay the time for the buffer solution to enter the labeling pad and thereby delay the release of the labeling agent on the labeling pad by selecting the material of the buffer pad and adjusting the buffer solution formula, making it possible for the sample to chromatograph independently on the detection pad prior to the labeling agent. In contrast, the use of the buffer pad and buffer solution in conventional products causes the labeling agent to be released too quickly, resulting in too short an independent chromatography time for the sample on the detection pad and low detection sensitivity.

[0037] The detection test strip of the present invention has a simple structure, is convenient to use, has strong anti-interference ability, can reduce the detection interference of IgG on IgM in the combined detection of IgG and IgM, has high detection sensitivity, and thus improves the accuracy of early detection of patient infections. Early diagnosis and early treatment can thereby reduce the spread of infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a structural diagram of a dengue IgG / IgM antibody detection test strip in the prior art.

[0039] Figure 2 is a structural diagram of a dengue IgG / IgM antibody detection test strip of the present invention.

[0040] Figure 3 is Figure 2 the exploded view of the detection test strip.

[0041] Figure 4 is a structural diagram of another dengue IgG / IgM antibody detection test strip of the present invention.

[0042] Figure 5 is the exploded view of the structure of the dengue IgG / IgM antibody detection plate of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0043] The technical solutions of the present invention are further described and illustrated below through specific embodiments.

[0044] Such as Figure 2 and Figure 3As shown in the figure, an IgG / IgM antibody detection test strip 10 of the present invention includes a buffer pad 1, a labeling pad 2, a sample pad 3, a detection pad 4, and an absorbent pad 8 that are sequentially overlapped and arranged from upstream to downstream. The buffer pad 1, the labeling pad 2, the sample pad 3, the detection pad 4, and the absorbent pad 8 are all adhered to the base card 5. The detection pad 4 includes a first detection line 41 and a second detection line 42. The first detection line 41 is coated with an anti-IgM antibody, and the second detection line 42 is coated with an anti-IgG antibody. The first detection line 41 is located downstream of the second detection line 42. The detection pad may also include a quality control line 43.

[0045] As Figure 4 shown in the figure, another IgG / IgM antibody detection test strip 10 of the present invention includes a labeling pad 2, a sample pad 3, a detection pad 4, and an absorbent pad 8 that are sequentially overlapped and arranged from upstream to downstream. The labeling pad 2, the sample pad 3, the detection pad 4, and the absorbent pad 8 are all adhered to the base card 5. The detection pad 4 includes a first detection line 41 and a second detection line 42. The first detection line 41 is coated with an anti-IgM antibody, and the second detection line 42 is coated with an anti-IgG antibody. The first detection line 41 is located downstream of the second detection line 42. The detection pad may also include a quality control line 43.

[0046] The material of the buffer pad 1 is selected from glass fiber materials or polyester film materials. Preferably, it is a glass fiber material or a polyester film material with relatively strong hydrophobicity, or a glass fiber material or a polyester film material treated with a hydrophobic reagent.

[0047] The material of the labeling pad 2 is selected from glass fiber, polyester film, etc. The material of the sample pad 3 is selected from glass fiber, etc. The material of the detection pad 4 is selected from nitrocellulose membranes, etc. The material of the absorbent pad 8 is selected from blotting paper, filter paper, etc.

[0048] As Figure 5 shown, the detection plate 20 includes a bottom plate 21 and an upper cover 22. Between the bottom plate and the upper cover, Figure 2 or Figure 4 shown in the figure, the detection test strip is installed. The upper cover 22 is provided with a buffer addition hole 23 corresponding to the buffer pad, a sample addition hole 24 corresponding to the sample pad 3 of the detection test strip, and a detection result observation hole 25 corresponding to the detection pad 4 of the detection test strip.

[0049] The detection test strip described in the present invention can be used to detect IgG, IgM, etc. in a sample. For example, a detection test strip for detecting dengue IgG, IgM, etc. in a blood sample. A detection kit for detecting dengue IgG / IgM antibodies includes the dengue IgG / IgM antibody detection test strip 10 described in the present invention and a buffer. The dengue IgG / IgM antibody detection kit may also be a detection kit that includes the dengue IgG / IgM antibody detection plate 20 described in the present invention and a buffer.

[0050] Example 1 Preparation and Detection Steps of Dengue IgG / IgM Antibody Detection Strip

[0051] As Figure 2 shown, the dengue IgG / IgM antibody detection strip 10 includes a buffer pad 1, a labeled pad 2, a sample pad 3, a detection pad 4, and an absorbent pad 8 that are respectively adhered to the base card 5 and are overlapped and stacked in sequence from upstream to downstream. In this embodiment, the width of the detection strip is 3.5 mm, and the overlapping area between each pad is 1 - 2 mm.

[0052] The labeled pad 2 is coated with type I, type II, type III, and type IV dengue recombinant antigen - colloidal gold complexes and biotin - colloidal gold complexes that are separately labeled.

[0053] Preparation method of the labeled pad:

[0054] (1) Rapidly add dengue virus recombinant antigen to the colloidal gold dispersion with a particle size of 40 - 60 nm and a concentration of 0.01 - 0.03 wr% at a ratio of 10 - 20 ug / mL, and quickly stir with a stirrer. After reacting for 15 - 30 minutes, rapidly add the blocking solution (1% BSA, 0.1% NaN 3 3) at a ratio of 10 - 15 uL / mL, react for 10 - 30 minutes, centrifuge at 8000 - 10000 rpm for 25 - 30 minutes, discard the supernatant to obtain the precipitate, and add the reconstitution solution (25 mM Tris buffer, 1% BSA, 0.1% NaN 3 3) to the precipitate at a ratio of 100 uL / mL, and ultrasonically reconstitute to respectively prepare type I dengue recombinant antigen - colloidal gold complex, type II dengue recombinant antigen - colloidal gold complex, type III dengue recombinant antigen - colloidal gold complex, and type IV dengue recombinant antigen - colloidal gold complex.

[0055] (2) Rapidly add biotin to the colloidal gold dispersion with a particle size of 40 - 60 nm and a concentration of 0.01 - 0.03 wr% at a ratio of 10 - 20 ug / mL, and quickly stir with a stirrer. After reacting for 15 - 30 minutes, rapidly add the blocking solution (1% BSA, 0.1% NaN 3 3) at a ratio of 10 - 15 uL / mL, react for 10 - 30 minutes, centrifuge at 8000 - 10000 rpm for 25 - 30 minutes, discard the supernatant to obtain the precipitate, and add the storage solution (25 mM Tris buffer, 1% BSA, 0.1% NaN 3 3) to the precipitate at a ratio of 100 uL / mL, and ultrasonically reconstitute to prepare biotin - colloidal gold complex.

[0056] (3) According to the titer, the above-mentioned type I dengue recombinant antigen-gold colloid complex, type II dengue recombinant antigen-gold colloid complex, type III dengue recombinant antigen-gold colloid complex, type IV dengue recombinant antigen-gold colloid complex and biotin-gold colloid complex are mixed and diluted with a diluent, and then evenly applied to the conjugate pad 2, and then dried at 37°C for 12 - 24 hours. The material of the conjugate pad is selected from glass fiber. The diluent is 25 mM Tris buffer, which contains 1% BSA, 20% sucrose, 0.1% NaN 3 .

[0057] Preparation method of the test pad:

[0058] The test pad 4 has a first test line (T1-line) 41, a second test line (T2-line) 42 and a quality control line (C-line) 43. The first test line 41 is coated with anti-human IgM antibody, the second test line is coated with anti-human IgG antibody, and the quality control line 43 is coated with streptavidin.

[0059] (1) The coating method of the first test line 41 is: Dilute the anti-human IgM antibody with a coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN 3 ) to 0.1 - 1 mg / mL and then coat, and then dry at 37°C for 12 - 24 hours.

[0060] (2) The coating method of the second test line 42 is: Dilute the anti-human IgG antibody with a coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN 3 ) to 0.1 - 1 mg / mL and then coat, and then dry at 37°C for 12 - 24 hours.

[0061] (3) The coating method of the quality control line 43 is: Dilute streptavidin with a coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN 3 ) to 1 - 5 mg / mL and then coat, and then dry at 37°C for 12 - 24 hours.

[0062] The material of the test pad 4 is selected from nitrocellulose membrane.

[0063] Preparation method of the sample pad 3:

[0064] Prepare a sample pad treatment buffer, the buffer is 25 mM Tris buffer, which contains 0.05% Tween 20, 1% BSA, 0.1 mg / mL anti-RBC, 0.1% NaN 3 , and evenly apply the buffer on the glass fiber, and then dry at 37°C for 12 - 24 hours.

[0065] The material of the sample pad 3 is selected from glass fiber.

[0066] The buffer pad 1 and the buffer solution:

[0067] The buffer pad 1 is made of a glass fiber or polyester film material with relatively strong hydrophobicity. In this example, a polyester film (Ahlstrom 6613) is selected. The buffer solution added to the buffer pad is 0.01M PBS buffer solution, which contains 0.2% Tween 20 and 0.1% NaN 3 .

[0068] The detection process of detecting dengue IgG / IgM antibodies using the test strip or test plate described in the present invention is as follows:

[0069] (1) Before detection, take out the test strip or test plate, and restore the sample and the buffer solution to room temperature.

[0070] (2) Place the test strip or test plate on a clean and flat desktop.

[0071] (3) Add 10 μL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample addition hole of the test plate.

[0072] (4) Add 2 drops (about 50 μL) of buffer solution to the buffer pad of the test strip or the buffer solution hole of the test plate.

[0073] (5) Wait for 15 minutes to read the detection result. Reading the result after 20 minutes is invalid.

[0074] Interpretation of the detection result:

[0075] (1) Negative: Only a red band appears in the control line C area, and no bands appear in the first detection line T1 area and the second detection line T2 area.

[0076] (2) Positive for dengue IgM antibody: A red band appears in the control line C area, a red band appears in the first detection line T1 area, and no red band appears in the second detection line T2 area.

[0077] (3) Positive for dengue IgG antibody: A red band appears in the control line C area, no red band appears in the first detection line T1 area, and a red band appears in the second detection line T2 area.

[0078] (4) Positive for dengue IgM / IgG antibody: Red bands appear in the control line C area, the first detection line T1 area, and the second detection line T2 area.

[0079] (5) Invalid: No red band appears in the control line C area. Insufficient sample volume or incorrect operation steps may be the reasons for the invalid result.

[0080] Example 2 Comparative Experiment

[0081] Comparative Example 1

[0082] like Figure 1 As shown, the conventional dengue IgG / IgM antibody test strip comprises a buffer pad 1, a marking pad 2, a detection pad 4 and a water-absorbing pad 8 which are overlapped and arranged in sequence from upstream to downstream. The buffer pad 1, the marking pad 2, the detection pad 4 and the water-absorbing pad 8 are all adhered to the bottom card 5.

[0083] The dengue IgG / IgM antibody test strip of Comparative Example 1 is substantially the same in structure, preparation method and detection process as the test strip described in Example 1, except that Comparative Example 1 does not include a sample pad, the sample is directly added to the label pad, and the material of the buffer pad and the composition of the buffer are different.

[0084] The material of the buffer pad is glass fiber or polyester film, and its hydrophobicity is weaker than that of the glass fiber or polyester film material of the buffer pad in Example 1. In this example, glass fiber (Ahlstrom 8964) is selected.

[0085] Buffer composition: 0.01M PBS buffer containing 1.0% Tween 20, 0.1% NaN 3 .

[0086] In this comparative example 1, during detection, the sample is directly added to the label pad, and the sample reaches the detection pad via the label pad, and the excess sample and reagent are absorbed by the absorption pad. In the detection process of dengue IgG and IgM positive samples at the same time, especially when detecting samples with strong positive IgG and weak positive IgM, the dengue IgG and IgM in the sample simultaneously contact and compete with the dengue recombinant antigen-colloidal gold complex on the label pad. Since the dengue IgM content in the blood sample is relatively low, it is at a competitive disadvantage, resulting in less dengue recombinant antigen-colloidal gold complex-dengue IgM antibody complex formed, and the sensitivity is reduced.

[0087] Comparative Example 2

[0088] The structure, preparation method and detection process of the dengue fever IgG / IgM antibody detection test strip of Comparative Example 2 are basically the same as those of the detection test strip described in Example 1, except that the material of the buffer pad of Comparative Example 2 is different.

[0089] The material of the buffer pad is glass fiber or polyester film, and its hydrophobicity is weaker than that of the glass fiber or polyester film material of the buffer pad in Example 1. In this example, glass fiber (Ahlstrom 8964) is selected.

[0090] Comparative Example 3

[0091] The dengue IgG / IgM antibody test strip of Comparative Example 3 is basically the same as the test strip described in Example 1 in terms of structure, preparation method, and detection process, except that the composition of the buffer solution in Comparative Example 3 is different.

[0092] Composition of the buffer solution: 0.01M PBS buffer solution, which contains 1.0% Tween 20 and 0.1% NaN 3 .

[0093] Test Example 1 of the present invention

[0094] The dengue IgG / IgM antibody test strip of Test Example 1 was prepared by the method described in Example 1.

[0095] As Figure 2 shown, for the dengue IgG / IgM antibody test strip of the present invention, the labeled pad 2 of the test strip is located upstream of the sample pad 3. After the sample is added to the sample pad, it chromatographs downstream along the test pad (i.e., the sample flows along the test pad towards the absorbent pad). At this time, the sample does not come into contact with the dengue recombinant antigen-gold colloid complex on the labeled pad 2. As the sample flows on the test pad, IgG and IgM in the sample successively bind to the anti-human IgG antibody pre-coated at the second test line 42 (T2 line) and the anti-human IgM antibody at the first test line 41 (T1 line) on the test pad and are fixed on the test pad. Since the content of IgM in blood samples is much less than that of IgG. On the test pad, the anti-human IgM antibody coated on the test pad is arranged downstream of the anti-human IgG antibody. When the sample chromatographs on the test pad, the sample first passes through the second test line 42 of the anti-human IgG antibody, and IgG is fixed on the second test line 42. In this way, because the content of IgG in the sample decreases, the relative content of IgM in the continuously flowing sample increases, thereby improving the binding efficiency of IgM in the sample to the anti-human IgM antibody at the first test line 41 of the test pad.

[0096] The detection process of Test Example 1 is the same as that of Example 1. The buffer solution was added to the buffer pad 1. With the addition and release of the buffer solution, the dengue recombinant antigen-gold colloid complex on the labeled pad 2 dissolves and begins to chromatograph along the test pad (such as nitrocellulose membrane). Due to the hydrophobicity of the buffer pad and the buffer solution, the time for the dengue recombinant antigen-gold colloid complex to reach the test pad is later than the time for the sample to reach the test pad. Subsequently, the dengue recombinant antigen-gold colloid complex successively binds to the dengue IgG (if there is dengue IgG in the sample) already bound to the second test line 42 and the dengue IgM bound to the first test line 41 on the test pad (if there is dengue IgG in the sample), thereby showing the test result.

[0097] In Test Example 1, the dengue recombinant antigen-gold colloid complex located on the marker pad 2 passed through the second test line 42 and the first test line 41 successively with the buffer. Since most of the IgG in the sample bound to the second test line 42, the interference of IgG in the sample with the binding of IgM to the dengue recombinant antigen-gold colloid complex on the first test line 41 of Test Example 1 of the present invention was reduced compared with the conventional product. And the absolute excess of the dengue recombinant antigen-gold colloid complex made the reduction of the dengue recombinant antigen-gold colloid complex due to passing through the second test line 42 negligible.

[0098] Test Example 1 of the present invention improves the sensitivity of IgM detection and does not affect the detection sensitivity and specificity of IgG. For the specific comparison results between Test Example 1 of the present invention and the comparative example, see Table 1.

[0099] Table 1: Comparison of the detection effects between Test Example 1 of the present invention and the comparative example

[0100]

[0101] Remarks: G0-G10 represent the strength of the line color. The larger the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0102] The dengue IgG / IgM antibody detection test strip is a qualitative detection product, and the detection results are divided into positive results and negative results. If the analyte in the sample is positive, the larger the value of the detection result measured by the test strip and the stronger the color, it indicates that the sensitivity of the test strip is higher and the performance is better. If the analyte in the sample is negative, the smaller the value of the detection result measured by the test strip and the weaker the color, it indicates that the specificity of the test strip is better and the performance is better.

[0103] Sample 1 is an IgG positive / IgM positive sample, and the detection results are as follows:

[0104] When detected using Comparative Example 1, IgG was positive with a strength of G10, and IgM was positive with a strength of G3; when detected using Comparative Example 2, IgG was positive with a strength of G10, and IgM was positive with a strength of G3.5; when detected using Comparative Example 3, IgG was positive with a strength of G10, and IgM was positive with a strength of G3.5; when detected using the present invention, IgG was still positive with a strength of G10, and IgM was positive with a strength of G5. The IgM signal strength was higher than that of the products of Comparative Example 1, Comparative Example 2, and Comparative Example 3. The IgM signal strengths of Comparative Example 2 and Comparative Example 3 were higher than that of Comparative Example 1.

[0105] Sample 2 is an IgG positive / IgM positive sample, and the detection results are as follows:

[0106] Using the detection of Comparative Example 1, IgG was positive with an intensity of G10, IgM was negative with an intensity of G2, and IgM was missed in the detection; using the detection of Comparative Example 2, IgG was positive with an intensity of G10, IgM was positive with an intensity of G3; using the detection of Comparative Example 3, IgG was positive with an intensity of G10, IgM was positive with an intensity of G3; using the detection of the present invention, IgG was still positive with an intensity of G10, IgM was positive with an intensity of G6, and IgM was not missed in the detection, and the signal intensity was higher than that of the products of Comparative Example 2 and Comparative Example 3.

[0107] The 3# sample was an IgG-positive / IgM-negative sample, and the test results were as follows:

[0108] Using Comparative Example 1, Comparative Example 2, Comparative Example 3 and the present invention for detection, IgG was positive with an intensity of G5, and IgM was negative. The test results were consistent.

[0109] The 4# sample was an IgG-negative / IgM-positive sample, and the test results were as follows:

[0110] Using the detection of Comparative Example 1, IgG was negative and IgM was positive with an intensity of G4; using the detection of Comparative Example 2, IgG was negative and IgM was positive with an intensity of G4.5; using the detection of Comparative Example 3, IgG was negative and IgM was positive with an intensity of G4.5; using the detection of the present invention, IgG was negative and IgM was positive with an intensity of G5, and the IgM signal intensity was higher than that of the products of Comparative Example 1, Comparative Example 2 and Comparative Example 3. At the same time, the IgM signal intensities of Comparative Example 2 and Comparative Example 3 were higher than that of the product of Comparative Example 1.

[0111] The 5# / 6# / 7# samples were IgG-negative / IgM-negative samples, and the test results were as follows:

[0112] Using the detection of Comparative Example 1, IgG was negative and IgM intensities were positive with G3 / G3 / G4 respectively, showing the situation of false positive IgM; using Comparative Example 2, Comparative Example 3 and the present invention for detection, IgG was negative and IgM was negative, and there was no phenomenon of false positive IgM.

[0113] According to the results in Table 1: The present invention improves the sensitivity and specificity of IgM detection and has no influence on IgG detection.

[0114] Example 3

[0115] The dengue IgG / IgM antibody detection test strip and its preparation method in this example were basically the same as those of Comparative Example 1 in Example 2, except that a sample pad was added.

[0116] The detection process of the dengue IgG / IgM antibody detection reagent is as follows:

[0117] (1)Before detection, take out the test strip or test plate, and restore the sample and buffer to room temperature.

[0118] (2)Place the test strip or test plate on a clean and flat tabletop.

[0119] (3)Add 10 uL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample well of the test plate.

[0120] (4)Add buffer to the test strip in two ways: wait or don't wait. Specifically, after waiting for 1 - 2 minutes, add 2 drops (about 50 uL) of buffer to the buffer pad of the test strip or the buffer well of the test plate; or without waiting, directly add 2 drops (about 50 uL) of buffer to the buffer pad of the test strip or the buffer well of the test plate, which is the detection process of Example 1.

[0121] (5)Read the test result after 15 minutes. Reading the result after 20 minutes is invalid.

[0122] Table 2: Comparison of the detection effect with different waiting times

[0123]

[0124] Note: G0 - G10 represent the strength of the line color. The larger the value, the stronger the color. Among them, G0 - G2: negative; G3 - G10: positive.

[0125] Sample 1 is an IgG - positive / IgM - positive sample, and the test results are as follows:

[0126] When using the non - waiting mode product for detection, IgG is positive with a strength of G10, and IgM is positive with a strength of G3; when using the waiting mode for detection, IgG is still positive with a strength of G10, and IgM is positive with a strength of G4.5, and the IgM signal strength is higher than that of the non - waiting mode product.

[0127] Sample 2 is an IgG - positive / IgM - positive sample, and the test results are as follows:

[0128] When using the non - waiting mode product for detection, IgG is positive with a strength of G10, and IgM is negative with a strength of G2, resulting in a missed detection of IgM; when using the waiting mode for detection, IgG is still positive with a strength of G10, and IgM is positive with a strength of G6.5, without missed detection of IgM, and the signal is stronger.

[0129] Sample 3 is an IgG - positive / IgM - negative sample, and the test results are as follows:

[0130] When using the non - waiting mode product for detection, IgG is positive with a strength of G5, and IgM is negative; when using the waiting mode for detection, IgG is positive with a strength of G5, and IgM is negative.

[0131] Sample 4 is an IgG negative / IgM positive sample, and the test results are as follows:

[0132] When using the non-waiting mode product for detection, IgG is negative and IgM is positive with an intensity of G4; when using the waiting mode for detection, IgG is negative and IgM is positive with an intensity of G5, and the IgM signal intensity is higher than that of the non-waiting mode product.

[0133] Samples 5 / 6 / 7 are IgG negative / IgM negative samples, and both are negative when detected using the two modes.

[0134] According to the results in Table 2: The waiting mode improves the sensitivity of IgM detection and has no effect on IgG detection.

[0135] Example 4

[0136] The dengue IgG / IgM antibody test strip and its preparation method in this example are basically the same as those in Example 1, except that the test strip does not include a buffer pad.

[0137] The detection process of the dengue IgG / IgM antibody detection reagent is as follows:

[0138] (1) Before detection, take out the test strip or test plate, and restore the sample and buffer to room temperature.

[0139] (2) Place the test strip or test plate on a clean and flat tabletop.

[0140] (3) Add 10 μL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample well of the test plate.

[0141] (4) Add buffer to the test strip in a waiting or non-waiting manner, respectively: After waiting for 1 - 2 minutes, drip 2 drops (about 50 μL) of buffer on the marking pad; or without waiting, directly drip 2 drops (about 50 μL) of buffer on the marking pad.

[0142] (5) Read the test results after 15 minutes. Reading the results after 20 minutes is invalid.

[0143] Table 3: Comparison of the detection effect with different waiting times

[0144]

[0145] Note: G0 - G10 represent the strength of the line color. The larger the value, the stronger the color. Among them, G0 - G2: negative; G3 - G10: positive.

[0146] Sample 1 is an IgG positive / IgM positive sample, and the test results are as follows:

[0147] Using the product detection in non-waiting mode, IgG is positive with intensity G10 and IgM is positive with intensity G3; using the waiting mode for detection, IgG is still positive with intensity G10 and IgM is positive with intensity G4, and the IgM signal intensity is higher than that in the non-waiting mode product.

[0148] Sample No. 2 is an IgG-positive / IgM-positive sample, and the test results are as follows:

[0149] Using the product detection in non-waiting mode, IgG is positive with intensity G10 and IgM is negative with intensity G2, and there is a situation of IgM missed detection; using the waiting mode for detection, IgG is still positive with intensity G10 and IgM is positive with intensity G5, and there is no situation of IgM missed detection, and the signal is relatively strong.

[0150] Sample No. 3 is an IgG-positive / IgM-negative sample, and the test results are as follows:

[0151] Using the product detection in non-waiting mode, IgG is positive with intensity G5 and IgM is negative with intensity G2, and there is a faint line shadow of IgM; using the waiting mode for detection, IgG is positive with intensity G5 and IgM is negative with intensity G2, and there is a faint line shadow of IgM.

[0152] Sample No. 4 is an IgG-negative / IgM-positive sample, and the test results are as follows:

[0153] Using the product detection in non-waiting mode, IgG is negative and IgM is positive with intensity G3; using the waiting mode for detection, IgG is negative and IgM is positive with intensity G4, and the IgM signal intensity is higher than that in the non-waiting mode product.

[0154] Samples No. 5 / 6 / 7 are IgG-negative / IgM-negative samples, and both modes of detection show negative results.

[0155] According to the results in Table 3, it shows that the waiting mode plays a certain role in improving the sensitivity of IgM detection and has no impact on IgG detection.

Claims

1. A test strip for detecting IgG / IgM antibodies, characterized in that: The test paper includes a marking pad, a sample pad, a detection pad and a water-absorbing pad which are adhered to a bottom card and overlapped in sequence from upstream to downstream; the detection pad includes a first detection line and a second detection line, the first detection line is coated with anti-IgM antibodies, the second detection line is coated with anti-IgG antibodies, the first detection line is located downstream of the second detection line, the marking pad includes a marker combined with an antigen, and the time when the marker on the marking pad reaches the detection pad is later than the time when the added sample reaches the detection pad.

2. The test paper according to claim 1, characterized in that Also included is a buffer pad for receiving a buffer, the buffer pad being located upstream of the label pad.

3. The test paper according to claim 2, characterized in that: The material of the buffer pad is hydrophobic.

4. The test paper according to claim 3, characterized in that: The cushion material is made of highly hydrophobic glass fiber or polyester film material.

5. The test strip according to any one of claims 1 to 4, characterized in that: The release rate of the marker on the marker pad is slower than the release rate of the sample on the sample pad.

6. The test strip according to any one of claims 1 to 4, characterized in that: The IgG / IgM antibody is a dengue fever IgG / IgM antibody.

7. The test strip according to claim 6, characterized in that: The label pad includes a label complex in which the dengue fever antigen is combined with a label.

8. The test paper according to claim 1, characterized in that: The detection pad of the test paper also includes a quality control line, which is coated with streptavidin; and the label pad is coated with a biotin-label compound.

9. A detection kit for detecting IgG / IgM antibodies, characterized in that: The detection kit comprises the detection test paper according to any one of claims 1 to 8 and a buffer solution.

10. The detection kit according to claim 9, characterized in that The buffer comprises 0.01M PBS buffer, wherein the PBS buffer contains 0.2% to 1% Tween 20 and 0.1% NaN3.

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