Detection test paper and detection kit for detecting IgG / IgM antibody

Abstract

The invention provides detection test paper and a detection kit for detecting an IgG / IgM antibody. The detection test paper comprises a marking pad, a sample pad, a detection pad and a water absorption pad which are adhered to a bottom card and are sequentially overlapped from upstream to downstream, the detection pad is coated with an IgG detection line and an IgM detection line, and the IgG detection line is located at the upstream of the IgM detection line; during detection, the sample reaches the detection line of the detection pad before the marker. The detection test paper is simple in structure, convenient to use, high in anti-interference capacity and high in detection sensitivity, and the detection kit can improve the accuracy, early diagnosis and early treatment of early diagnosis of diseases such as infectious diseases, so that transmission of the infectious diseases is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis, and in particular 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 transform into plasma cells and produce antibodies that specifically bind to the corresponding antigens to form immunoglobulins, including immunoglobulins such as IgM and IgG.

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

[0004] Abnormal IgM values ​​often indicate the possibility of infection. In the early stages of infection or during the active stages of certain autoimmune diseases, such as rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, etc., serum IgM levels may increase. Abnormal 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 for pregnancy tests to determine whether the fetus has intrauterine infection. In some diseases such as chronic liver diseases such as hepatitis A, hepatitis B, and hepatitis C, an increase in IgM may indicate that the virus is in an active stage.

[0005] IgM can be used as an early diagnostic indicator for infectious diseases, and its positive result usually indicates that the body is in the acute infection stage. IgM testing can help assist in the diagnosis of a variety of diseases, including autoimmune diseases, digestive system diseases, blood system diseases, respiratory system diseases, infectious diseases, and urinary system diseases.

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

[0007] Dengue fever is an acute insect-borne infectious disease caused by 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 confirm whether the patient is infected with dengue virus.

[0008] Antibody testing is done by detecting specific antibodies IgG and IgM in the patient's body. When a person is infected with the dengue virus, the body's immune system produces two types of antibodies, IgG and IgM, to fight the virus. IgM antibodies can be detected in patients with the first infection 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 the case of secondary infection.

[0009] like Figure 1 The test strip shown is a conventional product for detecting dengue antibodies using the lateral flow (LF) method. Figure 1 The test strip shown includes a buffer pad 1, a marking pad 2, a detection pad 4 (nitrocellulose membrane) and a water-absorbing pad 8 which are adhered to a base card 5 and arranged in sequence. The blood sample is directly added to the marking pad and reacts with the dengue fever recombinant antigen-colloidal gold complex on the marking pad to form a dengue fever recombinant antigen-colloidal gold-Dengue antibody complex. Buffer is added to the buffer pad to make the dengue fever recombinant antigen-colloidal gold-Dengue antibody complex move along the detection pad and combine with the anti-human IgM antibody or / and anti-human IgG antibody pre-coated on the detection line of the detection pad to develop color.

[0010] For products that detect Dengue IgG antibodies and Dengue IgM antibodies on one 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 low. Summary of the invention

[0011] In order to overcome the defects of the prior art, the present invention provides a test paper for detecting IgG / IgM antibodies, comprising a marking pad, a sample pad, a detection pad and a water-absorbing pad adhered to a bottom card and arranged in sequence from upstream to downstream; the detection pad comprises 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, and the marking pad comprises a marker combined with an antigen.

[0012] Furthermore, it also includes a buffer pad for receiving a buffer, wherein the buffer pad is located upstream of the labeling pad.

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

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

[0015] Furthermore, the release rate of the marker on the marker pad is slower than the release rate of the sample on the sample pad, that is, the time when the marker on the marker pad reaches the detection pad is later than the time when the added sample reaches the detection pad.

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

[0017] Furthermore, the label pad includes a labeling complex in which the dengue fever antigen is combined with a labeling substance.

[0018] More specifically, the dengue antigen is: type I, type II, type III, type IV dengue recombinant antigen. The marker is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a colloidal gold complex of type I, type II, type III, type IV dengue recombinant antigen that is individually labeled.

[0019] Furthermore, the detection pad of the test paper also includes a quality control line, which is coated with streptavidin; the label pad is coated with a biotin-label compound. The label is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a biotin-colloidal gold compound.

[0020] In the specific scheme of the dengue fever IgG / IgM antibody detection test paper of the present invention, the test paper adds a sample pad downstream of the marking pad, and the sample is directly added to the sample pad and flows along the detection pad. At this time, the sample is not in contact with the dengue fever recombinant antigen-colloidal gold complex. The IgG and IgM in the sample are successively combined with the anti-human IgG antibody and the anti-human IgM antibody pre-coated on the detection pad and fixed on the detection pad. Then, with the addition of buffer, the dengue fever recombinant antigen-colloidal gold complex fixed on the marking pad dissolves and begins to flow on the detection pad, and the dengue fever recombinant antigen-colloidal gold complex is successively combined with the Dengue-IgG antibody and the Dengue-IgM antibody fixed on the detection pad, so that the corresponding position of the detection pad is colored, and the purpose of the inspection is achieved.

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

[0022] Furthermore, the buffer comprises 0.01M PBS buffer, wherein the PBS buffer contains 0.2% to 1% Tween20 and 0.1% NaN3.

[0023] In a specific scheme of the dengue fever IgG / IgM antibody detection kit of the present invention, its use steps include: (1) directly adding a sample to a sample pad; (2) the sample flows from the sample pad to the detection pad, the sample chromatographs (flows) along the detection pad, and the IgG and IgM in the sample are successively combined with the anti-human IgG antibody and the anti-human IgM antibody pre-coated on the detection pad and fixed on the detection pad; (3) a buffer solution is added to the buffer solution pad and flows to the label pad, and the dengue fever recombinant antigen-colloidal gold complex fixed on the label pad is dissolved; (4) the dengue fever recombinant antigen-colloidal gold complex flows to the detection pad with the buffer solution and continues to flow, and on the detection pad, the dengue fever recombinant antigen-colloidal gold complex is successively combined with the Dengue-IgG antibody and the Dengue-IgM antibody that have been fixed on the detection pad by the anti-human IgG antibody and the anti-human IgM antibody, so that the corresponding position of the detection pad is colored.

[0024] The present invention also provides a detection method for detecting IgG / IgM antibodies, the method comprising: providing a detection test paper for detecting IgG / IgM antibodies, the detection test paper comprising a marking pad, a sample pad, a detection pad and a water-absorbing pad adhered to a bottom card and arranged in sequence from upstream to downstream; the detection pad comprises 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, and the marking pad comprises a marker combined with an antigen; adding a sample to be detected to the sample pad of the detection test paper; adding a buffer solution to the test paper to release the marker on the marking pad and flow to the detection pad; 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.

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

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

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

[0028] Furthermore, the release rate of the marker on the marker pad is slower than the release rate of the sample on the sample pad, that is, the time when the marker on the marker pad reaches the detection pad is later than the time when the added sample reaches the detection pad. Furthermore, after adding the sample to be tested to the sample pad of the detection test paper and waiting for a preset time, the buffer solution is added to the detection test paper.

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

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

[0031] Furthermore, the label pad includes a labeling complex in which the dengue fever antigen is combined with a labeling substance.

[0032] More specifically, the dengue antigen is: type I, type II, type III, type IV dengue recombinant antigen. The marker is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a colloidal gold complex of type I, type II, type III, type IV dengue recombinant antigen that is individually labeled.

[0033] Furthermore, the detection pad of the test paper also includes a quality control line, which is coated with streptavidin; the label pad is coated with a biotin-label compound. The label is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a biotin-colloidal gold compound.

[0034] Since the conventional IgG / IgM antibody detection product first combines the sample with the marker marked with the antigen on the label pad, the combined flow enters the detection pad for chromatography and reacts with the substances on the detection line of the detection pad. For example, the dengue fever antibody in the sample combines with the dengue fever recombinant antigen-colloidal gold complex on the label pad and then enters the nitrocellulose membrane of the detection pad for chromatography, and reacts with the anti-IgM antibody of the first detection line and the anti-IgG antibody of the second detection line on the detection pad, thereby displaying the detection result. This will lead to a decrease in the sensitivity of IgM.

[0035] The test strips and corresponding test kits and detection methods for detecting IgG / IgM antibodies of the present invention allow the sample enough time to flow and chromatograph independently on the test strips before the marker on the marker pad is released, and the sample reaches the detection line before the marker. For example, a sample pad is added downstream of the marker pad of the test strip, the sample is added to the sample pad, and the sample is allowed to enter the nitrocellulose membrane of the detection pad for chromatography before it is combined with the marker, and the substance on the detection line of the detection pad undergoes a binding reaction. Subsequently, the marker on the marker pad flows into the detection pad for chromatography under the drive of the buffer, and reacts with the analyte in the sample, such as IgG and IgM, to display the test result.

[0036] The test paper structure and detection method of the present invention can effectively weaken the interference of interfering substances in the sample before the sample contacts the marker. For example, the interference of IgG with a large content on IgM with a relatively small content in the sample is effectively weakened. The present invention will independently set up the sample pad and the marking pad, and can also make the sample pad treatment more selective without having to consider too much about the stability of related substances in the marker, such as active substances such as colloidal gold antigens. In addition, the present invention can also delay the time for the buffer to enter the marking pad by selecting the material of the buffer pad and adjusting the buffer formula, thereby delaying the release of the marker on the marking pad, so that the sample can be chromatographed independently on the detection pad before the marker. The use of the buffer pad and buffer of conventional products makes the marker release rate too fast, resulting in a short independent chromatography time of the sample on the detection pad, and low detection sensitivity.

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

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

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

[0040] Figure 3 yes Figure 2 Exploded view of the test strip.

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

[0042] Figure 5 It is a structural exploded view of the dengue fever IgG / IgM antibody detection plate of the present invention. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further illustrated and described below through specific implementation methods.

[0044] like Figure 2 and Figure 3As shown, an IgG / IgM antibody detection test paper 10 of the present invention comprises a buffer pad 1, a marking pad 2, a sample pad 3, a detection pad 4 and a water-absorbing pad 8 which are overlapped and stacked in sequence from upstream to downstream. The buffer pad 1, the marking pad 2, the sample pad 3, the detection pad 4 and the water-absorbing pad 8 are all adhered to the bottom card 5. The detection pad 4 comprises a first detection line 41 and a second detection line 42, the first detection line 41 is coated with anti-IgM antibodies, the second detection line 42 is coated with anti-IgG antibodies, and 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] like Figure 4 As shown, another IgG / IgM antibody detection test paper 10 of the present invention comprises a marking pad 2, a sample pad 3, a detection pad 4 and a water-absorbing pad 8 which are overlapped and superimposed in sequence from upstream to downstream. The marking pad 2, the sample pad 3, the detection pad 4 and the water-absorbing pad 8 are all adhered to the bottom card 5. The detection pad 4 comprises a first detection line 41 and a second detection line 42, the first detection line 41 is coated with anti-IgM antibodies, the second detection line 42 is coated with anti-IgG antibodies, and 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 material or polyester film material, preferably glass fiber material or polyester film material with strong hydrophobicity, or glass fiber material or polyester film material treated with a hydrophobic agent.

[0047] The material of the marking 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 membrane, etc. The material of the absorbent pad 8 is selected from absorbent paper, filter paper, etc.

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

[0049] The test paper of the present invention can be used for detecting IgG, IgM, etc. in a sample, for example, a test paper for detecting dengue IgG, IgM, etc. in a blood sample. The test kit for detecting dengue IgG / IgM antibodies comprises the dengue IgG / IgM antibody test paper 10 of the present invention and a buffer. The dengue IgG / IgM antibody test kit can also comprise the dengue IgG / IgM antibody test plate 20 of the present invention and a buffer.

[0050] Example 1 Preparation and detection steps of dengue fever IgG / IgM antibody test strips like Figure 2 The dengue IgG / IgM antibody test strip 10 shown comprises a buffer pad 1, a marking pad 2, a sample pad 3, a detection pad 4 and a water-absorbing pad 8, which are respectively adhered to a bottom card 5 and overlapped in sequence from upstream to downstream. In this embodiment, the width of the test strip is 3.5 mm, and the overlap area between the pads is 1-2 mm.

[0051] The label pad 2 is coated with dengue fever type I, type II, type III, type IV recombinant antigen-colloidal gold complex and biotin-colloidal gold complex which are individually labeled.

[0052] Preparation method of marking pad: (1) The dengue virus recombinant antigen is rapidly added to a colloidal gold dispersion having 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 the mixture is rapidly stirred with a stirrer. After reacting for 15-30 minutes, a blocking solution (1% BSA, 0.1% NaN3) is rapidly added at a ratio of 10-15 uL / mL, and the mixture is reacted for 10-30 minutes. The mixture is centrifuged at 8000-10000 rpm for 25-30 minutes, and the supernatant is discarded to obtain a precipitate. A reconstitution solution (25 mMTris buffer, 1% BSA, 0.1% NaN3) is added to the precipitate at a ratio of 100 uL / mL, and the mixture is reconstituted by ultrasonication to prepare a type I dengue fever recombinant antigen-colloidal gold complex, a type II dengue fever recombinant antigen-colloidal gold complex, a type III dengue fever recombinant antigen-colloidal gold complex, and a type IV dengue fever recombinant antigen-colloidal gold complex, respectively.

[0053] (2) Biotin was rapidly added to a 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 the mixture was rapidly stirred with a stirrer. After reacting for 15-30 minutes, blocking solution (1% BSA, 0.1% NaN3) was rapidly added at a ratio of 10-15 uL / mL, and the mixture was reacted for 10-30 minutes. The mixture was centrifuged at 8000-10000 rpm for 25-30 minutes, and the supernatant was discarded to obtain a precipitate. A preservation solution (25 mMTris buffer, 1% BSA, 0.1% NaN3) was added to the precipitate at a ratio of 100 uL / mL, and the mixture was re-dissolved by ultrasonication to prepare a biotin-colloidal gold complex.

[0054] (3) According to the titer, the above-mentioned dengue type I recombinant antigen-colloidal gold complex, dengue type II recombinant antigen-colloidal gold complex, dengue type III recombinant antigen-colloidal gold complex, dengue type IV recombinant antigen-colloidal gold complex and biotin-colloidal gold complex are mixed and diluted with a diluent, and then evenly applied on the marking pad 2, and then dried at 37°C for 12-24 hours. The material of the marking pad is selected from glass fiber. The diluent is a 25mM Tris buffer, which contains 1% BSA, 20% sucrose, and 0.1% NaN3.

[0055] Preparation method of test pad: The detection pad 4 has a first detection line (T1-line) 41, a second detection line (T2-line) 42 and a quality control line (C-line) 43. The first detection line 41 is coated with anti-human IgM antibody, the second detection line is coated with anti-human IgG antibody, and the quality control line 43 is coated with streptavidin.

[0056] (1) The coating method of the first detection line 41 is as follows: dilute the anti-human IgM antibody to 0.1-1 mg / mL with a coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN3) and then coat it, and then dry it at 37°C for 12-24 hours.

[0057] (2) The coating method of the second detection line 42 is as follows: dilute the anti-human IgG antibody to 0.1-1 mg / mL with a coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN3) and then coat it, and then dry it at 37°C for 12-24 hours.

[0058] (3) The coating method for quality control line 43 is as follows: dilute streptavidin to 1-5 mg / mL with coating buffer (0.01 M PBS buffer, 10% sucrose, 0.1% NaN3) and then dry at 37°C for 12-24 hours.

[0059] The material of the detection pad 4 is selected from nitrocellulose membrane.

[0060] Sample pad 3 preparation method: Prepare a sample pad treatment buffer, which is 25 mM Tris buffer containing 0.05% Tween 20, 1% BSA, 0.1 mg / mL anti-RBC, and 0.1% NaN3. Evenly apply the buffer on the glass fiber and dry it at 37°C for 12-24 hours.

[0061] The material of the sample pad 3 is selected from glass fiber.

[0062] Buffer pad 1 and buffer: The buffer pad 1 is made of glass fiber or polyester film material with strong hydrophobicity. In this example, polyester film (Ahlstrom 6613) is selected. The buffer added dropwise to the buffer pad is 0.01M PBS buffer, which contains 0.2% Tween 20 and 0.1% NaN3.

[0063] The detection process of using the test strip or test plate of the present invention to detect dengue IgG / IgM antibodies is as follows: (1) Before testing, remove the test strip or test plate and return the sample and buffer to room temperature.

[0064] (2) Place the test strip or test plate on a clean, flat table.

[0065] (3) Add 10uL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample well of the test plate.

[0066] (4) Add 2 drops of buffer (about 50uL) to the buffer pad of the test strip or the buffer well of the test plate.

[0067] (5) Wait 15 minutes to read the test results. Reading the results after 20 minutes will be invalid.

[0068] Explanation of test results: (1) Negative: A red stripe appears only in the quality control line C area, and no stripe appears in the first test line T1 and the second test line T2 area.

[0069] (2) Dengue IgM antibody positive: a red stripe appears in the quality control line C area, a red stripe appears in the first test line T1 area, and no red stripe appears in the second test line T2 area.

[0070] (3) Dengue IgG antibody positive: a red stripe appears in the C area of ​​the quality control line, no red stripe appears in the T1 area of ​​the first test line, and a red stripe appears in the T2 area of ​​the second test line.

[0071] (4) Dengue IgM / IgG antibody positive: Red stripes appear in the quality control line C area, the first test line T1 area, and the second test line T2 area.

[0072] (5) Invalid: No red band appears in the quality control line C area. Insufficient sample volume or incorrect operation steps may be the cause of invalid results.

[0073] Example 2 Comparative Experiment Comparative Example 1 like Figure 1As 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.

[0074] 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.

[0075] 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.

[0076] Buffer composition: 0.01 M PBS buffer containing 1.0% Tween 20, 0.1% NaN3.

[0077] 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.

[0078] Comparative Example 2 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.

[0079] 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.

[0080] Comparative Example 3 The structure, preparation method and detection process of the dengue fever IgG / IgM antibody detection test strip of Comparative Example 3 are basically the same as those of the detection test strip described in Example 1, except that the composition of the buffer solution of Comparative Example 3 is different.

[0081] Buffer composition: 0.01 M PBS buffer containing 1.0% Tween 20, 0.1% NaN3.

[0082] Test Example 1 of the present invention The dengue fever IgG / IgM antibody detection test paper of Test Example 1 was prepared by the method described in Example 1.

[0083] like Figure 2 As shown, the dengue fever IgG / IgM antibody test paper of the present invention has a label pad 2 located upstream of the sample pad 3. After the sample is added to the sample pad, it is chromatographed downstream along the test pad (i.e., the sample flows along the test pad toward the absorbent pad), and at this time, the sample does not contact the dengue fever recombinant antigen-colloidal gold complex on the label pad 2. As the sample flows on the test pad, the IgG and IgM in the sample are successively combined with the anti-human IgG antibody pre-coated at the second test line 42 (T2 line) of the test pad and the anti-human IgM antibody at the first test line 41 (T1 line) and fixed on the test pad. Since the content of IgM in the blood sample is much smaller than the content of IgG. On the detection pad, the anti-human IgM antibody coated on the detection pad is arranged downstream of the anti-human IgG antibody. When the sample is chromatographed on the detection pad, the sample first passes through the second detection line 42 of the anti-human IgG antibody to fix the IgG on the second detection line 42. In this way, since the content of IgG in the sample is reduced, the relative content of IgM in the sample that continues to flow increases, thereby improving the binding efficiency of IgM in the sample with the anti-human IgM antibody at the first detection line 41 of the detection pad.

[0084] The detection process of Test Example 1 is the same as that of Example 1. Buffer is added to the buffer pad 1. As the buffer is added and released, the dengue fever recombinant antigen-colloidal gold complex on the label pad 2 dissolves and begins to chromatograph along the detection pad (e.g., nitrocellulose membrane). Due to the hydrophobicity of the buffer pad and the buffer, the time when the dengue fever recombinant antigen-colloidal gold complex arrives at the detection pad is later than the time when the sample arrives at the detection pad, and then, the dengue fever recombinant antigen-colloidal gold complex successively binds to the dengue fever IgG bound to the second detection line 42 (if there is dengue fever IgG in the sample) and the dengue fever IgM bound to the first detection line 41 (if there is dengue fever IgG in the sample) on the detection pad, thereby displaying the test results.

[0085] In test example 1, the dengue fever recombinant antigen-colloidal gold complex located on the marking pad 2 passes through the second detection line 42 and the first detection line 41 with the buffer solution. Since most of the IgG in the sample is bound to the second detection line 42, compared with conventional products, the interference of IgG in the sample on the binding of IgM with the dengue fever recombinant antigen-colloidal gold complex on the first detection line 41 of test example 1 of the present invention is reduced. The absolute excess of the dengue fever recombinant antigen-colloidal gold complex makes the dengue fever recombinant antigen-colloidal gold complex reduced by passing through the second detection line 42 negligible.

[0086] The test example 1 improves the sensitivity of IgM detection, and does not affect the sensitivity and specificity of IgG detection. The specific comparison results of the test example 1 of the present invention and the comparative example are shown in Table 1.

[0087] Table 1: Comparison of the detection effects of test example 1 of the present invention and the comparative example

[0088] Note: G0-G10 represents the strength of the line color. The larger the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0089] Dengue IgG / IgM antibody test strips are qualitative test products. The test results are divided into positive and negative results. If the analyte in the sample is positive, the larger the value of the test result measured by the test strip and the stronger the color, the higher the sensitivity of the test strip and the better the performance. If the analyte in the sample is negative, the smaller the value of the test result measured by the test strip and the weaker the color, the better the specificity of the test strip and the better the performance.

[0090] Sample #1 is an IgG positive / IgM positive sample, and the test results are: When detected by Comparative Example 1, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3; when detected by Comparative Example 2, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3.5; when detected by Comparative Example 3, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3.5; when detected by the present invention, IgG was still positive with an intensity of G10, 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. The IgM signal intensity of Comparative Example 2 and Comparative Example 3 was higher than that of Comparative Example 1.

[0091] Sample 2# is IgG positive / IgM positive sample, the test results are: When detected using Comparative Example 1, IgG was positive with an intensity of G10, and IgM was negative with an intensity of G2, indicating that IgM was missed; when detected using Comparative Example 2, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3; when detected using Comparative Example 3, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3; when detected using the present invention, IgG was still positive with an intensity of G10, and IgM was positive with an intensity of G6, indicating that IgM was not missed, and the signal intensity was higher than that of the products of Comparative Examples 2 and 3.

[0092] Sample #3 is an IgG positive / IgM negative sample, and the test results are: Using comparative example 1, comparative example 2, comparative example 3 and the present invention for detection, IgG was positive with intensity G5, and IgM was negative. The detection results were consistent.

[0093] Sample #4 is an IgG negative / IgM positive sample, and the test results are: When tested with Comparative Example 1, IgG was negative and IgM was positive with an intensity of G4; when tested with Comparative Example 2, IgG was negative and IgM was positive with an intensity of G4.5; when tested with Comparative Example 3, IgG was negative and IgM was positive with an intensity of G4.5; when tested with 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 intensity of Comparative Example 2 and Comparative Example 3 was higher than that of the product of Comparative Example 1.

[0094] Samples 5# / 6# / 7# are IgG negative / IgM negative samples, and the test results are: When comparative example 1 was used for detection, IgG was negative, and the IgM intensities were positive for G3 / G3 / G4, respectively, and false positive IgM occurred; when comparative example 2, comparative example 3 and the present invention were used for detection, IgG was negative, IgM was negative, and no false positive IgM occurred.

[0095] The results in Table 1 show that the present invention improves the sensitivity and specificity of IgM detection and has no effect on IgG detection.

[0096] Example 3 The dengue IgG / IgM antibody detection test strip and preparation method of this embodiment are basically the same as those of Comparative Example 1 of Example 2, except that a sample pad is added.

[0097] The testing process of dengue IgG / IgM antibody test kit is as follows: (1) Before testing, remove the test strip or test plate and return the sample and buffer to room temperature.

[0098] (2) Place the test strip or test plate on a clean, flat table.

[0099] (3) Add 10uL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample well of the test plate.

[0100] (4) Adding buffer to the test strip by waiting or not waiting, respectively: after waiting for 1-2 minutes, add 2 drops of buffer (about 50uL) to the buffer pad of the test strip or the buffer well of the test plate; or without waiting, directly add 2 drops of buffer (about 50uL) to the buffer pad of the test strip or the buffer well of the test plate, that is, the detection process of Example 1.

[0101] (5) Read the test results after 15 minutes. Results read after 20 minutes are invalid.

[0102] Table 2: Comparison of waiting time on detection effect

[0103] Note: G0-G10 represents the strength of the line color. The larger the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0104] Sample #1 is an IgG positive / IgM positive sample, and the test results are: When testing with the no-wait mode product, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3; when testing with the wait mode, IgG was still positive with an intensity of G10, and IgM was positive with an intensity of G4.5. The IgM signal intensity was higher than that of the no-wait mode product.

[0105] Sample 2# is IgG positive / IgM positive sample, the test results are: When the product was tested in the non-waiting mode, IgG was positive with an intensity of G10, and IgM was negative with an intensity of G2, indicating that IgM was missed. When the product was tested in the waiting mode, IgG was still positive with an intensity of G10, and IgM was positive with an intensity of G6.5, indicating that IgM was not missed, and the signal was stronger.

[0106] Sample #3 is an IgG positive / IgM negative sample, and the test results are: When the product was tested in the no-wait mode, IgG was positive with an intensity of G5 and IgM was negative; when the product was tested in the wait mode, IgG was positive with an intensity of G5 and IgM was negative.

[0107] Sample #4 is an IgG negative / IgM positive sample, and the test results are: When the product was tested in the no-wait mode, IgG was negative and IgM was positive with an intensity of G4. When the product was tested in the wait mode, IgG was negative and IgM was positive with an intensity of G5. The IgM signal intensity was higher than that of the product in the no-wait mode.

[0108] Samples 5# / 6# / 7# were IgG-negative / IgM-negative samples, and were negative using both detection modes.

[0109] The results in Table 2 show that the waiting mode improves the sensitivity of IgM detection and has no effect on IgG detection.

[0110] Example 4

[0111] The dengue IgG / IgM antibody test strip and preparation method of this embodiment are substantially the same as those of embodiment 1, except that the test strip does not include a buffer pad.

[0112] The testing process of dengue IgG / IgM antibody test kit is as follows: (1) Before testing, remove the test strip or test plate and return the sample and buffer to room temperature.

[0113] (2) Place the test strip or test plate on a clean, flat table.

[0114] (3) Add 10uL of serum / plasma or whole blood sample to the sample pad of the test strip or the sample well of the test plate.

[0115] (4) Add buffer to the test strip by waiting or not waiting, respectively: wait for 1-2 minutes, then add 2 drops of buffer (about 50uL) on the marking pad; or do not wait, then add 2 drops of buffer (about 50uL) directly on the marking pad.

[0116] (5) Read the test results after 15 minutes. Results read after 20 minutes are invalid.

[0117] Table 3: Comparison of waiting time on detection effect

[0118] Note: G0-G10 represents the strength of the line color. The larger the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0119] Sample #1 is an IgG positive / IgM positive sample, and the test results are: When the no-wait mode product was used for testing, IgG was positive with an intensity of G10, and IgM was positive with an intensity of G3; when the waiting mode product was used for testing, IgG was still positive with an intensity of G10, and IgM was positive with an intensity of G4. The IgM signal intensity was higher than that of the no-wait mode product.

[0120] Sample 2# is IgG positive / IgM positive sample, the test results are: When the product was tested in the non-waiting mode, IgG was positive with an intensity of G10, and IgM was negative with an intensity of G2, indicating that IgM was missed. When the product was tested in the waiting mode, IgG was still positive with an intensity of G10, and IgM was positive with an intensity of G5, indicating that IgM was not missed, and the signal was stronger.

[0121] Sample #3 is an IgG positive / IgM negative sample, and the test results are: When testing using the no-wait mode product, IgG is positive with an intensity of G5, IgM is negative with an intensity of G2, and IgM has a faint line shadow; when testing using the wait mode product, IgG is positive with an intensity of G5, IgM is negative with an intensity of G2, and IgM has a faint line shadow.

[0122] Sample #4 is an IgG negative / IgM positive sample, and the test results are: When the product was tested in the no-wait mode, IgG was negative and IgM was positive with an intensity of G3. When the product was tested in the wait mode, IgG was negative and IgM was positive with an intensity of G4. The IgM signal intensity was higher than that of the product in the no-wait mode.

[0123] Samples 5# / 6# / 7# were IgG-negative / IgM-negative samples, and were negative using both detection modes.

[0124] The results in Table 3 show that the waiting mode plays a certain role in improving the sensitivity of IgM detection and has no effect 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, and the marking pad includes a marker that combines with an antigen.

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 buffer pad 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 paper 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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