Method for detecting antibody

Abstract

The present invention provides a method for detecting an antibody. The method comprises: providing a test strip used for detecting an antibody, wherein the test strip comprises a backing card, and a conjugate pad, a sample pad, a detection pad, and an absorbent pad which are attached to the backing card and sequentially arranged in an overlapping manner from the upstream portion to the downstream portion, the detection pad comprises a first detection line and a second detection line, the first detection line is located downstream of the second detection line, and the conjugate pad comprises a label conjugated to an antigen; adding a sample under detection onto the sample pad of the test strip; and adding a buffer solution onto the test strip to release the label on the conjugate pad and allow the label to flow toward the detection pad, wherein the label on the conjugate pad arrives at the detection pad later than the added sample arrives. The test strip of the present invention features a simple structure, strong anti-interference capability, and high detection sensitivity.

Description

A method for detecting antibodies Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to a method for detecting IgG / IgM antibodies. Background Technology

[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, forming immunoglobulins, including IgM and IgG.

[0003] Immunoglobulin M (IgM) is the first antibody to appear in the human body after infection. It plays a role in clearing viruses and fighting infection in the early stages of infection, and has functions such as lysing bacteria, activating complement, immunomodulation, and agglutination.

[0004] Abnormal IgM levels often indicate the possibility of infection. In the early stages of infection or during the active phase of certain autoimmune diseases, such as rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus, serum IgM levels may be elevated. Abnormal IgM levels are also related to daily habits; unhealthy habits such as chronic alcoholism or overeating can lead to abnormal IgM levels. IgM is also used in prenatal testing to determine if the fetus has an intrauterine infection. In certain diseases, such as chronic liver diseases like hepatitis A, B, and C, elevated IgM levels may indicate that the virus is in an active phase.

[0005] IgM can serve as an early diagnostic indicator for infectious diseases, and a positive result usually indicates that the body is in the acute infection phase. IgM testing helps in the diagnosis of various diseases, including autoimmune diseases, digestive system diseases, hematological diseases, respiratory system diseases, infectious diseases, and urinary system diseases.

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

[0007] Dengue fever is an acute vector-borne infectious disease caused by the dengue virus, primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes. Detection methods for dengue fever mainly include antigen testing, viral nucleic acid testing, and antibody testing. These methods help doctors diagnose whether a patient is infected with the dengue virus.

[0008] Antibody testing is performed 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. IgM antibodies can be detected 3-5 days after the onset of illness in patients with primary infection, and IgG antibodies can be detected one week after the onset of illness. The detection of these antibodies helps in the diagnosis of dengue fever and can be used as a reference in cases of reinfection.

[0009] The test strip shown in Figure 1 is a standard product for detecting dengue antibodies using lateral flow (LF) chromatography. The test strip in Figure 1 includes a buffer pad 1, a labeling pad 2, a test pad 4 (nitrocellulose membrane), and an absorbent pad 8, all sequentially arranged on a base card 5. Blood samples are added directly to the labeling pad, reacting with the dengue recombinant antigen-colloidal gold complex on the labeling pad to form a dengue recombinant antigen-colloidal gold-Dengue antibody complex. Buffer solution is added to the buffer pad, allowing the dengue recombinant antigen-colloidal gold-Dengue antibody complex to move along the test pad and bind to anti-human IgM antibody and / or 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 and Dengue IgM antibodies on a single test strip, the sensitivity of IgM will be lower due to interference from IgG, since the amount of IgM in blood is relatively small. When using the test strip structure and methods of conventional products for detection, the sensitivity of IgM will be lower. Summary of the Invention

[0011] To overcome the deficiencies of existing technologies, the present invention provides a method for detecting antibodies, the method comprising: providing a test strip for detecting antibodies, the test strip comprising a labeling pad, a sample pad, a test pad, and an absorbent pad sequentially overlapped from upstream to downstream on a base card; the test pad comprising a first test line and a second test line, the first test line being coated with an anti-IgM antibody, the second test line being coated with an anti-IgG antibody, the first test line being located downstream of the second test line; the labeling pad comprising a marker that binds to the antigen; adding a sample to be tested to the sample pad of the test strip; adding a buffer solution to the test strip, causing the marker on the labeling pad to be released and flow into the test pad; the time at which the marker on the labeling pad reaches the test pad is later than the time at which the added sample reaches the test pad.

[0012] Furthermore, it also includes a buffer pad for receiving buffer solution, the buffer pad being located upstream of the label pad.

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

[0014] Furthermore, the cushioning pad is made of highly hydrophobic glass fiber or polyester film material.

[0015] Furthermore, the release rate of the marker on the label pad is slower than the release rate of the sample on the sample pad; that is, the time it takes for the marker on the label pad to reach the detection pad is later than the time it takes for the added sample to reach the detection pad. Even further, after adding the sample to be tested to the sample pad of the test strip and waiting for a preset time, buffer solution is added to the test strip.

[0016] Further, add buffer solution to the buffer pad of the test strip.

[0017] Furthermore, the preset time is 1 to 2 minutes.

[0018] Furthermore, the IgG / IgM antibody is a dengue IgG / IgM antibody.

[0019] Furthermore, the labeling pad includes a labeling complex in which dengue antigen and the label are combined.

[0020] More specifically, the dengue antigens are: type I, type II, type III, and type IV recombinant dengue antigens. The labeling agents are selected from colloidal gold, latex, etc. More specifically, the labeling pad is coated with individually labeled type I, type II, type III, and type IV dengue recombinant antigen-colloidal gold complexes.

[0021] Furthermore, the test strip's detection pad also includes a control line coated with streptavidin; the label pad is coated with a biotin-labeled complex. The label is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a biotin-colloidal gold complex.

[0022] On the other hand, the present invention also provides a test strip for detecting antibodies, comprising a labeling pad, a sample pad, a test pad, and an absorbent pad that are adhered to a base card and sequentially overlapped from upstream to downstream; the test pad includes a first test line and a second test line, the first test line being coated with anti-IgM antibody, the second test line being coated with anti-IgG antibody, the first test line being located downstream of the second test line, and the labeling pad including a marker that binds to the antigen.

[0023] Furthermore, it also includes a buffer pad for receiving buffer solution, the buffer pad being located upstream of the label pad.

[0024] Furthermore, the buffer pad is made of a hydrophobic material.

[0025] Furthermore, the cushioning pad is made of highly hydrophobic glass fiber or polyester film material.

[0026] Furthermore, the release rate of the marker on the labeling pad is slower than the release rate of the sample on the sample pad; that is, the time it takes for the marker on the labeling pad to reach the detection pad is later than the time it takes for the added sample to reach the detection pad.

[0027] Furthermore, the IgG / IgM antibody is a dengue IgG / IgM antibody.

[0028] Furthermore, the labeling pad includes a labeling complex in which dengue antigen and the label are combined.

[0029] More specifically, the dengue antigens are: type I, type II, type III, and type IV recombinant dengue antigens. The labeling agents are selected from colloidal gold, latex, etc. More specifically, the labeling pad is coated with individually labeled type I, type II, type III, and type IV dengue recombinant antigen-colloidal gold complexes.

[0030] Furthermore, the test strip's detection pad also includes a control line coated with streptavidin; the label pad is coated with a biotin-labeled complex. The label is selected from colloidal gold, latex, etc. More specifically, the label pad is coated with a biotin-colloidal gold complex.

[0031] In the specific design of the dengue IgG / IgM antibody test strip of this invention, a sample pad is added downstream of the labeling pad. The sample is directly applied to the sample pad and flows along the test pad. At this point, the sample does not come into contact with the dengue recombinant antigen-colloidal gold complex. The IgG and IgM in the sample bind sequentially to the anti-human IgG antibody and anti-human IgM antibody pre-coated on the test pad and are thus immobilized on the test pad. Then, with the addition of buffer solution, the dengue recombinant antigen-colloidal gold complex immobilized on the labeling pad dissolves and begins to flow on the test pad. The dengue recombinant antigen-colloidal gold complex binds sequentially to the Dengue-IgG antibody and Dengue-IgM antibody immobilized on the test pad, causing color development at the corresponding positions on the test pad, thus achieving the purpose of detection.

[0032] Furthermore, the present invention also provides a detection kit for detecting IgG / IgM antibodies, the kit comprising the test strip and buffer solution described in the present invention.

[0033] Furthermore, the buffer solution comprises 0.01M PBS buffer, wherein the PBS buffer contains 0.2% to 1% Tween 20 and 0.1% NaN3.

[0034] The specific steps of the dengue IgG / IgM antibody detection kit of the present invention include: (1) adding the sample directly onto the sample pad; (2) the sample flows from the sample pad to the test pad, the sample is chromatographically (flows) along the test pad, and the IgG and IgM in the sample are successively bound to the anti-human IgG antibody and anti-human IgM antibody pre-coated on the test pad and thus fixed on the test pad; (3) adding buffer to the buffer pad and flowing to the label pad, the dengue recombinant antigen-colloidal gold complex fixed on the label pad is dissolved; (4) the dengue recombinant antigen-colloidal gold complex flows with the buffer to the test pad and continues to flow, on the test pad, the dengue recombinant antigen-colloidal gold complex successively binds to the Dengue-IgG antibody and Dengue-IgM antibody that have been fixed on the test pad by the anti-human IgG antibody and anti-human IgM antibody, so that the corresponding position of the test pad is colored. Beneficial effects

[0035] Conventional IgG / IgM antibody detection products first bind the sample to an antigen-labeled marker on a labeling pad. The bound material then flows into the test pad for chromatography and reacts with substances on the test lines. For example, dengue antibodies in the sample bind to a dengue recombinant antigen-colloidal gold complex on the labeling pad before entering the nitrocellulose membrane of the test pad for chromatography. There, they react with anti-IgM antibodies on the first test line and anti-IgG antibodies on the second test line, resulting in the detection result. This process can lead to reduced sensitivity for IgM antibodies.

[0036] The test strip, corresponding test kit, and detection method for detecting IgG / IgM antibodies of the present invention employ a method that allows the sample sufficient time to flow and chromatographically independently on the test strip before the label is released from the label pad, with the sample reaching the detection line before the label. For example, a sample pad is added downstream of the label pad on the test strip. The sample is placed on the sample pad and, before binding with the label, enters the nitrocellulose membrane of the detection pad for chromatography, where substances on the detection line of the detection pad undergo a binding reaction. Subsequently, the label on the label pad flows into the detection pad under the influence of the buffer solution for chromatography, reacting with the analytes in the sample, such as IgG and IgM, to display the detection result.

[0037] The test strip structure and detection method of this invention effectively reduce the interference of interfering substances in the sample before the sample comes into contact with the label. For example, the interference of a high concentration of IgG in the sample on a relatively low concentration of IgM is effectively reduced. This invention independently sets up the sample pad and the label pad, allowing for more selective sample pad processing without excessive consideration of the stability of related substances in the label, such as colloidal gold antigens and other active substances. Furthermore, this invention can delay the entry of the buffer into the label pad by selecting the buffer pad material and adjusting the buffer formulation, thereby delaying the release of the label on the label pad. This makes it possible for the sample to undergo independent chromatography on the test pad before the label. In contrast, the use of buffer pads and buffer solutions in conventional products results in excessively rapid label release, leading to insufficient independent chromatography time for the sample on the test pad and low detection sensitivity.

[0038] The test strip of this invention has a simple structure, is easy to use, and has strong anti-interference ability. In the combined detection of IgG and IgM, it can reduce the detection interference of IgG on IgM and has high detection sensitivity, thereby improving the accuracy of early detection of infection in patients. Early diagnosis and early treatment can reduce the spread of infectious diseases. Attached Figure Description

[0039] Figure 1 is a structural diagram of a dengue IgG / IgM antibody test strip in the prior art.

[0040] Figure 2 is a structural diagram of a dengue IgG / IgM antibody test strip according to the present invention.

[0041] Figure 3 is an exploded view of the test strip in Figure 2.

[0042] Figure 4 is a structural diagram of another dengue IgG / IgM antibody test strip of the present invention.

[0043] Figure 5 is an exploded view of the dengue IgG / IgM antibody detection plate of the present invention. Detailed Implementation

[0044] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0045] As shown in Figures 2 and 3, an IgG / IgM antibody test strip 10 of the present invention includes a buffer pad 1, a labeling pad 2, a sample pad 3, a test pad 4, and an absorbent pad 8, which are sequentially overlapped from upstream to downstream. The buffer pad 1, labeling pad 2, sample pad 3, test pad 4, and absorbent pad 8 are all adhered to a base card 5. The test pad 4 includes a first test line 41 and a second test line 42. The first test line 41 is coated with anti-IgM antibody, and the second test line 42 is coated with anti-IgG antibody. The first test line 41 is located downstream of the second test line 42. The test pad may also include a control line 43.

[0046] As shown in Figure 4, another IgG / IgM antibody test strip 10 of the present invention includes a labeling pad 2, a sample pad 3, a test pad 4, and an absorbent pad 8, which are sequentially overlapped from upstream to downstream. The labeling pad 2, sample pad 3, test pad 4, and absorbent pad 8 are all adhered to a base card 5. The test pad 4 includes a first test line 41 and a second test line 42. The first test line 41 is coated with anti-IgM antibody, and the second test line 42 is coated with anti-IgG antibody. The first test line 41 is located downstream of the second test line 42. The test pad may also include a control line 43.

[0047] 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 hydrophobic reagent.

[0048] The material of marking pad 2 is selected from glass fiber, polyester film, etc. The material of sample pad 3 is selected from glass fiber, etc. The material of test pad 4 is selected from nitrocellulose membrane, etc. The material of absorbent pad 8 is selected from absorbent paper, filter paper, etc.

[0049] As shown in Figure 5, the detection plate 20 includes a base plate 21 and a top cover 22. The test strip shown in Figure 2 or Figure 4 is installed between the base plate and the top cover. The top cover 22 has a buffer addition hole 23 corresponding to the buffer pad, a sample addition hole 24 corresponding to the sample pad 3 of the test strip, and a test result observation hole 25 corresponding to the test pad 4 of the test strip.

[0050] The test strip described in this invention can be used to detect IgG, IgM, etc., in samples, such as a test strip for detecting dengue IgG, IgM, etc., in blood samples. A test kit for detecting dengue IgG / IgM antibodies includes the dengue IgG / IgM antibody test strip 10 described in this invention and a buffer solution. The dengue IgG / IgM antibody test kit may also include the dengue IgG / IgM antibody detection plate 20 described in this invention and a buffer solution.

[0051] Example 1: Preparation and Detection Steps of Dengue Fever IgG / IgM Antibody Test Strip

[0052] As shown in Figure 2, the dengue IgG / IgM antibody test strip 10 includes a buffer pad 1, a labeling pad 2, a sample pad 3, a detection pad 4, and an absorbent pad 8, which are respectively adhered to the base card 5 and overlapped sequentially from upstream to downstream. In this embodiment, the width of the test strip is 3.5 mm, and the overlap area between each pad is 1-2 mm.

[0053] The label pad 2 is coated with individually labeled dengue recombinant antigen-colloidal gold complexes for types I, II, III, and IV, and biotin-colloidal gold complexes.

[0054] Method for preparing the marking pad:

[0055] (1) Add dengue virus recombinant antigen rapidly 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 μg / mL, and stir rapidly with a stirrer. After reacting for 15-30 minutes, add blocking solution (1% BSA, 0.1% NaN3) rapidly at a ratio of 10-15 μL / mL, react for 10-30 minutes, centrifuge at 8000-10000 rpm for 25-30 minutes, discard the supernatant to obtain the precipitate, add reconstitution solution (25 mM Tris buffer, 1% BSA, 0.1% NaN3) to the precipitate at a ratio of 100 μL / mL, and sonicate to reconstitute. 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 in sequence.

[0056] (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 μg / mL, and stirred rapidly 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 μL / mL, and reacted for 10-30 minutes. The mixture was then centrifuged at 8000-10000 rpm for 25-30 minutes. The supernatant was discarded to obtain the precipitate. A preservation solution (25 mM Tris buffer, 1% BSA, 0.1% NaN3) was added to the precipitate at a ratio of 100 μL / mL, and the mixture was reconstituted by sonication to prepare the biotin-colloidal gold complex.

[0057] (3) Based on their potency, the above-mentioned dengue recombinant antigen-colloidal gold complexes (Type I, II, III, IV, and biotin-colloidal gold) were mixed and diluted with a diluent, then evenly spread onto the labeling pad 2, and dried at 37°C for 12-24 hours. The labeling pad material was selected from glass fiber. The diluent was 25 mM Tris buffer, which contained 1% BSA, 20% sucrose, and 0.1% NaN3.

[0058] Method for preparing the test pad:

[0059] The test pad 4 has a first test line (T1-line) 41, a second test line (T2-line) 42, and a 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 control line 43 is coated with streptavidin.

[0060] (1) The coating method for the first detection line 41 is as follows: the anti-human IgM antibody is diluted with coating buffer (0.01M PBS buffer, 10% sucrose, 0.1% NaN3) to 0.1-1 mg / mL and then coated, and then dried at 37℃ for 12-24 hours.

[0061] (2) The coating method for the second detection line 42 is as follows: the anti-human IgG antibody is diluted with coating buffer (0.01M PBS buffer, 10% sucrose, 0.1% NaN3) to 0.1-1 mg / mL and then coated, and then dried at 37℃ for 12-24 hours.

[0062] (3) The coating method for quality control line 43 is as follows: dilute streptavidin with coating buffer (0.01M PBS buffer, 10% sucrose, 0.1% NaN3) to 1-5 mg / mL and then coat it, and then dry it at 37℃ for 12-24 hours.

[0063] The material of the test pad 4 is selected from nitrocellulose membrane.

[0064] Sample pad 3 preparation method:

[0065] Prepare a sample pad treatment buffer, which is a 25mM Tris buffer containing 0.05% Tween 20, 1% BSA, 0.1 mg / mL anti-RBC, and 0.1% NaN3. Spread the buffer evenly on the glass fiber and then dry it at 37°C for 12-24 hours.

[0066] The material of sample pad 3 is selected from glass fiber.

[0067] Buffer pad 1 and buffer solution:

[0068] The buffer pad 1 is made of a highly hydrophobic glass fiber or polyester film material. In this example, a polyester film (Ahlstrom 6613) is used. The buffer solution added to the buffer pad is 0.01M PBS buffer, which contains 0.2% Tween 20 and 0.1% NaN3.

[0069] The detection process for dengue IgG / IgM antibodies using the test strip or test plate described in this invention is as follows:

[0070] (1) Before testing, remove the test strip or test plate and bring the sample and buffer solution to room temperature.

[0071] (2) Place the test strip or test plate on a clean, flat table.

[0072] (3) Add 10uL of serum / plasma or whole blood sample to the sample pad or sample well of the test strip.

[0073] (4) Add 2 drops (about 50uL) of buffer solution to the buffer pad of the test strip or the buffer well of the test plate.

[0074] (5) Wait 15 minutes to read the test results. Results will be invalid after 20 minutes.

[0075] Interpretation of test results:

[0076] (1) Negative: Only the control line C area shows a red band, and no bands appear in the first test line T1 and the second test line T2 areas.

[0077] (2) Dengue fever IgM antibody positive: A red band appears in the control line C area, a red band appears in the first test line T1 area, and no red band appears in the second test line T2 area.

[0078] (3) Dengue IgG antibody positive: A red band appears in the control line C area, no red band appears in the first test line T1 area, and a red band appears in the second test line T2 area.

[0079] (4) Dengue fever IgM / IgG antibody positive: Red bands appear in the control line C area, the first test line T1 area, and the second test line T2 area.

[0080] (5) Invalid: No red band appeared in area C of the quality control line. Insufficient sample quantity or incorrect operation procedure may be the cause of invalid results.

[0081] Example 2 Comparative Experiment

[0082] Comparative Example 1

[0083] As shown in Figure 1, the standard dengue IgG / IgM antibody test strip includes a buffer pad 1, a labeling pad 2, a detection pad 4, and an absorbent pad 8, which are stacked sequentially from upstream to downstream. The buffer pad 1, labeling pad 2, detection pad 4, and absorbent pad 8 are all adhered to the base card 5.

[0084] The dengue IgG / IgM antibody test strip of Comparative Example 1 is basically the same as the test strip described in Example 1 in terms of structure, preparation method and detection process. The only difference is that Comparative Example 1 does not include a sample pad. The sample is directly added to the label pad. The material of the buffer pad and the composition of the buffer are different.

[0085] The buffer pad is made of glass fiber or polyester film, which is less hydrophobic than the glass fiber or polyester film material of the buffer pad in Example 1. In this example, glass fiber (Ahlstrom 8964) is selected.

[0086] Buffer composition: 0.01M PBS buffer containing 1.0% Tween 20 and 0.1% NaN3.

[0087] In Comparative Example 1, during testing, the sample was directly applied to the labeling pad, and the sample passed through the labeling pad to the test pad. Excess sample and reagent were absorbed by the absorption pad. In the detection of samples where both dengue IgG and IgM were positive, especially when detecting samples with strong IgG positivity and weak IgM positivity, both dengue IgG and IgM in the sample simultaneously came into contact with and competed with the dengue recombinant antigen-colloidal gold complex on the labeling pad. Because the dengue IgM content in blood samples was lower, they were at a competitive disadvantage, resulting in a lower formation of the dengue recombinant antigen-colloidal gold complex-dengue IgM antibody complex, thus reducing sensitivity.

[0088] Comparative Example 2

[0089] The dengue IgG / IgM antibody test strip of Comparative Example 2 is basically the same as the test strip described in Example 1 in terms of structure, preparation method and detection process, except that the material of the buffer pad of Comparative Example 2 is different.

[0090] The buffer pad is made of glass fiber or polyester film, which is less hydrophobic than the glass fiber or polyester film material of the buffer pad in Example 1. In this example, glass fiber (Ahlstrom 8964) is selected.

[0091] Comparative Example 3

[0092] 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 of Comparative Example 3 is different.

[0093] Buffer composition: 0.01M PBS buffer containing 1.0% Tween 20 and 0.1% NaN3.

[0094] Test Example 1 of the Invention

[0095] The dengue IgG / IgM antibody test strip in Test Example 1 was prepared using the method described in Example 1.

[0096] As shown in Figure 2, in the dengue IgG / IgM antibody test strip of the present invention, the labeling pad 2 is located upstream of the sample pad 3. After the sample is added to the sample pad, it flows downstream along the test pad for chromatography (i.e., the sample flows from the test pad towards the absorbent pad). At this point, the sample does not come into contact with the dengue recombinant antigen-colloidal gold complex on the labeling pad 2. As the sample flows on the test pad, the IgG and IgM in the sample bind sequentially to the anti-human IgG antibody pre-coated at the second test line 42 (T2 line) and the anti-human IgM antibody pre-coated at the first test line 41 (T1 line) and are thus immobilized on the test pad. Since the IgM content in blood samples is much lower than the IgG content... On the test pad, an anti-human IgM antibody coated on the test pad is placed downstream of an anti-human IgG antibody. When the sample is chromatographically analyzed on the test pad, the sample first passes through the second detection line 42 of the anti-human IgG antibody, causing IgG to be fixed on the second detection line 42. As the content of IgG in the sample decreases, the relative content of IgM in the continuing flow of the sample increases, thereby improving the binding efficiency of IgM in the sample to the anti-human IgM antibody at the first detection line 41 of the test pad.

[0097] The detection procedure for Test Example 1 is the same as in Example 1. Buffer solution is added to buffer pad 1. With the addition and release of buffer solution, the dengue recombinant antigen-colloidal gold complex on label pad 2 dissolves and begins to chromatographically precipitate along the test pad (e.g., a nitrocellulose membrane). Due to the hydrophobicity of the buffer pad and buffer solution, the dengue recombinant antigen-colloidal gold complex arrives at the test pad later than the sample. Subsequently, the dengue recombinant antigen-colloidal gold complex binds sequentially on the test pad to dengue IgG already bound to the second test line 42 (if dengue IgG is present in the sample) and dengue IgM bound to the first test line 41 (if dengue IgG is present in the sample), thereby displaying the test results.

[0098] In Test Example 1, the dengue recombinant antigen-colloidal gold complex located on the label pad 2 passed through the second detection line 42 and the first detection line 41 sequentially with the buffer solution. Since most of the IgG in the sample bound to the second detection line 42, compared to conventional products, the interference of IgG in the sample on the binding of IgM to the dengue recombinant antigen-colloidal gold complex was reduced at the first detection line 41 in Test Example 1 of this invention. Furthermore, the absolute excess of the dengue recombinant antigen-colloidal gold complex made the reduction in dengue recombinant antigen-colloidal gold complex due to passing through the second detection line 42 negligible.

[0099] Test Example 1 improves the sensitivity of IgM detection without affecting the sensitivity and specificity of IgG detection. Detailed comparison results between Test Example 1 and the comparative examples are shown in Table 1.

[0100] Table 1: Comparison of detection results between Test Example 1 and the comparative example of the present invention

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

[0102] The dengue IgG / IgM antibody test strip is a qualitative detection product. The test results are either positive or negative. If the analyte in the sample is positive, the higher the numerical value and the stronger the color of the test strip, the higher the sensitivity and the better the performance of the test strip. If the analyte in the sample is negative, the lower the numerical value and the weaker the color of the test strip, the better the specificity and the better the performance of the test strip.

[0103] Sample #1 was an IgG / IgM positive sample, and the test results were as follows:

[0104] Using Comparative Example 1, IgG was positive at intensity G10, and IgM was positive at intensity G3; using Comparative Example 2, IgG was positive at intensity G10, and IgM was positive at intensity G3.5; using Comparative Example 3, IgG was positive at intensity G10, and IgM was positive at intensity G3.5; using the test of this invention, IgG was still positive at intensity G10, and IgM was positive at intensity G5, with the IgM signal intensity higher than that 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.

[0105] Sample #2 was an IgG / IgM positive sample, and the test results were as follows:

[0106] Using Comparative Example 1, IgG was positive at intensity G10 and IgM was negative at intensity G2, indicating a false negative for IgM. Using Comparative Example 2, IgG was positive at intensity G10 and IgM was positive at intensity G3. Using Comparative Example 3, IgG was positive at intensity G10 and IgM was positive at intensity G3. Using the test of this invention, IgG was still positive at intensity G10 and IgM was positive at intensity G6, with no false negative for IgM, and the signal intensity was higher than that of Comparative Example 2 and Comparative Example 3.

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

[0108] Using Comparative Examples 1, 2, and 3, and the results of this invention, IgG was positive for intensity G5 in all cases, and IgM was negative in all cases. The test results were consistent.

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

[0110] Using Comparative Example 1, IgG was negative and IgM was positive at intensity G4; using Comparative Example 2, IgG was negative and IgM was positive at intensity G4.5; using Comparative Example 3, IgG was negative and IgM was positive at intensity G4.5; using the test results of this invention, IgG was negative and IgM was positive at intensity G5, with the IgM signal intensity higher than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3. Furthermore, the IgM signal intensity of Comparative Example 2 and Comparative Example 3 was higher than that of Comparative Example 1.

[0111] Samples #5, #6, and #7 were IgG negative and IgM negative, and the test results were as follows:

[0112] Using Comparative Example 1, IgG was negative, while IgM intensities were positive for G3 / G3 / G4, indicating a false positive for all IgM samples. Using Comparative Examples 2, 3, and the present invention, both IgG and IgM were negative, and no false positives for IgM were observed.

[0113] The results in Table 1 show that the present invention improves the sensitivity and specificity of IgM detection without affecting IgG detection.

[0114] Example 3

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

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

[0117] (1) Before testing, remove the test strip or test plate and bring the sample and buffer solution to room temperature.

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

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

[0120] (4) Add buffer solution to the test strip by waiting or not waiting, namely: wait 1-2 minutes and add 2 drops (about 50uL) of buffer solution to the buffer pad of the test strip or the buffer well of the test plate; or not wait and directly add 2 drops (about 50uL) of buffer solution 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 results after 15 minutes. Results are invalid after 20 minutes.

[0122] Table 2: Comparison of waiting time and detection results

[0123] Note: G0-G10 represent the intensity of the line color. The higher the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0124] Sample #1 was an IgG / IgM positive sample, and the test results were as follows:

[0125] When tested using the product in no-wait mode, IgG was positive at intensity G10 and IgM was positive at intensity G3. When tested using wait mode, IgG was still positive at intensity G10 and IgM was positive at intensity G4.5, with the IgM signal intensity being higher than that of the product in no-wait mode.

[0126] Sample #2 was an IgG / IgM positive sample, and the test results were as follows:

[0127] When testing with the product in no-wait mode, IgG was positive at intensity G10 and IgM was negative at intensity G2, indicating a missed detection of IgM. When testing with wait mode, IgG was still positive at intensity G10 and IgM was positive at intensity G6.5, indicating no missed detection of IgM and a strong signal.

[0128] Sample #3 was an IgG positive / IgM negative sample, and the test results were as follows:

[0129] When testing with the product in no-wait mode, IgG is positive for intensity G5 and IgM is negative; when testing with wait mode, IgG is positive for intensity G5 and IgM is negative.

[0130] Sample #4 was an IgG negative / IgM positive sample, and the test results were as follows:

[0131] When tested using the product in no-wait mode, IgG was negative and IgM was positive at intensity G4; when tested using wait mode, IgG was negative and IgM was positive at intensity G5, with the IgM signal intensity being higher than that of the product in no-wait mode.

[0132] Samples #5, #6, and #7 were IgG negative and IgM negative, respectively. Both tests were negative.

[0133] The results in Table 2 show that the waiting mode improved the sensitivity of IgM detection and had no effect on IgG detection.

[0134] Example 4

[0135] The dengue IgG / IgM antibody test strip and its preparation method in this embodiment are basically the same as those in Example 1, except that the test strip does not include a buffer pad.

[0136] The dengue IgG / IgM antibody detection process is as follows:

[0137] (1) Before testing, remove the test strip or test plate and bring the sample and buffer solution to room temperature.

[0138] (2) Place the test strip or test plate on a clean, flat table.

[0139] (3) Add 10 μL of serum / plasma or whole blood sample to the sample pad or sample well of the test strip.

[0140] (4) Add buffer to the test strip by waiting or not waiting, namely: wait 1-2 minutes and then add 2 drops (about 50uL) of buffer to the label pad; or not waiting and directly add 2 drops (about 50uL) of buffer to the label pad.

[0141] (5) Read the test results after 15 minutes. Results are invalid after 20 minutes.

[0142] Table 3: Comparison of waiting time and detection results

[0143] Note: G0-G10 represent the intensity of the line color. The higher the value, the stronger the color. Among them, G0-G2: negative; G3-G10: positive.

[0144] Sample #1 was an IgG / IgM positive sample, and the test results were as follows:

[0145] When tested using the product in no-wait mode, IgG was positive at intensity G10 and IgM was positive at intensity G3; when tested using wait mode, IgG was still positive at intensity G10 and IgM was positive at intensity G4, with the IgM signal intensity being higher than that of the product in no-wait mode.

[0146] Sample #2 was an IgG / IgM positive sample, and the test results were as follows:

[0147] When testing with the product in non-waiting mode, IgG was positive at intensity G10 and IgM was negative at intensity G2, indicating a missed detection of IgM. When testing with waiting mode, IgG was still positive at intensity G10 and IgM was positive at intensity G5, indicating no missed detection of IgM and a strong signal.

[0148] Sample #3 was an IgG positive / IgM negative sample, and the test results were as follows:

[0149] When tested in non-waiting mode, IgG was positive at intensity G5, IgM was negative at intensity G2, and IgM showed faint streaks; when tested in waiting mode, IgG was positive at intensity G5, IgM was negative at intensity G2, and IgM showed faint streaks.

[0150] Sample #4 was an IgG negative / IgM positive sample, and the test results were as follows:

[0151] When tested using the product in no-wait mode, IgG was negative and IgM was positive at intensity G3; when tested using wait mode, IgG was negative and IgM was positive at intensity G4, with the IgM signal intensity being higher than that of the product in no-wait mode.

[0152] Samples #5, #6, and #7 were IgG negative and IgM negative, respectively. Both tests were negative.

[0153] The results in Table 3 show that the waiting mode played a role in improving the sensitivity of IgM detection, but had no effect on IgG detection.

Claims

1. A method of detecting an antibody, characterized by, The method comprises: providing a test paper for detecting antibodies, the test paper comprising a mark pad, a sample pad, a detection pad and a water absorption pad which are sequentially arranged in a lap joint mode from upstream to downstream and are adhered to a base card; the detection pad comprises 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 mark pad comprises a label combined with an antigen; a sample to be detected is added to the sample pad of the test paper; a buffer solution is added to the test paper, so that the label on the mark pad is released and flows to the detection pad; and the time for the label on the mark pad to reach the detection pad is later than the time for the added sample to reach the detection pad.

2. The method of detecting an antibody according to claim 1, characterized by, The method further comprises a buffer solution pad for receiving the buffer solution, the buffer solution pad being located upstream of the mark pad.

3. The method of detecting an antibody according to claim 2, characterized in that, The material of the buffer solution pad is hydrophobic.

4. The method of detecting an antibody according to claim 3, characterized by, The material of the buffer solution pad is hydrophobic.

5. The method of detecting an antibody according to any one of claims 1 to 4, characterized in that, The material of the buffer solution pad is hydrophobic.

6. The method of detecting an antibody according to claim 5, wherein, The sample to be detected is added to the sample pad of the test paper and waits for a preset time, and then the buffer solution is added to the test paper.

7. The method of detecting an antibody according to claim 5, wherein The sample to be detected is added to the sample pad of the test paper and waits for a preset time, and then the buffer solution is added to the test paper.

8. The method of detecting an antibody according to claim 1, wherein The preset time is 1 to 2 minutes.

9. The method of detecting an antibody according to claim 8, characterized in that, The IgG / IgM antibody is a dengue IgG / IgM antibody.

10. The method of detecting an antibody according to claim 1, wherein The mark pad is coated with individually labeled type I, type II, type III and type IV dengue recombinant antigen-gold colloid complexes. The detection pad of the test paper further comprises a quality control line coated with streptavidin; and the mark pad is coated with a biotin-gold colloid complex. The detection pad of the test paper further comprises a quality control line coated with streptavidin; and the mark pad is coated with a biotin-gold colloid complex.

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