Hemoglobin detection test strip and application thereof

Abstract

The hemoglobin detection test strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially overlapped on the bottom plate; a colloidal gold labeled hemoglobin antigen and / or a colloidal gold labeled hemoglobin antibody and a colloidal gold labeled first antibody are / is sprayed on the combination pad; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a hemoglobin antigen and / or a hemoglobin antibody, and the quality control line is coated with a second antibody combined with the first antibody; the hemoglobin antigen comprises at least one of hemoglobin A, hemoglobin S and hemoglobin C, and correspondingly, the hemoglobin antibody comprises at least one of a hemoglobin A antibody, a hemoglobin S antibody and a hemoglobin C antibody. The hemoglobin mutation type can be accurately detected by utilizing the test strip, and the test strip has the advantages of high accuracy, high stability, good repeatability and the like, and has important significance on early screening and diagnosis of sickle-type cell anemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a hemoglobin test strip and its application. Background Art

[0002] Sickle cell anemia, also known as sickle cell anemia (SCD), is an autosomal recessive hereditary hemoglobinopathy. The red blood cells of patients present a sickle shape under the microscope, and their oxygen-carrying function is only half of that of normal red blood cells. Under hypoxia, these abnormal red blood cells will rupture, causing severe anemia and even death of patients. Sickle cell anemia mainly occurs in Africans and African Americans, and cases have also been reported in southern my country in recent years. Molecular biological studies have found that the cause of the disease is a mutation in the gene sequence encoding the sixth amino acid of the hemoglobin β subunit (HBB gene encoding), which can be specifically divided into HbS mutation (A20T), HbC mutation (G19A), HbD mutation (G364C), HbO mutation (G364A), etc. Among them, HbS mutation (A20T) and HbC mutation (G19A) are relatively common mutation types.

[0003] Screening and detecting mutant forms of sickle cell anemia is the best way to prevent and control birth defects and treat the disease as early as possible. However, there are few reports on the use of immunochromatography to diagnose sickle cell anemia, and this method still needs to be studied. Summary of the invention

[0004] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention provides a hemoglobin detection test strip and a preparation method thereof, a hemoglobin detection kit and a use thereof, and the test strip and the kit of the present invention can accurately detect whether sickle cell anemia and the specific mutation type, and have the advantages of high accuracy, high stability and good repeatability, etc., which is of great significance for the early screening and diagnosis of sickle cell anemia.

[0005] It should be noted that the present invention is completed based on the following findings of the inventors:

[0006] Since hemoglobin is a macromolecule, the sandwich ELISA method is usually adopted. Based on this, the inventors of the present application initially attempted to coat colloidal gold-labeled hemoglobin A (also referred to as "HbA" in this article), hemoglobin S (also referred to as "HbS" in this article), and hemoglobin C (also referred to as "HbC" in this article) protein antibodies on the conjugate pad of the test strip, and coat HbA protein capture antibody, HbS protein capture antibody, and HbC protein capture antibody on the nitrocellulose membrane. However, false positives or false negatives are likely to occur in the test results, affecting the test accuracy. Further, the inventors found through in-depth research that the competitive method can effectively avoid false positives and false negatives, and the test results have strong accuracy, high stability, and good repeatability.

[0007] The first aspect of the present invention provides a hemoglobin test strip. According to an embodiment of the present invention, the test strip includes a base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially overlapped on the base plate;

[0008] The conjugate pad is sprayed with colloidal gold-labeled hemoglobin antigen and / or colloidal gold-labeled hemoglobin antibody and colloidal gold-labeled first antibody;

[0009] The nitrocellulose membrane is provided with a test line and a control line. The test line is coated with hemoglobin antigen and / or hemoglobin antibody, and the control line is coated with a second antibody that binds to the first antibody;

[0010] The hemoglobin antigen includes at least one of hemoglobin A, hemoglobin S, and hemoglobin C. Correspondingly, the hemoglobin antibody includes at least one of hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody;

[0011] The hemoglobin antigen or hemoglobin antibody on the conjugate pad can specifically bind to the corresponding hemoglobin antigen or hemoglobin antibody on the nitrocellulose membrane to form an antigen-antibody complex.

[0012] The hemoglobin antigen in the present invention is hemoglobin A, hemoglobin S, or hemoglobin C; the hemoglobin antibodies in the present invention are all monoclonal antibodies.

[0013] The hemoglobin test strip provided by the present invention uses the principle of membrane chromatography competition method for detection:

[0014] Detection principle 1: When the sample contains the target antigen, the sample chromatographs forward by capillary action to the test line on the nitrocellulose membrane, and will specifically bind to the antibody on the test line, forming an antigen-antibody complex, resulting in the inability of the antigen labeled with colloidal gold on the conjugate pad to bind to the antibody on the test line, and the test line does not show color; when the sample does not contain the target antigen, the antigen labeled with colloidal gold on the conjugate pad chromatographs forward by capillary action to the test line and specifically binds to the antibody on the test line, and the test line shows color.

[0015] Detection principle 2: When the sample contains the target antigen, the target antigen specifically binds to the antibody labeled with colloidal gold on the conjugate pad, forming a colloidal gold-antibody-antigen complex, resulting in the inability of the antibody labeled with colloidal gold in the complex to bind to the antigen on the test line again, and the test line does not show color; when the sample does not contain the target antigen, the antibody labeled with colloidal gold on the conjugate pad chromatographs forward by capillary action to the test line and specifically binds to the antigen on the test line, and the test line shows color.

[0016] The first antibody labeled with colloidal gold sprayed on the conjugate pad can bind to the second antibody coated on the control line to show color, indicating that the detection reaction occurs normally and the quality of the test strip is normal.

[0017] Thus, the hemoglobin detection test strip according to the embodiment of the present invention can accurately detect the hemoglobin mutation type, thereby determining whether a person has sickle cell anemia. It has the advantages of strong accuracy, high stability and good repeatability, and is of great significance for the early screening and diagnosis of sickle cell anemia.

[0018] According to an embodiment of the present invention, the above test strip may further have the following additional technical features:

[0019] According to an embodiment of the present invention, the conjugate pad is sprayed with hemoglobin A labeled with colloidal gold, hemoglobin S labeled with colloidal gold or hemoglobin C labeled with colloidal gold. Correspondingly, the test line is divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody or a test line coated with hemoglobin C antibody; or the conjugate pad is sprayed with hemoglobin A antibody labeled with colloidal gold, hemoglobin S antibody labeled with colloidal gold or hemoglobin C antibody labeled with colloidal gold. Correspondingly, the test line is divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S or a test line coated with hemoglobin C.

[0020] According to an embodiment of the present invention, colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S, and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad. Correspondingly, the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody, and a test line coated with hemoglobin C antibody; or colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S antibody, and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad. Correspondingly, the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S, and a test line coated with hemoglobin C.

[0021] According to an embodiment of the present invention, when colloidal gold-labeled hemoglobin antigen and colloidal gold-labeled hemoglobin antibody are sprayed on the conjugate pad, and the test line is coated with hemoglobin antigen and hemoglobin antibody, the hemoglobin antigen includes at least two of hemoglobin A, hemoglobin S, and hemoglobin C, and the hemoglobin antibody includes at least two of hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody.

[0022] In some specific embodiments, colloidal gold-labeled hemoglobin A and colloidal gold-labeled hemoglobin S antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A antibody and a test line coated with hemoglobin S.

[0023] In some specific embodiments, colloidal gold-labeled hemoglobin A and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A antibody and a test line coated with hemoglobin C.

[0024] In some specific embodiments, colloidal gold-labeled hemoglobin A antibody and colloidal gold-labeled hemoglobin S are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A and a test line coated with hemoglobin S antibody.

[0025] In some specific embodiments, colloidal gold-labeled hemoglobin A antibody and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A and a test line coated with hemoglobin C antibody.

[0026] In some specific embodiments, colloidal gold-labeled hemoglobin S antibody and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin S and a test line coated with hemoglobin C antibody.

[0027] In some specific embodiments, colloidal gold-labeled hemoglobin S and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin S antibody and a test line coated with hemoglobin C.

[0028] In some specific embodiments, colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S antibody, and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S, and a test line coated with hemoglobin C antibody.

[0029] In some specific embodiments, colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S antibody, and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S, and a test line coated with hemoglobin C.

[0030] In some specific embodiments, colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S, and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody, and a test line coated with hemoglobin C.

[0031] In some specific embodiments, colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S antibody, and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S, and a test line coated with hemoglobin C antibody.

[0032] In some specific embodiments, colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S, and colloidal gold-labeled hemoglobin C antibody are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S antibody, and a test line coated with hemoglobin C.

[0033] In some specific embodiments, colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S, and colloidal gold-labeled hemoglobin C are sprayed on the conjugate pad, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S antibody, and a test line coated with hemoglobin C antibody.

[0034] According to an embodiment of the present invention, the first antibody is a chicken IgY antibody, and the second antibody is a goat anti-chicken antibody. Specifically, the first antibody cannot bind to the hemoglobin antigen.

[0035] According to an embodiment of the present invention, the test strip further includes a cartridge, and a base plate carrying a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad is fixed in the cartridge; a sample application hole and an observation window are provided on the surface of the cartridge, the sample application hole is disposed opposite to the sample pad, and the observation window is disposed opposite to the test line and the control line.

[0036] In a second aspect of the present invention, the present invention provides a method for preparing the hemoglobin test strip described above, including the following steps:

[0037] Step 1: Spraying a colloidal gold-labeled hemoglobin antigen solution and / or a colloidal gold-labeled hemoglobin antibody solution and a colloidal gold-labeled first antibody solution on the conjugate pad, and drying;

[0038] Step 2: Spraying a hemoglobin antigen coating solution and / or a hemoglobin antibody coating solution on the test line of the nitrocellulose membrane, and spraying a second antibody coating solution on the control line, and drying;

[0039] Step 3: Sequentially laminating a sample pad, the conjugate pad obtained in Step 1, the nitrocellulose membrane obtained in Step 2, and an absorbent pad on the base plate to obtain a test strip.

[0040] According to an embodiment of the present invention, the concentrations of the colloidal gold-labeled HbA protein solution, the colloidal gold-labeled HbS protein solution, the colloidal gold-labeled HbC protein solution, and the colloidal gold-labeled chicken IgY antibody solution are 0.005 - 0.15 mg / 100 μL, 0.005 - 0.21 mg / 100 μL, 0.005 - 0.02 mg / 100 μL, and 0.04 - 0.06 mg / 100 μL respectively, and the volume ratios of the mixing of each solution are: (20 - 10) : (10 - 3) : (5 - 1) : (5 - 1); the concentrations of the colloidal gold-labeled HbA antibody solution, the colloidal gold-labeled HbS antibody solution, and the colloidal gold-labeled HbC antibody solution are 0.005 - 0.15 mg / 100 μL, 0.005 - 0.21 mg / 100 μL, and 0.005 - 0.02 mg / 100 μL respectively, and the volume ratios of each solution to the chicken IgY antibody solution are (20 - 10) : (10 - 3) : (5 - 1) : (5 - 1).

[0041] According to an embodiment of the present invention, the concentrations of the HbA antibody coating solution, the HbS antibody coating solution, the HbC antibody coating solution, and the goat anti-chicken antibody coating solution are 0.5 - 1.5 mg / mL, 0.5 - 1.5 mg / mL, 0.5 - 1.5 mg / mL, and 0.5 - 1.5 mg / mL in sequence; the concentrations of the HbA antigen coating solution, the HbS antigen coating solution, and the HbC antigen coating solution are 0.5 - 1.5 mg / mL, 0.5 - 1.5 mg / mL, and 0.5 - 1.5 mg / mL in sequence.

[0042] According to an embodiment of the present invention, each of the coating liquids in the hemoglobin antigen coating liquid and / or the hemoglobin antibody coating liquid and the secondary antibody coating liquid independently includes at least one of the following: bovine serum albumin, PBS buffer, sucrose, and surfactant.

[0043] In a third aspect of the present invention, there is provided a hemoglobin detection kit, which includes the aforementioned hemoglobin detection test strip.

[0044] According to an embodiment of the present invention, the hemoglobin detection kit includes any one of (1), (2), and (3): (1) three of the aforementioned hemoglobin detection test strips, each of which is used to detect one of the aforementioned hemoglobin antigens; (2) two of the aforementioned hemoglobin detection test strips, where one test strip is used to detect two of the aforementioned hemoglobin antigens, and the other test strip is used to detect one hemoglobin antigen; (3) one of the aforementioned hemoglobin detection reagent strips, which is used to detect three of the aforementioned hemoglobin antigens.

[0045] The hemoglobin antigen of the present invention includes hemoglobin A, hemoglobin S, or hemoglobin C.

[0046] The hemoglobin detection kit provided by the present invention may include one hemoglobin detection test strip. For example, hemoglobin A, hemoglobin S, and hemoglobin C are provided on the binding pad of the test strip, and hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody are respectively coated on the detection lines; it may also include two hemoglobin detection test strips. For example, hemoglobin A and hemoglobin C are provided on the binding pad of one test strip, and hemoglobin A antibody and hemoglobin C antibody are respectively coated on the detection lines, and hemoglobin S is provided on the binding pad of the other test strip, and hemoglobin S antibody is coated on the detection line; it may also include three hemoglobin detection test strips. For example, hemoglobin A, hemoglobin S, and hemoglobin C are respectively provided on the binding pads of the three test strips, and hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody are respectively coated on the detection lines. All three kits can detect hemoglobin A, hemoglobin S, and hemoglobin C.

[0047] Using the kit according to the embodiment of the present invention can accurately detect the hemoglobin mutation type, thereby determining whether a person has sickle cell anemia. It has the advantages of strong accuracy, high stability, and good repeatability, and is of great significance for the early screening and diagnosis of sickle cell anemia.

[0048] In a specific embodiment, the kit further includes: a sample treatment solution;

[0049] The sample treatment solution includes: a buffer containing a lysing solution and / or a surfactant.

[0050] In a specific embodiment, the sample treatment solution includes PBS buffer solution, Tween 20, PEG20000, PEG4000, EDTA-2Na, SDS, and Proclin 300.

[0051] In the fourth aspect of the present invention, the present invention provides the use of the test strip provided in the first aspect or the kit provided in the third aspect in the preparation of a product for detecting sickle cell anemia. According to an embodiment of the present invention, the product is used for detecting sickle cell anemia. Thus, the test strip provided in the first aspect of the present invention or the kit provided in the third aspect of the present invention can accurately detect sickle cell anemia.

[0052] According to an embodiment of the present invention, the mutant types of sickle cell anemia include at least one of homozygous mutation of hemoglobin S, homozygous mutation of hemoglobin C, heterozygous mutation of hemoglobin C and hemoglobin S, heterozygous mutation of hemoglobin A and hemoglobin C, and heterozygous mutation of hemoglobin A and hemoglobin S.

[0053] The additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0055] Figure 1 and Figure 2 shows a schematic structural diagram of a test strip according to an embodiment of the present invention;

[0056] Figure 3 shows an analysis diagram of the results of a test strip according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0058] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0059] Hemoglobin test strip

[0060] The present invention provides a hemoglobin detection test strip. Refer to Figure 1 , in a specific embodiment of the present invention, the hemoglobin detection test strip includes a bottom plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad are sequentially lapped; the conjugate pad is sprayed with colloidal gold-labeled HbA protein, HbS protein, HbC protein and chicken IgY antibody; on the nitrocellulose membrane, there are a detection line (T1 line) coated with HbA antibody, a detection line (T2 line) coated with HbS antibody, a detection line (T3 line) coated with HbC antibody, and a quality control line (C line) coated with goat anti-chicken antibody.

[0061] The bottom plate can be a PVC bottom plate. The PVC bottom plate refers to a plate produced from polyvinyl chloride material. Specifically, it can be mainly made of polyvinyl chloride and its copolymer resin, added with auxiliary materials such as fillers, plasticizers, stabilizers, and colorants, and produced on a sheet-like continuous substrate through a coating process or a calendering, extrusion or extrusion process.

[0062] The sample pad can be made of non-woven material (cellulose fiber). Its main function is to help the test sample be evenly distributed on the conjugate pad, prevent overflow by controlling the liquid flow rate of the sample entering the conjugate pad. After being integrated with other auxiliary reagents, it can also increase the sample viscosity (adjust the fluidity), prevent non-specific binding, improve the physical and chemical properties of the sample, make it compatible with the formation of immune complexes on the detection line; and promote the uniform flow of the sample along the membrane.

[0063] The conjugate pad can be made of glass fiber, mainly used to evenly transfer the test sample and the test reagent to the NC membrane, and has characteristics such as low non-specific binding, consistent flow characteristics, consistent bed volume, and low exudation.

[0064] In the present invention, the spraying amount of the mixed solution on the conjugate pad is 3 - 8 μL / cm.

[0065] The nitrocellulose membrane (referred to as NC membrane for short) is used as the carrier of the detection line and the quality control line (referred to as C / T line for short), and is also the place where the immune reaction occurs. As the solid-phase support medium for immune complexes, the NC membrane can directly interpret the test results.

[0066] On the nitrocellulose membrane, the spraying amount of each coating solution is 0.5 - 2 μL / cm.

[0067] The main function of the absorbent pad is to suck the carrier liquid / buffer solution on the nitrocellulose membrane from the detection line and the quality control line to the end of the test strip.

[0068] According to an embodiment of the present invention, the coating solution is selected from PBS buffer containing bovine serum albumin, surfactant and sucrose. The PBS buffer can provide a stable reaction system, bovine serum albumin (BSA) and sucrose can protect the antibody, and the surfactant can enhance the binding degree of the antibody.

[0069] It should be noted that the present invention does not strictly limit the type of surfactant, and surfactants commonly used in the art for coating test strips can be used, such as Tween, Triton, SDS, etc.

[0070] For hemoglobin detection kit

[0071] The present invention provides a hemoglobin detection kit. According to an embodiment of the present invention, the kit includes: including (1) or (2) or (3);

[0072] (1), three of the aforementioned test strips, each test strip for detecting one of the hemoglobin antigens;

[0073] (2), two of the aforementioned test strips, one of which is used to detect two of the hemoglobin antigens, and the other test strip is used to detect one hemoglobin antigen;

[0074] (3), one of the aforementioned reagent strips, and the test strip is used to detect three of the hemoglobin antigens.

[0075] The hemoglobin detection kit of the present invention can present three product forms: three test strips respectively detect hemoglobin A, hemoglobin S and hemoglobin C; one of the two test strips detects two of the proteins of hemoglobin A, hemoglobin S and hemoglobin C, and the other test strip detects the remaining one hemoglobin; one test strip simultaneously detects three hemoglobins of hemoglobin A, hemoglobin S and hemoglobin C.

[0076] According to an embodiment of the present invention, the kit further includes: a sample treatment solution. By using the sample treatment solution to treat the sample, for example, lysing red blood cells to collect the hemoglobin therein for detecting sickle cell anemia.

[0077] According to an embodiment of the present invention, the sample treatment solution is selected from buffer solutions. If the sample is only diluted with water, the release amount of hemoglobin in water is low, which is not conducive to subsequent immunoassay. Therefore, the inventor uses a buffer solution to dilute the sample, which is beneficial to providing the pH value required for the reaction system. Specifically, the buffer solution is selected from PBS buffer solution. In some embodiments, the sample treatment solution is selected from buffer solutions containing chemical reagents.

[0078] Since hemoglobin is mainly present inside cells, if only water or buffer solution is used to treat the sample, other tools or instruments are needed to break the cells in order to release the hemoglobin therein, which increases the detection time and complexity. Therefore, a lysis agent is added to break the red blood cells so as to collect the hemoglobin therein. Specifically, the lysis agent can be a substance commonly used in the art for lysing red blood cells, such as SDS and EDTA-2Na. Adding a surfactant can increase the ionic strength and contribute to the lysis reaction. The surfactant can be a surfactant commonly used in the art for cells, such as Tween, PEG20000 or PEG4000, etc.

[0079] Specifically, the sample treatment solution contains PBS buffer solution, 0.6%-2% of Tween20, 0.05%-2% of PEG20000, 0.05%-2% of PEG4000, 0.01%-0.1% of EDTA-2Na, 0.08%-0.5% of SDS and 0.5%-1.5% of Proclin300. The mass content is preferably 0.6% of Tween20, 0.14% of PEG20000, 0.17% of PEG4000, 0.05% of EDTA-2Na, 0.10% of SDS and 1.0% of Proclin300.

[0080] Beneficial effects

[0081] (1) Using the test strip and kit of the present invention, the sample pretreatment is simple or no pretreatment is required.

[0082] (2) Using the test strip and kit of the present invention is convenient and fast. The reaction generally only takes 5-15 minutes to obtain results, while other methods, such as PCR, take 1-2 hours and require supporting equipment, etc. It is convenient and fast to operate and is not restricted by the site. It is fast, specific and highly sensitive, and the sensitivity can reach 0.4 mg / mL or lower level.

[0083] (3) Using the test strip and kit of the present invention has a low use cost. The detection does not require special equipment and reagents. The result determination is intuitive, and the negative and positive results are significantly colored and can be easily judged by the naked eye.

[0084] (4) Using the test strip and kit of the present invention has specificity; it has a wide application range and can still be used under different detection conditions; it can detect multiple items simultaneously, improving the utilization rate of the sample, especially when the sample is difficult to obtain, the advantage is more significant.

[0085] (5) The test strip and kit of the present invention have stable properties. The kit or test strip can be stored for a long time and can be stored at room temperature for more than 2 years without signal attenuation.

[0086] (6) Since colloidal gold is red, there is no need to add coloring reagents, eliminating the carcinogenic substrates of enzyme labels, reducing the harm to experimental personnel and environmental pollution in the workplace, being safe and environmentally friendly, and meeting the requirements for building an environmentally friendly society.

[0087] The following will explain the solution of the present invention in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchases.

[0088] Example 1

[0089] 1. Reagents and materials

[0090] 1.1 Preparation of sample treatment solution: Take 7.14 g of solid Na2HPO4·12H2O, 1.36 g of solid NaH2PO4·2H2O, 1.76 g of solid NaCl, 1.4 g of solid PEG20000, 1.7 g of solid PEG4000, 0.5 g of solid EDTA-2Na, 1.0 g of solid SDS, 6 mL of Tween-20, 1 mL of Proclin300, and 993 mL of ultrapure water and add them to a 1000 mL beaker in sequence. Then use a magnetic stirrer to stir and mix evenly to completely dissolve the solids.

[0091] 1.2 Preparation of colloidal gold-labeled HbA protein solution:

[0092] 1) Take 50 μL of colloidal gold solution and mix it evenly with 50 μL of boric acid-citric acid buffer solution to obtain a colloidal gold-labeled solution.

[0093] 2) Take 0.08 mg of HbA protein solution and add it to the colloidal gold-labeled solution, and place it on a thermostatic shaking metal bath to shake to obtain HbA protein-colloidal gold complex 1.

[0094] 3) Take 5 μL of 10% BSA solution and add it to the above HbA protein-colloidal gold complex 1, and place it on a thermostatic shaking metal bath to shake HbA protein-colloidal gold complex 2.

[0095] 4) Place the above HbA protein-colloidal gold complex 2 solution in a refrigerated centrifuge for high-speed centrifugation.

[0096] 5) Discard the supernatant after centrifugation, take the precipitate and mix it thoroughly with 80 μL of colloidal gold re-solution to obtain a HbA protein colloidal gold-labeled solution.

[0097] 1.3 Preparation of colloidal gold-labeled HbS protein solution:

[0098] 1) Mix 50 μL of colloidal gold solution with 50 μL of boric acid-citric acid buffer solution evenly to obtain a colloidal gold labeling solution.

[0099] 2) Add 0.1 mg of HbS protein solution to the colloidal gold labeling solution, and place it on a thermostatic shaking metal bath to shake to obtain HbS protein-colloidal gold complex 1.

[0100] 3) Add 5 μL of 10% BSA solution to HbS protein-colloidal gold complex 1, and place it on a thermostatic shaking metal bath to shake to obtain HbS protein-colloidal gold complex 2.

[0101] 4) Centrifuge the above HbS protein-colloidal gold complex 2 at high speed in a refrigerated centrifuge.

[0102] 5) Discard the supernatant after centrifugation, take the precipitate and mix it evenly with 80 μL of colloidal gold reconstitution solution to obtain an HbS protein colloidal gold labeling solution.

[0103] 1.4 Preparation of colloidal gold-labeled HbC protein solution:

[0104] 1) Mix 40 μL of colloidal gold solution with 60 μL of citrate-citric acid buffer solution evenly to obtain a colloidal gold labeling solution.

[0105] 2) Add 0.013 mg of HbC protein solution to the colloidal gold labeling solution, and place it on a thermostatic shaking metal bath to shake to obtain HbC protein-colloidal gold complex 1.

[0106] 3) Add 5 μL of 10% BSA solution to the above HbC protein-colloidal gold complex 1, and place it on a thermostatic shaking metal bath to shake to obtain HBC protein-colloidal gold complex 2.

[0107] 4) Centrifuge the above HbC protein-colloidal gold complex 2 at high speed in a refrigerated centrifuge.

[0108] 5) Discard the supernatant after centrifugation, take the precipitate and mix it evenly with 40 μL of colloidal gold reconstitution solution to obtain an HbC protein colloidal gold labeling solution.

[0109] 1.5 Preparation of colloidal gold-labeled chicken IgY antibody solution:

[0110] 1) Mix 50 μL of colloidal gold solution with 50 μL of buffer solution evenly to obtain a colloidal gold labeling solution.

[0111] 2) Add 0.045 mg of IgY protein solution to the colloidal gold labeling solution, and place it on a thermostatic shaking metal bath to shake to obtain IgY colloidal gold complex 1.

[0112] 3) Add 5 μL of 10% BSA solution to the above IgY colloidal gold complex 1, and place it on a thermostatic shaking metal bath for shaking to obtain IgY colloidal gold complex 2.

[0113] 4) Centrifuge the above IgY colloidal gold complex 2 at high speed in a refrigerated centrifuge.

[0114] 5) Discard the supernatant after centrifugation, take 80 μL of the colloidal gold complex solution, and mix well to obtain the IgY colloidal gold labeling solution.

[0115] 1.6 Preparation of the coating solution:

[0116] Coating solution preparation: Weigh 7.5 g of BSA, 3 g of PVP30, and 0.5 g of sucrose, add them to 500 mL of 0.02 M PBS buffer solution, and mix well to obtain the coating solution.

[0117] Preparation of the HbA antibody coating solution: Mix the coating buffer with the HbA antibody to obtain an HbA antibody coating solution with a concentration of 0.8 mg / mL.

[0118] Preparation of the HbS antibody coating solution: Mix the coating buffer with the HbS antibody in equal volume to obtain an HbS antibody coating solution with a concentration of 0.5 mg / mL.

[0119] Preparation of the HbC antibody coating solution: Mix the coating buffer with the HbC antibody to obtain an HbC antibody coating solution with a concentration of 0.7 mg / mL.

[0120] Preparation of the goat anti-chicken antibody coating solution: Mix the coating buffer with the goat anti-chicken antibody to obtain a goat anti-chicken antibody coating solution with a concentration of 0.7 mg / mL.

[0121] 2. The steps for preparing the test strip are as follows:

[0122] 1) Add 80 μl of the sample treatment solution to the disposable sampling tube.

[0123] 2) Add 10 μl of the whole blood sample, draw it repeatedly 5 - 10 times or stir and mix well, and let it stand at room temperature for 15 minutes.

[0124] 3) A sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are successively overlapped on the PVC bottom plate, and the specific structure is as Figure 1 shown. Mix the HbA protein colloidal gold labeling solution, the HbS protein colloidal gold labeling solution, the HbC protein colloidal gold labeling solution, and the chicken IgY antibody colloidal gold labeling solution according to a volume ratio of 10:5:4:3, and spray them on the conjugate pad at a spraying amount of 6.5 μL / cm.

[0125] As Figure 2As shown in the figure, HbA antibody coating solution (T1 line), HbS antibody coating solution (T2 line), HbC antibody coating solution (T3 line) and goat anti-chicken antibody coating solution (C line) were respectively sprayed on the nitrocellulose membrane (spraying volume: 0.9 μL / cm), and after dehumidifying for 4 hours, it was dried for later use.

[0126] 4) Place the test strip into a disposable sampling tube, ensuring that the sample pad is immersed in the liquid surface and the conjugate pad does not contact the liquid. Start timing when the liquid begins to move upward from the lower edge of the conjugate pad (NC membrane). Observe the result visually between 10 - 15 minutes. As Figure 3 shown in the figure, if no color develops on the test line (T line), it indicates that the corresponding antigen (protein) is present in the sample; if color develops on the test line (T line), it indicates that the corresponding antigen (protein) is not present in the sample; if color develops on the control line (C line), it indicates that the test strip is normal, otherwise it indicates that the test strip is abnormal and the result is invalid; if all three test lines and the control line show color, it indicates that the result is invalid.

[0127] 5) Extract genomic DNA from the treatment solution obtained in step 1, item 2) for PCR detection.

[0128] The results are shown in Tables 1 and 2, and some of the experimental results are shown in Table 3. Among them, AA represents the HbA homozygous wild genotype, CC represents the HbC homozygous mutant genotype, SS represents the HbS homozygous mutant genotype, CS represents the HbS and HbC heterozygous mutant genotype, AS represents the HbS heterozygous mutant genotype, and AC represents the HbC heterozygous mutant genotype.

[0129] It can be seen that the detection results using the kit of the present invention are consistent with the PCR amplification results, indicating that the method of the present invention can accurately determine whether HbA protein, HbS protein, and HbC protein are contained in the sample, and thus realize the detection of sickle cell anemia.

[0130] Table 1 Sample Information

[0131] Sample type Sample size (pcs) AA 40 SS 5 CC 1 AS 15 AC 6 SC 7 Total amount 74

[0132] Table 2 Comparison and Statistics of the Results of the Reagent Strip and PCR Amplification in this Example

[0133]

[0134] Table 3 Partial Data of the Comparison between the Reagent Strip and PCR Amplification Results in this Example

[0135]

[0136]

[0137] Example 2

[0138] In the test strip of this embodiment, the conjugate pad is sprayed with colloidal gold-labeled HbA antibody, colloidal gold-labeled HbS antibody, colloidal gold-labeled HbC antibody, and colloidal gold-labeled chicken IgY antibody; the nitrocellulose membrane is coated with HbA protein (T1 line), HbS protein (T2 line), HbC protein (T3 line), and goat anti-chicken antibody (C line).

[0139] The preparation method, required reagents and materials of the reagent strip in this embodiment are the same as those in Embodiment 1. The difference from Embodiment 1 is only that:

[0140] 1. Reagents and Materials

[0141] 1.1 Preparation of colloidal gold-labeled HbA antibody solution:

[0142] 1) Take 40 μL of colloidal gold solution and mix it evenly with 60 μL of boric acid-citric acid buffer solution to obtain a colloidal gold-labeled solution.

[0143] 2) Add 0.006 mg of HbA antibody to the colloidal gold-labeled solution, and place it on a thermostatic shaking metal bath to shake to obtain HbA antibody-colloidal gold complex 1.

[0144] 3) Take 5 μL of 10% CaseinNa solution and add it to the above HbA antibody-colloidal gold complex 1 solution, and place it on a thermostatic shaking metal bath to shake HbA antibody-colloidal gold complex 2.

[0145] 4) Place the above HbA antibody-colloidal gold complex 2 solution in a refrigerated centrifuge for high-speed centrifugation.

[0146] 5) Discard the supernatant after centrifugation, take 80 μL of colloidal gold complex solution, and mix it well to obtain a colloidal gold-labeled HbA antibody solution.

[0147] 1.2 Preparation of colloidal gold-labeled HbS antibody solution:

[0148] 1) Take 40 μL of colloidal gold solution and mix it evenly with 60 μL of boric acid-citric acid buffer solution to obtain a colloidal gold-labeled solution.

[0149] 2) Add 0.005 mg of HbS antibody to the colloidal gold-labeled solution, and place it on a thermostatic shaking metal bath to shake to obtain HbS antibody-colloidal gold complex 1.

[0150] 3) Take 5 μL of 10% CaseinNa solution and add it to the above HbS antibody-colloidal gold complex 1, and place it on a thermostatic shaking metal bath to shake to obtain HbS antibody-colloidal gold complex 2.

[0151] 4) Place the above HbS antibody-colloidal gold complex 2 solution in a refrigerated centrifuge for high-speed centrifugation.

[0152] 5) Discard the supernatant after centrifugation, take 80 μL of colloidal gold reconstitution solution, and after thorough mixing, obtain the colloidal gold-labeled solution of HbS antibody.

[0153] 1.3 Preparation of colloidal gold-labeled HbC protein solution:

[0154] 1) Take 40 μL of colloidal gold solution and mix it evenly with 60 μL of boric acid-citric acid buffer solution to obtain a colloidal gold-labeled solution.

[0155] 2) Take 0.006 mg of HbC protein solution and add it to the colloidal gold-labeled solution, and place it on a thermostatic shaking metal bath to shake to obtain HbC antibody-colloidal gold complex 1.

[0156] 3) Take 5 μL of 10% CaseinNa solution and add it to the above-mentioned colloidal gold-labeled solution, and place it on a thermostatic shaking metal bath to shake to obtain HbC antibody-colloidal gold complex 2.

[0157] 4) Place the above-mentioned HbC antibody-colloidal gold complex 2 in a refrigerated centrifuge for high-speed centrifugation.

[0158] 5) Discard the supernatant after centrifugation, take 40 μL of colloidal gold reconstitution solution, and after thorough mixing, obtain the colloidal gold-labeled solution of HbC antibody.

[0159] 1.4 Preparation of coating solution:

[0160] The preparation of the coating buffer is the same as that in Example 1.

[0161] Preparation of coating solution containing HbA protein: Mix the coating buffer with HbA protein to obtain a HbA protein coating solution with a concentration of 0.8 mg / mL.

[0162] Preparation of coating solution containing HbS protein: Mix the coating buffer with HbS protein to obtain a HbS protein coating solution with a concentration of 0.8 mg / mL.

[0163] Preparation of coating solution containing HbC protein: Mix the coating buffer with HbC protein to obtain a HbC protein coating solution with a concentration of 0.8 mg / mL.

[0164] The samples in this example are the same as those in Example 1 (as shown in Table 1), and the detection results are shown in Table 4. Among them, some experimental results are shown in Table 5. Among them, AA represents the HbA homozygous wild genotype, CC represents the HbC homozygous mutant genotype, SS represents the HbS homozygous mutant genotype, CS represents the HbS and HbC heterozygous mutant genotype, AS represents the HbS heterozygous mutant genotype, and AC represents the HbC heterozygous mutant genotype.

[0165] It can be seen that the detection results using the kit of the present invention are consistent with the PCR amplification results, indicating that the method of the present invention can accurately determine whether HbA protein, HbS protein, and HbC protein are contained in a sample, thereby realizing the detection of sickle cell anemia.

[0166] Table 4 Comparison and statistics of the test strip and PCR amplification results in this example

[0167]

[0168] Table 5 Partial data of the comparison between the test strip and PCR amplification results in this example

[0169] Sample serial number PCR result Kit result Sample serial number PCR result Kit result SCD-1 AA AA SCD-24 SS SS SCD-2 AS AS SCD-25 AS AS SCD-3 AA AA SCD-26 AS AS SCD-4 SS SS SCD-27 AS AA SCD-5 AS AS SCD-28 AC AC SCD-6 AS AS SCD-29 SC SC SCD-7 AC AC SCD-30 AC AC SCD-8 SC SC SCD-31 SS SS SCD-9 AA AA SCD-32 AS AS SCD-10 SC SC SCD-33 SS SS SCD-11 AC AC SCD-34 AA AA SCD-12 AS AS SCD-35 AS AS SCD-13 AA AA SCD-36 CC CC SCD-14 AS AS SCD-37 AS AS SCD-15 AS AS SCD-38 SC SC SCD-16 SC SC SCD-39 SS SS SCD-17 AA AA SCD-40 AA AA SCD-18 AC AC SCD-41 AS AS SCD-19 AC AC SCD-42 AS AS SCD-20 AA AA SCD-43 AS AS SCD-21 SC SC SCD-44 AA AA SCD-22 SC SC SCD-45 AA AA SCD-23 AA AA SCD-46 AA AA

[0170] Example 3

[0171] In the test strip of this example, the conjugate pad is sprayed with colloidal gold-labeled HbA protein, colloidal gold-labeled HbS antibody, colloidal gold-labeled HbC protein, and colloidal gold-labeled chicken IgY antibody; the nitrocellulose membrane is coated with HbA antibody (T1 line), HbS protein (T2 line), HbC antibody (T3 line), and goat anti-chicken antibody (C line).

[0172] In this example, the preparation method of the test strip, the required reagents and materials are the same as those in Examples 1 and 2.

[0173] The samples are the same as those in Example 1 (as shown in Table 1), and the detection results are shown in Table 6. Among them, some experimental results are shown in Table 7. Among them, AA represents the HbA homozygous wild genotype, CC represents the HbC homozygous mutant genotype, SS represents the HbS homozygous mutant genotype, CS represents the HbS and HbC heterozygous mutant genotype, AS represents the HbS heterozygous mutant genotype, and AC represents the HbC heterozygous mutant genotype.

[0174] It can be seen that the detection results using the kit of the present invention are consistent with the PCR amplification results, indicating that the method of the present invention can accurately determine whether HbA protein, HbS protein, and HbC protein are contained in a sample, thereby realizing the diagnosis of sickle cell anemia.

[0175] Table 6 Comparison and statistics of the test strip and PCR amplification results in this example

[0176]

[0177] Table 7 Partial data of the comparison between the test strip and PCR amplification results in this example

[0178]

[0179]

[0180] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A hemoglobin test strip, characterized in that, The test strip includes a base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially overlapped; The conjugate pad is sprayed with colloidal gold-labeled hemoglobin antigen and / or colloidal gold-labeled hemoglobin antibody, and colloidal gold-labeled first antibody; The nitrocellulose membrane is provided with a test line and a control line. The test line is coated with hemoglobin antigen and / or hemoglobin antibody, and the control line is coated with a second antibody that binds to the first antibody; The hemoglobin antigen includes at least one of hemoglobin A, hemoglobin S, and hemoglobin C. Correspondingly, the hemoglobin antibody includes at least one of hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody; The hemoglobin antigen or hemoglobin antibody on the conjugate pad can specifically bind to the corresponding hemoglobin antigen or hemoglobin antibody on the nitrocellulose membrane to form an antigen-antibody complex.

2. The test strip according to claim 1, characterized in that, The conjugate pad is sprayed with colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S, or colloidal gold-labeled hemoglobin C. Correspondingly, the test line is divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody, or a test line coated with hemoglobin C antibody; or The conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S antibody, or colloidal gold-labeled hemoglobin C antibody. Correspondingly, the test line is divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S, or a test line coated with hemoglobin C; 3. The test strip according to claim 1, wherein, The conjugate pad is sprayed with colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S, and colloidal gold-labeled hemoglobin C. Correspondingly, the test line is divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody, and a test line coated with hemoglobin C antibody; or The conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S antibody, and colloidal gold-labeled hemoglobin C antibody. Correspondingly, the test line is divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S, and a test line coated with hemoglobin C; 4. The test strip according to claim 1, wherein When the conjugate pad is sprayed with colloidal gold-labeled hemoglobin antigen and colloidal gold-labeled hemoglobin antibody, and the test line is coated with hemoglobin antigen and hemoglobin antibody, the hemoglobin antigen includes at least two of hemoglobin A, hemoglobin S, and hemoglobin C, and the hemoglobin antibody includes at least two of hemoglobin A antibody, hemoglobin S antibody, and hemoglobin C antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A and colloidal gold-labeled hemoglobin S antibody, and the test line is divided into a test line coated with hemoglobin A antibody and a test line coated with hemoglobin S; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A and colloidal gold-labeled hemoglobin C antibody, and the test line is divided into a test line coated with hemoglobin A antibody and a test line coated with hemoglobin C; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody and colloidal gold-labeled hemoglobin S, and the test lines are divided into a test line coated with hemoglobin A and a test line coated with hemoglobin S antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody and colloidal gold-labeled hemoglobin C, and the test lines are divided into a test line coated with hemoglobin A and a test line coated with hemoglobin C antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin S antibody and colloidal gold-labeled hemoglobin C, and the test lines are divided into a test line coated with hemoglobin S and a test line coated with hemoglobin C antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin S and colloidal gold-labeled hemoglobin C antibody, and the test lines are divided into a test line coated with hemoglobin S antibody and a test line coated with hemoglobin C; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S antibody and colloidal gold-labeled hemoglobin C, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S and a test line coated with hemoglobin C antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S antibody and colloidal gold-labeled hemoglobin C antibody, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S and a test line coated with hemoglobin C; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A, colloidal gold-labeled hemoglobin S and colloidal gold-labeled hemoglobin C antibody, and the test lines are divided into a test line coated with hemoglobin A antibody, a test line coated with hemoglobin S antibody and a test line coated with hemoglobin C; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S antibody and colloidal gold-labeled hemoglobin C, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S and a test line coated with hemoglobin C antibody; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S and colloidal gold-labeled hemoglobin C antibody, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S antibody and a test line coated with hemoglobin C; Preferably, the conjugate pad is sprayed with colloidal gold-labeled hemoglobin A antibody, colloidal gold-labeled hemoglobin S and colloidal gold-labeled hemoglobin C, and the test lines are divided into a test line coated with hemoglobin A, a test line coated with hemoglobin S antibody and a test line coated with hemoglobin C antibody.

5. The test strip according to claim 1, wherein, The first antibody is a chicken IgY antibody, and the second antibody is a goat anti-chicken antibody.

6. The test strip according to claim 1, wherein The test strip further includes a cartridge, and the bottom plate carrying the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad is fixed in the cartridge; The surface of the cassette is provided with a sample adding hole and an observation window. The sample adding hole is arranged opposite to the sample pad, and the observation window is arranged opposite to the test line and the control line.

7. A method for preparing the hemoglobin test strip according to any one of claims 1 to 6, characterized in that, It includes the following steps: Step 1: Spraying a colloidal gold-labeled hemoglobin antigen solution and / or a colloidal gold-labeled hemoglobin antibody solution and a colloidal gold-labeled first antibody solution on the conjugate pad, and drying. Step 2: Spraying a hemoglobin antigen coating solution and / or a hemoglobin antibody coating solution on the test line of the nitrocellulose membrane, and spraying a second antibody coating solution on the control line, and drying. Step 3: Sequentially overlapping the sample pad, the conjugate pad obtained in Step 1, the nitrocellulose membrane obtained in Step 2, and the absorbent pad on the bottom plate to obtain a test strip.

8. The preparation method according to claim 7, wherein The coating solutions in the hemoglobin antigen coating solution and / or the hemoglobin antibody coating solution and the second antibody coating solution each independently include at least one of the following: bovine serum albumin, PBS buffer, sucrose, and surfactant.

9. A hemoglobin detection kit, characterized in that, It includes the hemoglobin detection test strip according to any one of claims 1 to 6. Optionally, the hemoglobin detection kit includes any one of (1), (2), and (3): (1) Three of the hemoglobin detection test strips, each of the hemoglobin detection test strips being used to detect one of the hemoglobin antigens. (2) Two of the hemoglobin detection test strips, one of the test strips being used to detect two of the hemoglobin antigens, and the other test strip detecting one hemoglobin antigen. (3) One of the hemoglobin detection test strips, the hemoglobin detection test strip being used to detect three of the hemoglobin antigens.

10. The kit according to claim 9, characterized in that, It further includes: A sample treatment solution; The sample treatment solution includes: a buffer containing a lysis solution and / or a surfactant. Preferably, the sample treatment solution includes a PBS buffer solution, Tween 20, PEG 20000, PEG 4000, EDTA-2Na, SDS, and Proclin 300.

11. Use of the hemoglobin detection test strip according to any one of claims 1 to 6 or the hemoglobin detection kit according to claim 9 or 10 in the preparation of a product for detecting sickle cell anemia.

12. The use according to claim 11, wherein The mutant types of the sickle cell anemia include at least one of homozygous mutation of hemoglobin S, homozygous mutation of hemoglobin C, heterozygous mutation of hemoglobin C and hemoglobin S, heterozygous mutation of hemoglobin A and hemoglobin C, and heterozygous mutation of hemoglobin A and hemoglobin S.

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