Method for simultaneously detecting various hemoglobin in vitro and application thereof

Abstract

The invention discloses a method for simultaneously detecting various hemoglobin in vitro and application thereof. The method comprises the following steps: (1) preparing a detection test strip; (2) dropwise adding human whole blood or a whole blood diluted sample on the reaction pad of the test strip; and (3) dropwise adding a sample flushing fluid on the flushing pad of the test strip, enabling the sample flushing fluid to sequentially flow to the absorption pad along the marking pad, the cracking pad and the reaction pad of the test strip, and after 4-6 minutes of complete reaction, interpreting a detection result according to the color development states of a Ctl point and a plurality of detection points on the reaction pad. The invention provides a rapid, simple and low-cost hemoglobin detection method, can simultaneously detect various hemoglobin such as HbA, HbS and HbC, and is suitable for rapid screening and clinical diagnosis of abnormal hemoglobin diseases such as sickle cell anemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a method for simultaneously detecting multiple hemoglobins in vitro and an application thereof. Background Art

[0002] Hemoglobin (Hb) is the core protein molecule responsible for oxygen transport in red blood cells. It is typically composed of four polypeptide chains. Normal adult hemoglobin (HbA) consists of two α chains and two β chains, resulting in a stable structure and complete function. However, mutations in globin genes can lead to structural abnormalities or impaired function of the hemoglobin molecule, leading to a range of inherited disorders collectively known as hemoglobinopathies. Sickle cell disease (SCD) is a prime example of these diseases, primarily caused by a mutation at amino acid position 6 in the β-globin gene. For example, when glutamic acid mutates to valine, HbS forms, causing red blood cells to become sickle-shaped under hypoxic conditions, predisposing to serious consequences such as vascular occlusion, severe pain, infection, and organ damage. Mutations from glutamic acid to lysine form HbC, which can cause mild anemia or worsen the condition in combination with other hemoglobin variants. Other hemoglobin variants, such as HbE and HbD, are also associated with specific genetic backgrounds and have a higher incidence in certain populations.

[0003] The clinical manifestations of abnormal hemoglobin diseases are complex and diverse, and their severity is closely related to the type and content of hemoglobin variants. Therefore, the development of a method that can quickly and accurately detect multiple hemoglobin variants such as HbA, HbS, and HbC is not only of great significance for the early screening and diagnosis of sickle cell disease, but also provides a scientific basis for genetic counseling, disease prevention, and the formulation of personalized treatment plans. With the continuous development of in vitro diagnostic technology, a variety of technical means for hemoglobin detection have been developed. These methods have their own advantages and disadvantages in sensitivity, specificity, and application scenarios, but they also have certain limitations. The following is a detailed analysis of the main types of existing technologies and their characteristics:

[0004] 1. Electrophoresis: Electrophoresis is one of the earliest traditional methods used for hemoglobin testing. Its basic principle is to use an electric field to separate charged hemoglobin molecules based on their charge and molecular weight in a medium such as a gel or filter paper. Common electrophoretic methods include agarose gel electrophoresis and cellulose acetate membrane electrophoresis, which are widely used in clinical laboratories to differentiate hemoglobin variants such as HbA, HbS, and HbC. The advantage of electrophoresis lies in its high resolution, which can clearly distinguish different hemoglobin types, making it particularly suitable for analyzing complex samples. However, this method also has significant drawbacks: First, the operation is cumbersome, requiring specialized personnel in a laboratory setting using dedicated electrophoresis equipment; second, the test time is long, typically taking several hours, making it unsuitable for rapid screening. Furthermore, electrophoresis requires high sample quality; impurities in the sample or environmental factors (such as temperature and humidity) can interfere with the separation effect, resulting in unstable results. Finally, the interpretation of results relies on the technician's experience and is somewhat subjective. These limitations make electrophoresis difficult to meet the demand for efficient and convenient testing in modern medicine.

[0005] 2. High-Performance Liquid Chromatography (HPLC): High-performance liquid chromatography (HPLC) is a high-precision analytical technique that separates molecules based on size and charge. In recent years, it has become the gold standard for hemoglobin testing. HPLC uses specific ion-exchange or reversed-phase columns to separate hemoglobin components and, combined with a UV detector, quantifies the levels of variants such as HbA, HbS, and HbC. This method offers high sensitivity and reproducibility, providing precise hemoglobin ratio data and thus has wide application in clinical diagnosis and research. However, HPLC has its drawbacks: its equipment is expensive and maintenance-intensive, making it difficult to implement in resource-limited primary healthcare settings. Furthermore, HPLC testing requires complex sample pretreatment steps, such as red blood cell lysis, centrifugation, and dilution, which not only increases the complexity of the procedure but also prolongs the test time, typically taking tens of minutes per test. Furthermore, the method requires high technical expertise, and improper operation can lead to biased results. Therefore, HPLC is more suitable for laboratory settings rather than point-of-care testing (POCT).

[0006] 3. Mass spectrometry: Mass spectrometry can accurately identify the molecular weight and structural variations of hemoglobin molecules by detecting the mass-to-charge ratio of hemoglobin molecules. It is an ideal tool for research analysis and diagnosis of complex cases. For example, liquid chromatography-mass spectrometry (LC-MS) can distinguish tiny differences in hemoglobin structure and provide support for the identification of rare variants. However, the application of mass spectrometry technology is limited in many aspects: first, its equipment is extremely expensive and complicated to operate, and is limited to high-end research institutions or large hospitals; second, mass spectrometry has extremely high requirements for sample purity and requires multiple steps of purification, which increases the complexity of detection; in addition, this method does not have high-throughput detection capabilities and is difficult to meet the needs of routine clinical screening. Therefore, although mass spectrometry technology has important value in scientific research, it is less popular in daily clinical testing.

[0007] 4. Genetic testing technology: Genetic testing technology directly analyzes the sequence of the globin gene through methods such as polymerase chain reaction (PCR) or gene sequencing, and confirms the genetic basis of hemoglobin variation at the molecular level. For example, by detecting specific mutation sites in the β-globin gene, the presence of variants such as HbS and HbC can be accurately determined. This method is highly specific and can provide clear genetic information, so it has important applications in genetic counseling and prenatal diagnosis. However, genetic testing also has limitations: first, its detection cycle is long, usually taking several hours to several days, and it cannot achieve rapid diagnosis; second, the cost of equipment and reagents is high, which is not suitable for large-scale screening; in addition, this method can only reflect information at the gene level and cannot directly detect the expression level or functional status of hemoglobin, so its application in clinical diagnosis is subject to certain restrictions.

[0008] 5. Lateral Flow Immunoassay (LFIA): In recent years, lateral flow immunoassay (LFIA) has garnered widespread attention in the field of point-of-care testing due to its ease of use, low cost, and intuitive results. Existing technologies have attempted to apply LFIA to hemoglobin detection, for example, by utilizing specific antibodies to recognize the target hemoglobin and forming a visible detection line on a test strip. Common LFIA detection methods currently include competitive and sandwich methods. The competitive method achieves detection through competitive binding between an antigen and a labeled antibody, but its sensitivity is low and prone to false-negative results, especially in low-concentration samples. The sandwich method utilizes a capture antibody and a labeled antibody to bind to the antigen, offering relatively high sensitivity, but can induce a "hook effect" at high antigen concentrations, leading to signal attenuation or even disappearance. Furthermore, existing LFIA test strips often utilize a single detection line design, making it difficult to simultaneously detect multiple hemoglobins. The detection process often requires sample pretreatment (such as red blood cell lysis), adding additional steps. When detecting complex samples, existing LFIA methods may also affect the accuracy of the results due to signal cross-interference.

[0009] In summary, the existing hemoglobin detection technology has the following major problems in practical application:

[0010] 1. Operational complexity and equipment dependence: Methods such as electrophoresis, HPLC, and mass spectrometry require specialized equipment and complex sample pretreatment steps, making them difficult to operate and unsuitable for primary healthcare institutions or emergency medical scenarios.

[0011] 2. Low detection efficiency: Existing technologies are mostly single-target detection, which can only analyze one hemoglobin type at a time. It is impossible to detect multiple variants such as HbA, HbS, and HbC at the same time, resulting in a time-consuming and costly detection process.

[0012] 3. Insufficient sensitivity and accuracy: Although the existing LFIA-based methods are easy to operate, the competitive method has low sensitivity, the sandwich method is easily interfered by the hook effect, and the detection area design (such as linear layout) may cause signal overlap or cross-interference, affecting the reliability of the result interpretation.

[0013] 4. Limited applicability: The equipment cost and operation requirements of high-end technologies (such as HPLC and mass spectrometry) limit their promotion in resource-poor areas, and the performance of existing LFIA methods in high-throughput screening and complex sample detection is still unsatisfactory.

[0014] These problems make existing technologies significantly insufficient in the rapid screening and early diagnosis of abnormal hemoglobinopathies, especially in scenarios where fast, convenient, and low-cost testing is required, and there is a lack of an efficient solution. Summary of the Invention

[0015] The present invention aims to overcome the defects of the prior art and provide a method for the simultaneous in vitro detection of multiple hemoglobins.

[0016] Another object of the present invention is to provide a test strip for simultaneous in vitro detection of multiple hemoglobins.

[0017] The technical solutions of the present invention are as follows:

[0018] A method for simultaneously detecting multiple hemoglobins in vitro comprises the following steps:

[0019] (1) A test strip is prepared, the test strip comprising a substrate and a flushing pad, a labeling pad, a lysis pad, a reaction pad, and an absorption pad disposed on the substrate and contacting each other in sequence along a chromatography direction, wherein:

[0020] The labeling pad is pre-coated with tracer-labeled anti-Hb antibodies.

[0021] The lysis pad is pre-coated with dried saponin.

[0022] The reaction pad is provided with a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the above-mentioned tracer-labeled anti-Hb antibody, and the several detection points are respectively coated with antibodies corresponding to the hemoglobin.

[0023] (2) adding human whole blood or diluted whole blood sample to the reaction pad;

[0024] (3) Add a sample rinse solution containing Tween 20 to the above-mentioned rinse pad, so that the sample rinse solution flows along the labeling pad, lysis pad and reaction pad to the absorption pad in sequence. After waiting for 4-6 minutes for the reaction to be complete, the test results are interpreted according to the color development status of the Ctl point and several detection points on the reaction pad.

[0025] In a preferred embodiment of the present invention, the tracer is nano-metal particles or colored latex particles.

[0026] In a preferred embodiment of the present invention, the reaction pad is a reaction pad made of nitrocellulose.

[0027] In a preferred embodiment of the present invention, the Ctl spot and the plurality of detection spots are arranged in an array on the reaction pad.

[0028] In a preferred embodiment of the present invention, the plurality of detection points include a point A, a point S and a point C, point A is coated with anti-HbA antibodies, point S is coated with anti-HbS antibodies, and point C is coated with anti-HbC antibodies.

[0029] A test strip for simultaneous in vitro detection of multiple hemoglobins comprises a substrate and a flushing pad, a labeling pad, a lysis pad, a reaction pad and an absorption pad arranged on the substrate and contacting each other in sequence along a chromatography direction, wherein:

[0030] The labeling pad is pre-coated with tracer-labeled anti-Hb antibodies.

[0031] The lysis pad is pre-coated with dried saponin.

[0032] The reaction pad is provided with a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the above-mentioned tracer-labeled anti-Hb antibody, and the several detection points are respectively coated with antibodies corresponding to the hemoglobin.

[0033] In a preferred embodiment of the present invention, the tracer is nano-metal particles or colored latex particles.

[0034] In a preferred embodiment of the present invention, the reaction pad is a reaction pad made of nitrocellulose.

[0035] In a preferred embodiment of the present invention, the Ctl spot and the plurality of detection spots are arranged in an array on the reaction pad.

[0036] In a preferred embodiment of the present invention, the plurality of detection points include a point A, a point S and a point C, point A is coated with anti-HbA antibodies, point S is coated with anti-HbS antibodies, and point C is coated with anti-HbC antibodies.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention realizes in-situ lysis of the sample by providing a lysis pad on the test strip, eliminating the need for pre-lysis treatment of the sample, simplifying the operation steps and improving detection efficiency.

[0039] 2. The present invention adopts a detection sequence of capture first and then labeling. The hemoglobin in the sample first binds to the capture antibody on the reaction membrane, and the excess hemoglobin is washed into the absorption pad. Then the labeled antibody forms a sandwich complex with the bound hemoglobin, avoiding the hook effect caused by high concentration of antigen and improving the sensitivity and accuracy of the detection.

[0040] 3. The present invention adopts a point-shaped detection point design, and the detection points are arranged in an array on the reaction membrane, which reduces cross-interference and steric hindrance between detection points and ensures the independence and accuracy of the results.

[0041] 4. The detection process of the present invention only requires one sample addition, and the results can be interpreted within 5 minutes. It does not require any equipment assistance, is easy to operate, and is suitable for various clinical scenarios.

[0042] 5. The test strip of the present invention has a simple structure, is easy to produce, has low cost, and is suitable for areas with limited resources.

[0043] 6. The test strips of the invention can be stored at room temperature, have a long shelf life, and are easy to store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the structure of the test strip for simultaneous in vitro detection of multiple hemoglobins in Example 1 of the present invention. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0046] Example 1

[0047] like Figure 1 As shown, a test strip for simultaneous in vitro detection of multiple hemoglobins includes a substrate 10 and a washing pad 11, a labeling pad 12, a lysis pad 13, a reaction pad 14 and an absorption pad 15 arranged on the substrate 10 and contacting each other in sequence along the chromatography direction.

[0048] The marking pad 12 is a glass fiber pad, which is pre-coated and solidified with a tracer-labeled anti-Hb antibody. The tracer in this embodiment is preferably colloidal gold, that is, the tracer-labeled anti-Hb antibody is an anti-human Hb antibody-colloidal gold complex. The preparation method of the marking pad 12 comprises: quickly adding anti-human hemoglobin monoclonal antibody at a ratio of 15-50 μg / mL to a colloidal silica gold dispersion with a particle size of 20-60 nm and a concentration of 0.01-0.05 wt%, quickly mixing, reacting for 4-15 minutes, quickly adding blocking solution at a ratio of 10-15 μL / mL, reacting for 4-15 minutes, centrifuging at 8000-10000 rpm for 5-10 minutes, discarding the supernatant to obtain a precipitate, and adding a reconstitution solution (3.9325 g Trizma base, 2.86 g sodium citrate, 1.739 ml concentrated hydrochloric acid, 5 g casein, 1 g polyvinyl pyrrolidone, 60 g sucrose, 5 g bovine serum albumin, 1 g sodium azide, and dilute to 1 L with ultrapure water), and re-dissolved by ultrasonication to obtain an anti-human Hb antibody-colloidal gold complex; the prepared anti-human Hb antibody-colloidal gold complex was evenly applied on a glass fiber mat, and dried at 25-40° C. for 2-6 hours to obtain a labeling pad 12;

[0049] The cracking pad 13 has a thickness of 0.2-0.5 mm and a gram weight of 60-90 g / m 2The preparation method of the pyrolysis pad 13 comprises: applying 4-10g / L saponin solution evenly on the hydrophilic glass fiber mat, with a volume to area ratio of about 0.5-0.8mL / cm 2 , and dried at 25-45° C. for 2-6 h to obtain a pyrolysis pad 13.

[0050] The reaction pad 14 is a nitrocellulose membrane having a Ctl point 141 and three detection points (A point 142, S point 143, and C point) for detecting different hemoglobins. The Ctl point 141 is coated with a protein that can bind to the above-mentioned tracer-labeled anti-Hb antibody, the A point 142 is coated with an anti-HbA antibody, the S point 143 is coated with an anti-HbS antibody, and the C point 144 is coated with an anti-HbC antibody. The Ctl point 141, the A point 142, the S point 143, and the C point are arranged in a spatial rhombus. The preparation method of the reaction pad 14 includes: diluting anti-HbA antibody, anti-HbS antibody, and anti-HbC antibody to 0.25-2 mg / mL with a coating buffer, coating the antibody onto point A 142, point S 143, and point C 144 of a nitrocellulose membrane, and then drying the antibody at a temperature of 20-32° C. and a humidity of less than 30% for 40 minutes to 2 hours; diluting goat anti-mouse IgG antibody to 1-3 mg / mL with a coating buffer, coating the antibody onto point Ctl 141 of the nitrocellulose membrane, and then drying the antibody at a temperature of 20-32° C. and a humidity of less than 30% for 40 minutes to 2 hours.

[0051] The antibodies mentioned above are shown in the following table:

[0052]

[0053] The specific process of using the test strips for simultaneous in vitro detection of multiple hemoglobins prepared in this embodiment is as follows:

[0054] (1) dropping 10-15 μL of human whole blood or diluted human whole blood sample to be tested onto the reaction pad 14;

[0055] (2) Add 140 μL of sample flushing solution (10 mM PBS containing 3% Tween 20) to the flushing pad 11, allowing the sample flushing solution to gradually flow toward the absorption pad 15 along the chromatography direction;

[0056] (3) Wait 3-5 minutes for the result to be read. If the result exceeds 15 minutes, the result will be invalid. The following table is provided for reference:

[0057]

[0058]

[0059] The detection principle of this embodiment is that immune complexes (anti-human Hb antibody-colloidal gold complex-hemoglobin antigen-hemoglobin antibody) form at points A 142, S 143, C 144, and Ct1 141, leading to color development. Specifically, if point A 142 develops color, it indicates that the sample contains HbA; if point S 143 develops color, it indicates that the sample contains HbS; if point C 144 develops color, it indicates that the sample contains HbC. Ct1 141, serving as a control point, must develop color; otherwise, the test strip will be invalid.

[0060] When the test strip of this embodiment is used for the simultaneous detection of HbA, HbS, and HbC, the red blood cells in the sample can be lysed by the lysis pad on the lysis pad 13 to release hemoglobin, thereby eliminating the need for the prior art to perform lysis. When the test strip of this embodiment detects hemoglobin, only one sample addition is required to determine whether the sample contains HbA / HbS / HbC within 5 minutes, thereby reducing the risk of manual operation errors. It has high sensitivity, does not require any equipment assistance, can be stored at room temperature, has a long shelf life, and is more suitable for clinical blood type rapid detection scenarios.

[0061] In addition, existing test strips all adopt the method of dripping the sample to be tested on the sample pad. When the antigen concentration is too high, it is easy for excessive hemoglobin to not bind to the anti-human Hb antibody-colloidal gold complex but directly bind to the hemoglobin antibody on the reaction pad 14 along the chromatography direction, resulting in the hemoglobin antibody binding site on the reaction pad 14 being occupied and no longer able to bind to the hemoglobin-anti-human Hb antibody-colloidal gold complex, thus low sensitivity. This embodiment adopts the method of directly dripping the sample to be tested on the reaction pad 14. The sample rinse solution containing Tween 20 passes through the labeling pad 12 and the lysis pad 13, dissolving and mixing the anti-human Hb antibody-colloidal gold complex from the labeling pad 12 and the saponin from the lysis pad 13. After reaching the reaction pad 14, the red blood cells in the sample to be tested are lysed under the synergistic action of saponin and Tween 20 to release hemoglobin, wherein different hemoglobins respectively bind to the goat anti-mouse IgG antibody labeled on the Ctl point 141, the anti-HbA antibody coated on the A point 142, and the S point 143. The anti-HbS antibody coated with 43 and the anti-HbC antibody coated with C dot 144 combine to form corresponding immune complexes, which then respectively combine with the anti-human Hb antibody-colloidal gold complex from the labeling pad 12 to form different anti-human Hb antibody-colloidal gold complex-hemoglobin antigen complexes. Excess unbound different hemoglobins will simultaneously chromatograph into the absorption pad 15, thereby solving the problem that excess antigens in the sample to be tested first combine with the antibodies on the reaction pad 14 and develop weak or no color, thereby improving the detection sensitivity.

[0062] In this example, during the red blood cell lysis process, the interaction between the saponin in the lysis pad and the surfactant Tween 20 in the rinse solution promotes red blood cell lysis through a synergistic effect of hydrophobic interaction and membrane disruption, micelle formation and solubilization, effects on membrane proteins, and osmotic pressure. This mechanism is described in detail below:

[0063] Hydrophobic interactions and membrane disruption: Saponin is an amphiphilic molecule whose hydrophobic aglycone interacts with cholesterol in the erythrocyte membrane to form an insoluble complex. This interaction disrupts the erythrocyte membrane structure, increasing its permeability and creating defects such as pores. This allows substances within the erythrocyte, such as hemoglobin, to leak out, leading to erythrocyte lysis. The surfactant Tween 20 typically has both hydrophilic and hydrophobic groups. The hydrophobic groups can insert into the lipid bilayer of the erythrocyte membrane, reducing its stability and further enhancing its permeability. Together with saponin, they disrupt the integrity of the erythrocyte membrane and promote erythrocyte lysis.

[0064] Micelle formation and solubilization: The hydrophobic groups of the surfactant Tween 20 aggregate to form micelles. During red blood cell lysis, the micelles of the surfactant Tween 20 solubilize lipid molecules in the red blood cell membrane, disrupting the membrane structure and function and accelerating red blood cell lysis. Furthermore, the presence of saponins may affect the formation and properties of the micelles of the surfactant Tween 20, thereby altering its effect on the red blood cell membrane and further improving the efficiency of red blood cell lysis.

[0065] Effects on Membrane Proteins: The surfactant Tween 20 can also interact with proteins on the red blood cell membrane, altering their conformation and function. This interaction may lead to aggregation or denaturation of membrane proteins, further disrupting the normal physiological function of the red blood cell membrane and making it more susceptible to lysis. The presence of saponins may also indirectly affect the stability of membrane proteins, acting together with the surfactant to enhance the destructive effect on the red blood cell membrane and promote red blood cell lysis.

[0066] Synergistic Osmotic Pressure Effect: The synergistic effect of the surfactant Tween 20 and saponin in the lysis pad may also be related to the osmotic pressure effect. Surfactants can change the surface tension and osmotic pressure of the solution, increasing the osmotic pressure difference between the inside and outside of red blood cells. This causes water to quickly enter the red blood cells, causing them to swell and rupture. The destructive effect of saponin on the red blood cell membrane further increases the permeability of the cell membrane, making it easier for water to enter the cells and accelerating the swelling and lysis process.

[0067] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for simultaneous in vitro detection of multiple hemoglobins, characterized by: The steps include: (1) A test strip is prepared, the test strip comprising a substrate and a flushing pad, a labeling pad, a lysis pad, a reaction pad, and an absorption pad disposed on the substrate and contacting each other in sequence along a chromatography direction, wherein: The labeling pad is pre-coated with tracer-labeled anti-Hb antibodies. The lysis pad is pre-coated with dried saponin. The reaction pad is provided with a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the above-mentioned tracer-labeled anti-Hb antibody, and the several detection points are respectively coated with antibodies corresponding to the hemoglobin. (2) adding human whole blood or diluted whole blood sample to the reaction pad; (3) Add a sample rinse solution containing Tween 20 to the above-mentioned rinse pad, so that the sample rinse solution flows along the labeling pad, lysis pad and reaction pad to the absorption pad in sequence. After waiting for 4-6 minutes for the reaction to be complete, the test results are interpreted according to the color development status of the Ctl point and several detection points on the reaction pad.

2. The method for simultaneous in vitro detection of multiple hemoglobins according to claim 1, wherein: The tracer is nano metal particles or colored latex particles.

3. The method for simultaneous in vitro detection of multiple hemoglobins according to claim 1, wherein: The reaction pad is made of nitrocellulose.

4. The method for simultaneous in vitro detection of multiple hemoglobins according to claim 1, wherein: The Ctl point and the plurality of detection points are arranged in an array on the reaction pad.

5. The method for simultaneous in vitro detection of multiple hemoglobins according to any one of claims 1 to 4, characterized in that: The plurality of detection points include a point A, a point S and a point C. Point A is coated with anti-HbA antibodies, point S is coated with anti-HbS antibodies, and point C is coated with anti-HbC antibodies.

6. A test strip for simultaneous in vitro detection of multiple hemoglobins, characterized by: The invention comprises a substrate and a washing pad, a labeling pad, a lysis pad, a reaction pad and an absorption pad which are arranged on the substrate and contacted with each other in sequence along the chromatography direction, wherein: The labeling pad is pre-coated with tracer-labeled anti-Hb antibodies. The lysis pad is pre-coated with dried saponin. The reaction pad is provided with a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the above-mentioned tracer-labeled anti-Hb antibody, and the several detection points are respectively coated with antibodies corresponding to the hemoglobin.

7. The test strip for simultaneous in vitro detection of multiple hemoglobin levels according to claim 6, wherein: The tracer is nano metal particles or colored latex particles.

8. The test strip for simultaneous in vitro detection of multiple hemoglobin levels according to claim 6, wherein: The reaction pad is made of nitrocellulose.

9. The test strip for simultaneous in vitro detection of multiple hemoglobin levels according to claim 6, wherein: The Ctl point and the plurality of detection points are arranged in an array on the reaction pad.

10. The test strip for simultaneous in vitro detection of multiple hemoglobin levels according to any one of claims 6 to 9, characterized in that: The plurality of detection points include a point A, a point S and a point C. Point A is coated with anti-HbA antibodies, point S is coated with anti-HbS antibodies, and point C is coated with anti-HbC antibodies.

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