A multi-hemoglobin simultaneous in-vitro test strip

Abstract

The utility model discloses a variety of hemoglobin in vitro simultaneous detection test strip, including a substrate and set on the substrate and along the chromatography direction contact setting one washing pad, one mark pad, one lysis pad, one reaction pad and one absorption pad. The utility model discloses can detect HbA, HbS and HbC simultaneously and so on variety of hemoglobin, is applicable to the rapid screening and clinical diagnosis of abnormal hemoglobinopathy such as sickle cell anemia, and is simple to operate, and the cost is low.

Description

Technical Field

[0001] This utility model specifically relates to a test strip for simultaneous in vitro detection of multiple hemoglobins. Background Technology

[0002] Hemoglobin (Hb) is the core protein molecule in red blood cells responsible for oxygen transport. It is typically composed of four polypeptide chains, with normal adult hemoglobin (HbA) consisting of two α chains and two β chains, exhibiting a stable structure and complete function. However, mutations in globin genes can lead to abnormal hemoglobin molecular structure or impaired function, resulting in a series of hereditary diseases collectively known as abnormal hemoglobinopathies. Sickle cell disease (SCD) is a typical example, primarily caused by a mutation at the 6th amino acid position of the β-globin gene. For instance, when glutamate mutates to valine, HbS is formed, causing red blood cells to morphology into a sickle shape under hypoxic conditions, easily leading to serious consequences such as vascular occlusion, severe pain, infection, and organ damage. When glutamate mutates to lysine, HbC is formed, potentially causing mild anemia or exacerbating the condition in conjunction with other hemoglobin variants. Furthermore, 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 hemoglobinopathies are complex and diverse, and their severity is closely related to the type and amount of hemoglobin variants. Therefore, developing a rapid and accurate method for detecting multiple hemoglobin variants such as HbA, HbS, and HbC is not only crucial for the early screening and diagnosis of sickle cell disease, but also provides a scientific basis for genetic counseling, disease prevention, and the development of personalized treatment plans. With the continuous development of in vitro diagnostic technology, various techniques for hemoglobin detection have been developed. These methods each have their advantages and disadvantages in terms of sensitivity, specificity, and application scenarios, but they also have certain limitations. The following is a detailed analysis of the main types and characteristics of existing technologies:

[0004] 1. Electrophoresis: Electrophoresis is one of the earliest traditional methods used for hemoglobin detection. Its basic principle is to use an electric field to separate charged hemoglobin molecules in a medium such as a gel or filter paper based on differences in charge and molecular weight. Common electrophoresis methods include agarose gel electrophoresis and cellulose acetate membrane electrophoresis. These techniques are widely used in clinical laboratories to distinguish hemoglobin variants such as HbA, HbS, and HbC. The advantage of electrophoresis is its high resolution, which can clearly distinguish different types of hemoglobin, making it particularly suitable for analyzing complex samples. However, this method also has significant drawbacks: First, the operation is relatively cumbersome, requiring professionals in a laboratory environment using specialized electrophoresis equipment; second, the detection time is long, usually several hours, which cannot meet the needs of rapid screening; in addition, electrophoresis requires high sample quality, and impurities in the sample or environmental factors (such as temperature and humidity) may interfere with the separation effect, leading to unstable results; finally, result interpretation relies on the experience of technicians and has a certain degree of subjectivity. These limitations make electrophoresis technology difficult to adapt to the demands of modern medicine for efficient and convenient detection.

[0005] 2. High-Performance Liquid Chromatography (HPLC): HPLC is a high-precision analytical technique based on differences in molecular size and charge, and has gradually become the gold standard method for hemoglobin detection in recent years. HPLC separates hemoglobin components using specific ion-exchange or reversed-phase columns and combines this with a UV detector to quantitatively analyze the content of variants such as HbA, HbS, and HbC. This method has high sensitivity and good repeatability, providing accurate hemoglobin ratio data, and therefore has wide applications in clinical diagnosis and research. However, the disadvantages of HPLC cannot be ignored: its equipment is expensive, maintenance costs are high, and it is difficult to promote in resource-limited primary healthcare institutions; in addition, HPLC detection requires complex sample pretreatment steps, such as red blood cell lysis, centrifugation, and dilution, which not only increases the difficulty of operation but also prolongs the detection time, usually taking tens of minutes per test; at the same time, this method requires highly skilled technicians, and improper operation may lead to inaccurate results. Therefore, HPLC is more suitable for laboratory environments than for point-of-care testing (POCT) scenarios.

[0006] 3. Mass Spectrometry: Mass spectrometry, by detecting the mass-to-charge ratio of hemoglobin molecules, can accurately identify molecular weight and structural variations, making it an ideal tool for research analysis and the diagnosis of complex cases. For example, liquid chromatography-mass spectrometry (LC-MS) can resolve minute structural differences in hemoglobin, supporting the identification of rare variants. However, the application of mass spectrometry is limited in several ways: First, the equipment is extremely expensive and the operation is complex, limiting its use to high-end research institutions or large hospitals; second, mass spectrometry requires extremely high sample purity, necessitating multi-step purification processes, increasing the complexity of detection; furthermore, this method lacks high-throughput detection capabilities, making it difficult to meet the needs of routine clinical screening. Therefore, despite its significant value in scientific research, mass spectrometry has a low adoption rate in routine clinical testing.

[0007] 4. Genetic Detection Technology: Genetic detection technology uses methods such as polymerase chain reaction (PCR) or gene sequencing to directly analyze the sequence of globin genes, confirming the genetic basis of hemoglobin variations 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, thus having important applications in genetic counseling and prenatal diagnosis. However, genetic detection also has limitations: First, its testing cycle is long, usually requiring several hours to several days, making rapid diagnosis impossible; second, the equipment and reagent costs are high, making it unsuitable for large-scale screening; furthermore, this method only reflects information at the gene level and cannot directly detect the expression level or functional status of hemoglobin, thus limiting its application in clinical diagnosis.

[0008] 5. Lateral Flow Immunoassay (LFIA): In recent years, lateral flow immunoassay (LFIA) has received widespread attention in the field of point-of-care testing due to its advantages such as ease of operation, low cost, and intuitive results. Existing technologies have attempted to apply LFIA to hemoglobin detection, for example, by using specific antibodies to identify target hemoglobins and forming a visible test line on the test strip. Currently, common LFIA detection modes include competitive and sandwich methods. The competitive method achieves detection through the competitive binding of antigen and labeled antibody, but its sensitivity is low and it is prone to false negative results, especially in low-concentration samples. The sandwich method uses a "sandwich" binding of capture antibody and labeled antibody to bind the antigen, resulting in relatively high sensitivity, but under high antigen concentration conditions, it may trigger a hook effect, leading to signal weakening or even disappearance. Furthermore, existing LFIA test strips mostly use a single test line design, making it difficult to simultaneously detect multiple hemoglobins, and the detection process usually requires sample pretreatment (such as red blood cell lysis), increasing the operational steps. Existing LFIA methods may also affect the accuracy of results due to signal cross-interference when detecting complex samples.

[0009] In summary, existing hemoglobin detection technologies have the following main problems in practical applications:

[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 type of hemoglobin at a time and cannot detect multiple variants such as HbA, HbS, and HbC at the same time, resulting in long detection time and high cost.

[0012] 3. Insufficient sensitivity and accuracy: Although existing methods based on LFIA are easy to operate, the competitive method has low sensitivity, the sandwich method is easily affected by the hook effect, and the design of the detection area (such as a 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-scarce areas, while the performance of existing LFIA methods in high-throughput screening and complex sample detection is still not ideal.

[0014] These issues highlight the significant shortcomings of existing technologies in the rapid screening and early diagnosis of abnormal hemoglobinopathies, particularly in scenarios requiring rapid, convenient, and low-cost testing, where an efficient solution is lacking. Utility Model Content

[0015] The purpose of this invention is to overcome the defects of the existing technology and provide a test strip for simultaneous in vitro detection of multiple hemoglobins.

[0016] The technical solution of this utility model is as follows:

[0017] A multi-hemoglobin in vitro simultaneous detection test strip includes a substrate and a washing pad, a labeling pad, a lysis pad, a reaction pad, and an absorption pad disposed on the substrate and sequentially contacted along the chromatography direction.

[0018] The labeled pad is pre-coated with tracer-labeled anti-Hb antibodies.

[0019] The lysis pad is pre-coated with dried saponin.

[0020] The reaction pad has a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the anti-Hb antibody labeled by the above tracer, and the several detection points are coated with antibodies corresponding to the hemoglobin.

[0021] In a preferred embodiment of this invention, the tracer is a nano-metal particle or a colored latex particle.

[0022] More preferably, the tracer is nanocolloidal gold.

[0023] In a preferred embodiment of this invention, the reaction pad is a reaction pad made of nitrocellulose.

[0024] In a preferred embodiment of this invention, the Ctl point and the plurality of detection points are arranged in an array on the reaction pad.

[0025] In a preferred embodiment of this utility model, the plurality of detection points include a point A, a point S and a point C, point A is coated with anti-HbA antibody, point S is coated with anti-HbS antibody and point C is coated with anti-HbC antibody.

[0026] More preferably, the array is arranged in a spatial rhombus shape.

[0027] The beneficial effects of this utility model are:

[0028] 1. This utility model achieves in-situ lysis of samples by setting a lysis pad on the test strip, eliminating the need for pre-lysis treatment of the samples, simplifying the operation steps and improving the detection efficiency.

[0029] 2. This invention adopts a detection sequence of capture followed by labeling. Hemoglobin in the sample first binds to the capture antibody on the reaction membrane. Excess hemoglobin is washed into the absorption pad. Subsequently, the labeling 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 detection.

[0030] 3. This utility model adopts a dotted detection point design, in which the detection points are arranged in a row on the reaction membrane, reducing cross interference and spatial steric hindrance between detection points, and ensuring the independence and accuracy of the results.

[0031] 4. The detection process of this utility model only requires one sample addition, and the results can be interpreted within 5 minutes. No equipment assistance is required, the operation is simple, and it is suitable for various clinical scenarios.

[0032] 5. The test strip of this utility model has a simple structure, is easy to produce, and has low cost, making it suitable for areas with limited resources.

[0033] 6. The utility model test strip can be stored at room temperature, has a long shelf life, and is easy to store and transport. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the structure of the test strip for simultaneous in vitro detection of multiple hemoglobins in Embodiment 1 of this utility model. Detailed Implementation

[0035] The technical solution of this utility model will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0036] Example 1

[0037] 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 disposed on the substrate 10 and sequentially contacted along the chromatography direction.

[0038] The labeled pad 12 is a glass fiber pad pre-coated with a tracer-labeled anti-Hb antibody. In this embodiment, the tracer is preferably colloidal gold, meaning the tracer-labeled anti-Hb antibody is an anti-human Hb antibody-colloidal gold complex. The preparation method of the labeled pad 12 includes: rapidly adding anti-human hemoglobin monoclonal antibody at a ratio of 15-50 μg / mL to a colloidal silicon gold dispersion with a particle size of 20-60 nm and a concentration of 0.01-0.05 wt%, rapidly mixing, reacting for 4-15 min, then rapidly adding blocking buffer at a ratio of 10-15 μL / mL, reacting for 4-15 min, centrifuging at 8000-10000 rpm for 5-10 min, discarding the supernatant to obtain a precipitate, and adding reconstitution solution (3.9325 g Trizma) to the precipitate at a ratio of 100-400 μL / mL. The following ingredients were prepared: base, 2.86g sodium citrate, 1.739ml concentrated hydrochloric acid, 5g casein, 1g polyvinylpyrrolidone, 60g sucrose, 5g bovine serum albumin, and 1g sodium azide (to be diluted to 1L with ultrapure water). The mixture was ultrasonically reconstituted to obtain an anti-human Hb antibody-colloidal gold complex. The prepared anti-human Hb antibody-colloidal gold complex was evenly coated onto a glass fiber pad and dried at 25-40℃ for 2-6 hours to obtain labeling pad 12.

[0039] The pyrolysis pad 13 has a thickness of 0.2-0.5 mm and a basis weight of 60-90 g / m³. 2 A hydrophilic glass fiber pad, pre-coated with dried saponin. The preparation method of this lysis pad 13 includes: uniformly coating a 4-10 g / L saponin solution onto the hydrophilic glass fiber pad, with a volume-to-area ratio of approximately 0.5-0.8 mL / cm². 2 Dry at 25-45℃ for 2-6 hours to obtain pyrolysis pad 13.

[0040] The reaction pad 14 is a nitrocellulose membrane with a Ctl point 141 and three detection points (A point 142, S point 143 and C point) for detecting different hemoglobins. Ctl point 141 is coated with a protein that can bind to the tracer-labeled anti-Hb antibody. A point 142 is coated with anti-HbA antibody. S point 143 is coated with anti-HbS antibody. C point is coated with anti-HbC antibody. Ctl point 141, A point 142, S point 143 and C point 144 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 coating buffer and coating them onto points A142, S143 and C144 of a nitrocellulose membrane, and then drying them at a temperature of 20-32℃ and a humidity of <30% for 40 min-2 h; diluting goat anti-mouse IgG antibody to 1-3 mg / mL with coating buffer and coating it onto point Ctl 141 of a nitrocellulose membrane, and then drying it at a temperature of 20-32℃ and a humidity of <30% for 40 min-2 h.

[0041] The specific antibodies mentioned above are shown in the table below:

[0042]

[0043]

[0044] The specific process for using the in vitro simultaneous detection test strip for multiple hemoglobins prepared in this embodiment is as follows:

[0045] (1) Add 10-15 μL of human whole blood or diluted human whole blood sample to the reaction pad 14;

[0046] (2) Add 140 μL of sample rinsing solution (using 10 mM PBS as solvent and containing 0.3% Tween 20) to the rinsing pad 11 and let the sample rinsing solution flow gradually towards the absorption pad 15 along the chromatography direction.

[0047] (3) Wait 3-5 minutes for the interpretation results. Interpretations exceeding 15 minutes are invalid. Refer to the table below for interpretation guidelines:

[0048] Point A 142 S point 143 Point C 144 Ctl.141 Hb antigen Remark Color development No color development No color development Color development HbAA normal hemoglobin Color development Color development No color development Color development HbAS sickle cell phenotype or carrier Color development No color development Color development Color development HbAC Hemoglobin C traits or carriers No color development Color development No color development Color development HbSS sickle cell anemia No color development Color development Color development Color development HbSC sickle cell anemia and hemoglobin C disease No color development No color development Color development Color development HbCC Hemoglobin C disease Color development Color development Color development Color development Invalid test / / / / No color development Invalid test /

[0049] The detection principle of this embodiment is as follows: an immune complex (a complex consisting of anti-human Hb antibody, colloidal gold complex, hemoglobin antigen, and hemoglobin antibody) is formed at points A142, S143, C144, and Ctl141, which then develops color. Specifically, if point A142 develops color, it indicates the presence of HbA in the sample; if point S143 develops color, it indicates the presence of HbS in the sample; and if point C144 develops color, it indicates the presence of HbC in the sample. Point Ctl141 serves as a control point and must develop color; otherwise, the test strip is invalid.

[0050] When the test strip of this embodiment is used for simultaneous detection of HbA, HbS, and HbC, the red blood cells in the sample can be lysed through the lysis pad on the lysis pad 13, releasing hemoglobin. This reduces the need for lysis operations required in existing technologies. 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, reducing the risk of human error. It has high sensitivity, requires no equipment assistance, can be stored at room temperature, has a long shelf life, and is more suitable for rapid clinical blood typing scenarios.

[0051] Furthermore, existing test strips all involve adding the sample to the sample pad. When the antigen concentration is too high, excessive hemoglobin may not bind to the anti-human Hb antibody-colloidal gold complex and instead binds directly to the hemoglobin antibody on the reaction pad 14 along the chromatographic direction. This results in the hemoglobin antibody binding sites on the reaction pad 14 being occupied and unable to bind to the hemoglobin-anti-human Hb antibody-colloidal gold complex, thus leading to low sensitivity. This embodiment, however, uses a method of directly adding the sample to the reaction pad 14. The sample washing solution containing Tween 20, after passing through the labeling pad 12 and lysis pad 13, dissolves and mixes the anti-human Hb antibody-colloidal gold complex from the labeling pad 12 and the saponin from the lysis pad 13. Upon reaching the reaction pad 14, the red blood cells in the sample lyse under the synergistic effect of saponin and Tween 20, releasing hemoglobin. Different hemoglobins bind to the goat anti-mouse IgG antibody labeled at point Ctl 141, the anti-HbA antibody coated at point A 142, and the antibody at point S 142, respectively. The anti-HbS antibody coated at point 43 and the anti-HbC antibody coated at point C144 bind to form corresponding immune complexes. These immune complexes then bind to the anti-human Hb antibody-colloidal gold complex from the labeled pad 12 to form different anti-human Hb antibody-colloidal gold complex-hemoglobin antigen complexes. Excess unbound different hemoglobins will simultaneously chromatographically enter the absorbent pad 15. This solves the problem of excessive antigen in the test sample binding to the antibody on the reaction pad 14 first, resulting in weak or no color development, thus making the detection sensitivity higher.

[0052] In this embodiment, during erythrocyte lysis, the interaction between the saponins in the lysis pad and the surfactant Tween 20 in the rinsing solution promotes erythrocyte lysis through a synergistic effect of hydrophobic interactions, membrane structure disruption, micelle formation and solubilization, influence on membrane proteins, and osmotic pressure. This mechanism is explained in detail below:

[0053] Hydrophobic interactions and membrane structure disruption: Saponins are amphoteric molecules, with their aglycone portion being hydrophobic. They can interact with cholesterol in the erythrocyte membrane to form an insoluble complex. This interaction disrupts the structure of the erythrocyte membrane, leading to increased permeability and defects such as pores, allowing substances within the erythrocyte, such as hemoglobin, to leak out, thus causing erythrocyte lysis. The surfactant Tween 20 typically possesses both hydrophilic and hydrophobic groups. Its hydrophobic group can insert into the lipid bilayer of the erythrocyte membrane, reducing membrane stability and further enhancing membrane permeability. Together with saponins, it disrupts the integrity of the erythrocyte membrane, promoting erythrocyte lysis.

[0054] Micelle formation and solubilization: The hydrophobic groups of surfactant Tween 20 aggregate to form micelle structures. During erythrocyte lysis, Tween 20 micelles can solubilize lipid molecules in the erythrocyte membrane into the micelles, thereby disrupting the membrane structure and function and accelerating erythrocyte lysis. Simultaneously, the presence of saponins may also affect the formation and properties of Tween 20 micelles, thus altering their effect on the erythrocyte membrane and further increasing the erythrocyte lysis efficiency.

[0055] Effects on membrane proteins: The surfactant Tween 20 can also interact with proteins on the erythrocyte membrane, altering their conformation and function. This interaction may lead to the aggregation or denaturation of membrane proteins, further disrupting the normal physiological function of the erythrocyte membrane and making it more susceptible to lysis. The presence of saponins may also indirectly affect the stability of membrane proteins, working in conjunction with surfactants to enhance the damaging effect on the erythrocyte membrane and promote erythrocyte lysis.

[0056] Synergistic effect of osmotic pressure: In the lysis pad, the synergistic effect of surfactants Tween 20 and saponins 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 inside and outside the red blood cells, causing water to rapidly enter the red blood cells, leading to cell swelling and rupture. The disruptive effect of saponins on the red blood cell membrane further increases cell membrane permeability, making it easier for water to enter the cell and accelerating the swelling and lysis process of the red blood cells.

[0057] The above description is only a preferred embodiment of the present utility model, and therefore cannot be used to limit the scope of the present utility model. All equivalent changes and modifications made in accordance with the scope of the present utility model patent and the contents of the specification should still fall within the scope of the present utility model.

Claims

1. A test strip for simultaneous in vitro detection of multiple hemoglobins, characterized in that: It includes a substrate and a washing pad, a marking pad, a lysis pad, a reaction pad, and an absorption pad disposed on the substrate and sequentially contacted along the chromatography direction, wherein, The labeled pad is pre-coated with tracer-labeled anti-Hb antibodies. The lysis pad is pre-coated with dried saponin. The reaction pad has a Ctl point and several detection points for detecting different hemoglobins. The Ctl point is coated with a protein that can bind to the anti-Hb antibody labeled by the above tracer, and the several detection points are coated with antibodies corresponding to the hemoglobin.

2. The in vitro simultaneous detection test strip for multiple hemoglobins as described in claim 1, characterized in that: The tracer is a nano-metal particle or a colored latex particle.

3. The in vitro simultaneous detection test strip for multiple hemoglobins as described in claim 2, characterized in that: The tracer is nano-colloidal gold.

4. The in vitro simultaneous detection test strip for multiple hemoglobins as described in claim 1, characterized in that: The reaction pad is made of nitrocellulose.

5. The in vitro simultaneous detection test strip for multiple hemoglobins as described in claim 1, characterized in that: The Ctl point and the plurality of detection points are arranged in an array on the reaction pad.

6. A test strip for simultaneous in vitro detection of multiple hemoglobins as described in any one of claims 1 to 5, characterized in that: The detection points include point A, point S, and point C. Point A is coated with anti-HbA antibody, point S is coated with anti-HbS antibody, and point C is coated with anti-HbC antibody.

7. The in vitro simultaneous detection test strip for multiple hemoglobins as described in claim 6, characterized in that: The array is arranged in a spatial diamond shape.

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