Novel multi-combination lateral immune quantitative detection kit
By separating the control line and the test line on the lateral flow immunoassay strip and using the test line/control line ratio quantification method, the problems of cross-interference and quantitative instability in multi-item testing are solved, and efficient and accurate multi-target simultaneous detection is achieved.
Patent Information
- Application Number
- CN202520479876.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2035-03-19
AI Technical Summary
Traditional lateral flow immunoassay strips are prone to interference from reaction residues on the test line when performing multi-item tests, and the quantitative results are unstable, affecting the accuracy of the test.
The control line and the test line are separated and placed on both sides of the sample pad. The quantitative method of the test line/control line ratio is used. The total amount of marker is reflected by the control line, which reduces cross-interference and quantitative fluctuation between the test lines.
It has achieved improvements in the stability and accuracy of multi-item testing, with a 300% increase in testing efficiency, a 40% increase in quantitative stability, a false detection rate of less than 0.1%, an intra-batch coefficient of variation of ≤5%, and an inter-batch coefficient of variation of ≤8%.
Smart Images
Figure CN223940949U_ABST
Abstract
Description
Technical Field
[0001] This utility model relates to the field of immunoassay technology, specifically to a novel multi-purpose lateral flow immunoassay kit, suitable for lateral flow immunoassay devices that can simultaneously detect multiple targets. Background Technology
[0002] Traditional lateral flow immunoassay strips often employ a sandwich or competitive single-detection mode, with the control line typically located behind the test line. This makes the control signal susceptible to interference from residues remaining on the test line during chromatography, especially in multi-item assays where cross-influence exists between test lines. Furthermore, existing quantitative methods rely solely on the color intensity of the test line, but uneven distribution of labeled antibodies on the strip can lead to fluctuations in quantitative results, affecting detection stability. Therefore, a technical solution is urgently needed that enables simultaneous multi-item detection with stable quality control and accurate quantification. Utility Model Content
[0003] To address the aforementioned technical problems, this invention provides a novel multi-purpose unidirectional immunoassay kit. By separating the control line and the test line and placing them on opposite sides of the sample sheet, the kit can solve the problems of cross-interference and quantitative stability in multi-item detection through the test line / control line ratio quantification method, thereby achieving high-precision simultaneous detection of multiple targets.
[0004] This utility model provides a novel multi-component lateral immunoassay kit to solve the above-mentioned technical problems, comprising:
[0005] A microplate, wherein each well of the microplate is coated with freeze-dried gold label;
[0006] The test strip includes a base plate, a sample pad, a first chromatographic membrane, a second chromatographic membrane, an absorbent pad, and an absorbent pad 2. The sample pad is placed on the base plate. The first and second chromatographic membranes are respectively located on the left and right sides of the sample pad. The first absorbent pad is located on the left side of the first chromatographic membrane, and the second absorbent pad is located on the right side of the second chromatographic membrane. The first chromatographic membrane has a control line C coated with anti-antibody. The second chromatographic membrane has multiple detection lines coated with protein-coupled complete antigens corresponding to the detection items.
[0007] Furthermore, it also includes a bottom cover and a top cover, which together form a cavity for accommodating the test strip. The top cover is provided with a sample application hole corresponding to the sample pad, a detection window one corresponding to the first chromatographic membrane, and a detection window two corresponding to the second chromatographic membrane.
[0008] Furthermore, the gold-labeled lyophilized product contains homologous monoclonal antibodies labeled with colloidal gold or fluorescent microspheres for various detection items.
[0009] Furthermore, the first and second chromatographic membranes are made of nitrocellulose membranes, and the sample pad is made of glass cellulose or polyurethane material.
[0010] Furthermore, the detection lines include T1 line, T2 line, T3 line and T4 line, and the protein-coupled complete antigen on each detection line is a competitive antigen.
[0011] Furthermore, the detection lines are formed by diluting the BSA-conjugated complete antigen corresponding to the detection item in the gold-labeled lyophilized solution with PBS and sequentially coating it onto the second chromatography membrane. The T1 line, T2 line, T3 line and T4 line are parallel to each other and spaced 0.4 cm apart.
[0012] Furthermore, the quality control line C is formed by diluting the rabbit anti-mouse IgG anti-antibody to a suitable concentration with PBS and streaking it in the middle of the chromatography membrane at a concentration of 1.4 μl / cm.
[0013] Furthermore, the base plate is made of PVC material, with the left side of the first absorbent pad and the sample pad mounted on the first chromatography membrane by 1-2 mm, and the right side of the second absorbent pad and the sample pad mounted on the second chromatography membrane by 1-2 mm.
[0014] The beneficial effects of this invention are as follows: the gold-labeled lyophilized antibody in the microplate is a colloidal gold or fluorescent microsphere-labeled homologous monoclonal antibody, and each antibody can correspond to different detection items. The base plate of the test strip supports the chromatography membrane, sample pad, and absorbent pad. The sample pad is used for sample absorption and preliminary filtration. A control line C is set on chromatography membrane one, coated with anti-antibody, to capture the gold-labeled antibody and its conjugate with the target antigen in the sample, ensuring that the control signal only reflects the total amount of labeled substance. Multiple detection lines are set on chromatography membrane two, which can be set according to the number of detection items, allowing simultaneous detection of multiple items. The detection lines are coated with protein-coupled complete antigens corresponding to the detection items. When the gold-labeled antibody and its conjugate with the target antigen in the sample pass through the detection lines, they bind with the corresponding protein-coupled complete antigens and develop color. The absorbent pad provides the driving force for the chromatography of the sample. A standard curve is constructed based on the ratio of the color intensity of the detection line to the color intensity of the control line, reducing batch-to-batch differences and improving quantitative accuracy.
[0015] This invention places the control line C and the test line at opposite ends of the sample pad. During the reaction, the sample solution is split between the two sides. The control line C binds only to the various labeled monoclonal antibodies in the sample solution, unaffected by the test line or the target analyte in the sample. It is only related to the amount of the labeled analyte, forming a stable control line. Therefore, after chromatography, the color intensity of the test lines for different detection items is linear with respect to the control line, and they do not interfere with each other. This effectively simplifies the preparation mode for multi-item detection, increasing the efficiency of multi-item detection by more than 300%. At the same time, the method of quantitatively creating curves using the color intensity of the test line / control line effectively reduces the influence of the antibody label distribution on each test strip when using only the color intensity of the test line. The intra-batch coefficient of variation (CV) is ≤5%, and the inter-batch CV is ≤8%, which is 40% more stable than the traditional single-line quantitative method. The color stability of the control line reaches 99%, and the false detection rate is less than 0.1%, improving the accuracy and stability of detection.
[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description
[0017] Figure 1 This is a schematic diagram of the structure of the test strip of this utility model;
[0018] Figure 2 This is a schematic diagram of the structure of the top cover of this utility model.
[0019] In the attached diagram: 1-base plate, 2-sample pad, 3-chromatographic membrane one, 4-chromatographic membrane two, 5-absorbent pad one, 6-absorbent pad two, 7-control line C, 8-detection line, 9-top cover, 91-sample loading hole, 92-detection window one, 93-detection window two, 10-bottom cover. Detailed Implementation
[0020] Referring to the accompanying drawings, the specific embodiments of this utility model will be described in detail.
[0021] Reference Figures 1 to 2 This utility model provides a novel multi-component lateral immunoassay kit, comprising:
[0022] A microplate, wherein each well of the microplate is coated with gold-labeled lyophilized material; the gold-labeled lyophilized material contains homologous monoclonal antibodies labeled with colloidal gold or fluorescent microspheres for various detection items, and each antibody can correspond to different detection items.
[0023] Specifically, it uses the chloroauric acid reduction method to prepare colloidal gold labels containing monoclonal antibodies for four detectable substances: including detection items 1-4. The colloidal gold labels are mixed with buffer solution in a certain proportion, and blocking agents such as bovine serum albumin (BSA) are added to prevent non-specific adsorption. Unbound gold-labeled complexes are removed by centrifugation or ultrafiltration. The gold-labeled complex solution is then diluted to a suitable concentration and added to each well of a microplate. After freeze-drying, the microplate is quickly sealed in a dry inert gas environment such as nitrogen. A desiccant is added and the plate is stored away from light to prevent moisture absorption and deterioration.
[0024] The test strip includes a base plate 1, a sample pad 2, a first chromatographic membrane 3, a second chromatographic membrane 4, a first absorbent pad 5, and a second absorbent pad 6. The base plate 1 of the test strip in the microplate is used to support the chromatographic membrane, the sample pad 2, and the absorbent pad. The sample pad 2 is used for sample absorption and preliminary filtration, and the absorbent pad is used to provide driving force for the chromatography of the sample solution.
[0025] The sample pad 2 is disposed on the base plate 1. The first chromatographic membrane 3 and the second chromatographic membrane 4 are respectively disposed on the left and right sides of the sample pad 2. The first absorbent pad 5 is disposed on the left side of the first chromatographic membrane 3, and the second absorbent pad 6 is disposed on the right side of the second chromatographic membrane 4. Further, the first chromatographic membrane 3 and the second chromatographic membrane 4 are made of nitrocellulose membrane, and the sample pad 2 is made of glass cellulose or polyurethane material. The base plate 1 is made of PVC material. The first absorbent pad 5 and the left side of the sample pad 2 are mounted on the first chromatographic membrane 3 by 1-2 mm, and the second absorbent pad 6 and the right side of the sample pad 2 are mounted on the second chromatographic membrane 4 by 1-2 mm.
[0026] The first chromatographic membrane 3 has a control line C7 coated with anti-antibody to capture the gold-labeled antibody and its conjugate with the target antigen in the sample solution, ensuring that the control signal only reflects the total amount of the label. The second chromatographic membrane 4 has multiple detection lines 8 coated with protein-coupled complete antigens corresponding to the detection items. The number of detection items can be set according to the number of detection items, allowing simultaneous detection of multiple items. When the gold-labeled antibody and its conjugate with the target antigen in the sample solution flow through the detection line 8, they bind with the corresponding protein-coupled complete antigen and develop color. In this embodiment, there are four detection lines 8, which can simultaneously detect four detection items. The detection lines 8 include T1 line, T2 line, T3 line, and T4 line. The protein-coupled complete antigen on each detection line 8 is a competitive antigen, allowing simultaneous detection of multiple items without interference between the competing reactions.
[0027] Specifically, chromatography membrane 3 and chromatography membrane 4 are adhered to the base plate 1 on both sides of the sample pad 2. Rabbit anti-mouse IgG anti-antibody is diluted to a suitable concentration with PBS and streaked in the middle of chromatography membrane 3 at a concentration of 1.4 μl / cm. Then, BSA-conjugated complete antigen corresponding to the detection item in the gold-labeled lyophilized solution is diluted with PBS and sequentially coated onto chromatography membrane 4, wherein the T1, T2, T3, and T4 lines are parallel to each other and spaced 0.4 cm apart. The coated chromatography membranes are then dried in a 41°C forced-air drying oven for 24 hours to ensure that the anti-antibody and antigen are firmly adsorbed onto the membrane surface.
[0028] Furthermore, it also includes a bottom cover 10 and an upper cover 9, which together form a cavity for accommodating the test strip. The upper cover 9 is provided with a sample application hole 91 corresponding to the sample pad 2, a detection window 92 corresponding to the first chromatographic membrane 3, and a detection window 93 corresponding to the second chromatographic membrane 4. The sample solution is added through the sample application hole 91, the first detection window 92 is used to observe the color development of the control line C7, and the second detection window 93 is used to observe the color development of the detection line 8.
[0029] The sample to be tested was crushed, extracted, diluted and mixed, and then dropped into the gold-labeled microplate wells to fully bind with the gold-labeled antibody. The mixture was then dropped into sample well 91. After reacting for 15 minutes, the gold-labeled antibody and its conjugate with the target antigen in the mixture flowed through the left control line C7 via chromatography, where the gold-labeled antibody bound to rabbit anti-mouse IgG and showed color. The right-end chromatography solution moved to the detection lines 8T1-T4 and bound to their corresponding antigens and showed color. The fluorescence signal intensity of the T1-T4 lines and the C line was then read. The T / C ratios were calculated and substituted into the pre-calibrated standard curve to obtain the concentration of the target substance.
[0030] By employing this invention, the control line C7 and the detection line 8 are placed at opposite ends of the sample pad 2. During the reaction, the sample solution is split between the two sides. The control line C7 binds only to the various labeled monoclonal antibodies in the sample solution and is unaffected by the detection line 8 or the target analyte in the sample. It is only related to the amount of the labeled analyte, forming a stable control line. Therefore, after chromatography, the color intensity of the detection line 8 for different detection items is linear with respect to the control line and does not interfere with each other. This effectively simplifies the preparation mode for multi-item detection. At the same time, since the method of quantitatively preparing the curve using the color intensity of the detection line 8 with the control line is adopted, the influence of the amount of antibody labeled analyte on each test strip is effectively reduced when only the color intensity of the detection line 8 is used to prepare the curve, thus improving the accuracy and stability of the detection.
[0031] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.
[0032] The above description is merely a preferred embodiment of the present utility model and is not intended to limit the present utility model. Although the present utility model has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present utility model should be included within the protection scope of the present utility model.
Claims
1. A novel multi-synthetic lateral immunoassay kit, characterized in that, include: A microplate, wherein each well of the microplate is coated with freeze-dried gold label; The test strip includes a base plate (1), a sample pad (2), a first chromatographic membrane (3), a second chromatographic membrane (4), an absorbent pad (5), and an absorbent pad (6). The sample pad (2) is placed on the base plate (1). The first chromatographic membrane (3) and the second chromatographic membrane (4) are respectively placed on the left and right sides of the sample pad (2). The first absorbent pad (5) is placed on the left side of the first chromatographic membrane (3), and the second absorbent pad (6) is placed on the right side of the second chromatographic membrane (4). The first chromatographic membrane (3) has a control line C (7) coated with anti-antibody. The second chromatographic membrane (4) has multiple detection lines (8) coated with protein-coupled complete antigens corresponding to the detection items.
2. The novel multi-synthetic unidirectional immunoassay kit according to claim 1, characterized in that, It also includes a bottom cover (10) and a top cover (9), which together form a cavity for accommodating the test strip. The top cover (9) is provided with a sample application hole (91) corresponding to the sample pad (2), a detection window (92) corresponding to the first chromatographic membrane (3), and a detection window (93) corresponding to the second chromatographic membrane (4).
3. The novel multi-synthetic unidirectional immunoassay kit according to claim 1, characterized in that, The gold-labeled lyophilized products contain homologous monoclonal antibodies labeled with colloidal gold or fluorescent microspheres for various detection items.
4. The novel multi-synthetic unidirectional immunoassay kit according to claim 1, characterized in that, The first chromatographic membrane (3) and the second chromatographic membrane (4) are made of nitrocellulose membrane, and the sample pad (2) is made of glass cellulose or polyurethane material.
5. The novel multi-synthetic lateral immunoassay kit according to claim 1, characterized in that, The detection lines (8) include T1 line, T2 line, T3 line and T4 line, and the protein-coupled complete antigen on each detection line (8) is a competitive antigen.
6. The novel multi-synthetic unidirectional immunoassay kit according to claim 5, characterized in that, The detection line (8) is formed by diluting the BSA-conjugated complete antigen corresponding to the detection item in the gold-labeled lyophilized solution with PBS and sequentially coating it onto the chromatography membrane (4). The T1 line, T2 line, T3 line and T4 line are parallel to each other and spaced 0.4 cm apart.
7. The novel multi-synthetic lateral immunoassay kit according to claim 1, characterized in that, The quality control line C(7) is formed by diluting rabbit anti-mouse IgG anti-antibody to a suitable concentration with PBS and streaking it in the middle of the chromatography membrane (3) at a concentration of 1.4 μl / cm.
8. The novel multi-synthetic lateral immunoassay kit according to claim 1, characterized in that, The base plate (1) is made of PVC material. The left side of the absorbent pad (5) and the sample pad (2) are mounted on the first chromatography membrane (3) by 1-2 mm, and the right side of the absorbent pad (6) and the sample pad (2) are mounted on the second chromatography membrane (4) by 1-2 mm.