Serum amyloid protein A determination kit

Abstract

The utility model relates to a serum amyloid protein A determination kit. The kit comprises a shell and a test strip, the test strip comprises a support plate, a sample pad, a combination pad, a reaction film and absorbent paper, the shell comprises an upper cover and a lower cover, and the upper cover is provided with a sample adding hole and an observation window; colloidal carbon marked by a first serum amyloid protein A monoclonal antibody is sprayed on the combination pad; the reaction film is provided with a detection line and a quality control line, the detection line is sprayed with a second serum amyloid protein A monoclonal antibody, and the quality control line is sprayed with a goat-anti-mouse second antibody; the sample pad, the combination pad, the reaction film and the absorbent paper are fixed on the surface of the support plate; the conjugate pad is overlapped with one end, close to the detection line, of the reaction film, the sample pad is overlapped with the other end of the conjugate pad, the absorbent paper is overlapped with one end, close to the quality control line, of the reaction film, and all overlapping distances are within a preset distance range; the test strip is fixed in the shell; the sample adding hole is formed above the sample pad; the observation window is arranged above the reaction film and is large enough to expose the detection line and the quality control line on the reaction film, so that the measurement sensitivity is improved.

Description

Technical Field

[0001] This application relates to the field of medical device technology, and in particular to a serum amyloid A assay kit. Background Technology

[0002] Infectious diseases such as respiratory infections, gastrointestinal infections, and post-traumatic infections are highly susceptible to missed or misdiagnosis if diagnosed solely based on patient symptoms and imaging findings without the use of inflammatory markers. Serum amyloid A (SAA), a novel inflammatory marker, is a protein primarily synthesized by the liver, composed of 103 to 104 amino acids. It plays a crucial role in pathophysiological processes such as inflammatory responses, immune regulation, intercellular signaling, and lipid metabolism. Plasma concentrations peak within 4 to 6 hours of the initial inflammatory phase and gradually decline as inflammation subsides. In acute infections, particularly bacterial and viral infections, serum A levels are significantly elevated in the early stages of disease. Serum A has also been found to be involved in the occurrence, development, diagnosis, and prognosis of chronic non-biological inflammatory diseases such as diabetes, coronary heart disease, rheumatic immune diseases, tumors, and transplant rejection. However, serum amyloid A originates from a variety of cell types and involves a wide range of diseases, resulting in a lack of specificity in clinical applications. But with the continuous advancement of testing technology, the detection and dynamic monitoring of serum amyloid A will become more convenient.

[0003] Currently known kits for detecting serum amyloid A have significant drawbacks, such as insufficient sensitivity and high cost. Utility Model Content

[0004] Therefore, it is necessary to provide a serum amyloid A assay kit to address the aforementioned technical problems, thereby resolving issues such as insufficient sensitivity and high cost of serum amyloid A assay kits in related technologies.

[0005] This application provides a serum amyloid A assay kit, which includes an outer shell and a test strip for detecting serum amyloid A; the test strip includes a support plate, a sample pad, a conjugation pad, a reaction membrane, and absorbent paper; the outer shell includes an upper cover and a lower cover, and the upper cover is provided with a sample application hole and an observation window;

[0006] The conjugation pad is coated with colloidal carbon labeled with a first serum amyloid A monoclonal antibody;

[0007] The reaction membrane is provided with a detection line and a control line. The detection line is coated with a second serum amyloid A monoclonal antibody, and the control line is coated with goat anti-mouse secondary antibody.

[0008] The sample pad, the conjugate pad, the reaction membrane, and the absorbent paper are sequentially fixed to the surface of the support plate; the conjugate pad overlaps with one end of the reaction membrane near the detection line, and the overlap distance between the conjugate pad and the reaction membrane is within a preset distance range; the sample pad overlaps with the other end of the conjugate pad, and the overlap distance between the sample pad and the conjugate pad is within the preset distance range; the absorbent paper overlaps with one end of the reaction membrane near the quality control line, and the overlap distance between the absorbent paper and the reaction membrane is within the preset distance range.

[0009] The test strip is fixed inside the outer casing; the sample application port is located above the sample pad; the observation window is located above the reaction membrane, and the size of the observation window is sufficient to expose the detection line and the control line on the reaction membrane.

[0010] In one embodiment, the lower cover is provided with a slot for fixing the test strip.

[0011] In one embodiment, the reaction membrane is a nitrocellulose membrane.

[0012] The aforementioned serum amyloid A assay kit includes an outer casing and a test strip for serum amyloid A. The test strip includes a support plate, a sample pad, a conjugate pad, a reaction membrane, and absorbent paper. The outer casing includes a top cover and a bottom cover, with the top cover having a sample application well and an observation window. The conjugate pad is coated with colloidal carbon labeled with a first serum amyloid A monoclonal antibody. The reaction membrane has a detection line and a control line; the detection line is coated with a second serum amyloid A monoclonal antibody, and the control line is coated with goat anti-mouse secondary antibody. The kit also includes a sample pad, a conjugate pad, a reaction membrane, and absorbent paper. The components are sequentially fixed to the surface of the support plate; the conjugate pad overlaps with one end of the reaction membrane near the detection line, with the overlap distance between the conjugate pad and the reaction membrane within a preset range; the sample pad overlaps with the other end of the conjugate pad, with the overlap distance between the sample pad and the conjugate pad within a preset range; the absorbent paper overlaps with one end of the reaction membrane near the control line, with the overlap distance between the absorbent paper and the reaction membrane within a preset range; the test strip is fixed inside the outer casing; the sample application port is located above the sample pad; the observation window is located above the reaction membrane, and the size of the observation window is sufficient to expose the detection line and control line on the reaction membrane. In this application, the sample solution added to the sample pad from the sample application port is chromatographically deposited towards the absorbent paper by capillary action under the drag of the absorbent paper. This application enhances the chromatographic efficiency between the various structures on the support plate by allowing the various structures on the support plate to overlap by a certain distance, thereby effectively improving the detection sensitivity. On the other hand, this application improves the assay sensitivity by spraying colloidal carbon labeled with a first serum amyloid A monoclonal antibody onto the conjugate pad, whereas commercially available serum amyloid A assay kits typically use colloidal gold labeled with a serum amyloid A monoclonal antibody. Colloidal carbon offers higher color intensity and labeling efficiency than colloidal gold. Furthermore, colloidal carbon is less expensive to manufacture than colloidal gold, thus effectively reducing the cost of the serum amyloid A assay kit. Attached Figure Description

[0013] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0014] Figure 1 This is a schematic diagram of the structure of the reagent strip in the serum amyloid A assay kit provided according to the embodiments of this application. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this application clearer, the application is described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0016] Obviously, the accompanying drawings described below are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0017] In this application, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0018] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms “a,” “an,” “an,” “the,” and similar words used in this application do not indicate quantity limitation and may indicate singular or plural. The terms “comprising,” “including,” “having,” and any variations thereof used in this application are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or device that includes a series of steps or modules (units) is not limited to the listed steps or units, but may also include steps or units not listed, or may include other steps or units inherent to these processes, methods, products, or devices. The terms “connected,” “linked,” “coupled,” and similar words used in this application are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. “Multiple” used in this application refers to two or more. “And / or” describes the relationship between related objects, indicating that three relationships may exist; for example, “A and / or B” can represent: A alone, A and B simultaneously, and B alone. The character " / " generally indicates that the preceding and following objects are in an "or" relationship. The terms "first," "second," and "third" used in this application are merely to distinguish similar objects and do not represent a specific ordering of the objects.

[0019] This application provides a serum amyloid A assay kit, which includes a shell and, as shown in the example, a serum amyloid A assay kit. Figure 1 The test strip shown is for detecting serum amyloid A. The outer casing includes an upper cover and a lower cover, with the upper cover having a sample application hole and an observation window. The test strip includes a support plate 11, a sample pad 12, a conjugate pad 13, a reaction membrane 14, and absorbent paper 15. The conjugate pad 13 is coated with colloidal carbon labeled with a first serum amyloid A monoclonal antibody. The reaction membrane 14 has a detection line 16 and a control line 17. The detection line 16 is coated with a second serum amyloid A monoclonal antibody, and the control line 17 is coated with goat anti-mouse secondary antibody. The sample pad 12, conjugate pad 13, reaction membrane 14, and absorbent paper 15 are sequentially fixed to the surface of the support plate 11. The conjugate pad 13 overlaps with one end of the reaction membrane 14 near the detection line 16, and the overlap distance between the conjugate pad 13 and the reaction membrane 14 is within a preset distance range; the sample pad 12 overlaps with the other end of the conjugate pad 13, and the overlap distance between the sample pad 12 and the conjugate pad 13 is within a preset distance range; the absorbent paper 15 overlaps with one end of the reaction membrane 14 near the control line 17, and the overlap distance between the absorbent paper 15 and the reaction membrane 14 is within a preset distance range; the test strip is fixed inside the outer casing; the sample application port is located above the sample pad 12; the observation window is located above the reaction membrane 14, and the size of the observation window is sufficient to expose the detection line 16 and the control line 17 on the reaction membrane 14.

[0020] In this application, the support plate 11 can support the sample pad 12, the conjugate pad 13, the reaction membrane 14 and the absorbent paper 15. Specifically, the support plate 11 can be a PVC board or a PVA board.

[0021] Alternatively, polyester cellulose membrane can be used as the raw material for conjugation pad 13. The conjugation pad 13 is coated with colloidal carbon labeled with a monoclonal antibody against first serum amyloid A. Specifically, the conjugation pad 13 is manufactured as follows:

[0022] Step 1: Take 1 mL of colloidal carbon solution, add 10 μg of first serum amyloid A monoclonal antibody and mix evenly. Mix at room temperature for 3 hours to allow the first serum amyloid A monoclonal antibody to fully bind to the colloidal carbon, thus completing the labeling of the first serum amyloid A monoclonal antibody.

[0023] Step 2: Add 30 μL of 1% BSA (bovine serum albumin) to the labeled solution obtained in Step 1 to make the BSA concentration 0.03%, and block at room temperature for 30 minutes to block the exposed sites on the colloidal carbon.

[0024] Step 3: Centrifuge the solution obtained in Step 2, discard the supernatant, and resuspend it in a reconstitution solution to a volume of 50 μL. The reconstitution solution may consist of Tris, BSA, sucrose, Triton 100, PEG, and PC300, with the following concentrations: Tris concentration 0.2 mol / L, BSA concentration 1.5%, sucrose concentration 10%, Triton 100 concentration 0.5%, PEG concentration 0.5%, and PC300 concentration 0.2%. Tris in the reconstitution solution acts as a buffer to adjust the ionic strength and pH of the solution. Tris can be replaced by PB (phosphate buffer), BUP (citric acid), carbonate, or borax, which have the same function. BSA in the reconstitution solution acts as an inert protein to protect the protein activity of the solution and maintain colloidal stability. BSA can be replaced by OVA (ovalbumin) or Casein Na, which have the same function. Sucrose in the reconstitution solution protects the protein activity of the solution and improves stability. Sucrose can be replaced by trehalose, glycerol, and various oligosaccharides, which have the same function. Triton 100 in the reconstitution solution acts as an surfactant to improve hydrophilicity and color enhancement. Triton 100 can be replaced by surfactants such as TW20, S9, S5, S17, and S22. PEG in the reconstitution solution acts as a macromolecular polymer to make the molecules in the solution uniformly dispersed. It can be replaced by PVP or PVA, which have the same function. PC300 in the reconstitution solution acts as a preservative. PC300 can be replaced by NaN3, which has the same function.

[0025] Step 4: Coat the conjugate pad 13 with a conjugate pad treatment solution. The conjugate pad treatment solution can be composed of Tris, S9, PEG, BSA, sucrose, and trehalose. The concentration of Tris is 0.01 mol / L, the concentration of S9 is 0.5%, the concentration of PEG is 0.5%, the concentration of BSA is 1%, the concentration of sucrose is 5%, and the concentration of trehalose is 2%. The functions of each component in the conjugate pad treatment solution are as described above. Similarly, other solutions with the same function can be used to replace the components in the conjugate pad treatment solution. Then, the solution obtained in Step 3 is sprayed onto the conjugate pad 13 treated with the conjugate pad treatment solution using a three-dimensional etching and gold spraying machine, and then dried in a forced-air drying oven at 37°C for 12 hours.

[0026] In this application, glass fiber can be used as the raw material for sample pad 12. The sample pad 12 is manufactured as follows: the sample pad 12 is coated with a sample pad treatment solution, which can be composed of Tris, PVP, and Ficoll70 (sucrose 70). The concentration of Tris is 0.01 mol / L, the concentration of PVP is 0.5%, and the concentration of Ficoll70 is 0.5%. Tris acts as a buffer to adjust the ionic strength and pH of the solution; PVP acts as a macromolecular polymer to ensure uniform molecular distribution in the solution; and Ficoll70 acts as an inert protein to protect protein activity. Similarly, other solutions with the same function can be used to replace the components in the sample pad treatment solution.

[0027] In addition, in this application, nitrocellulose membrane can be used as the raw material for reaction membrane 14. The reaction membrane 14 is prepared as follows: Second serum amyloid A monoclonal antibody is diluted to a concentration of 0.15 mg / mL with coating buffer, and goat anti-mouse secondary antibody is diluted to a concentration of 0.25 mg / mL. Then, the second serum amyloid A monoclonal antibody diluted with coating buffer is streaked onto reaction membrane 14 using a three-dimensional streaking and gold spraying device to form a detection line 16, and the diluted goat anti-mouse secondary antibody is streaked onto reaction membrane 14 using a three-dimensional streaking and gold spraying device to form a control line 17. The coating buffer can be composed of PB, NaCl, sucrose, and PC300. PB acts as a buffer to adjust the ionic strength and pH of the solution; NaCl further adjusts the ionic strength of the solution; sucrose protects the protein activity of the solution and improves stability; and PC300 acts as a preservative. Similarly, other solutions with the same function can be used to replace the components in the coating buffer.

[0028] The test strip 10 is manufactured as follows: A reaction membrane 14 is fixed to a support plate 11; a conjugate pad 13 is fixed to the reaction membrane 14 near the detection line 16, with the conjugate pad 13 overlapping the reaction membrane 14, and the overlap distance between the conjugate pad 13 and the reaction membrane 14 is within a preset distance range; a sample pad 12 is fixed to the other end of the conjugate pad 13, with the sample pad 12 overlapping the conjugate pad 13, and the overlap distance between the sample pad 12 and the conjugate pad 13 is within a preset distance range; absorbent paper 15 is fixed to the reaction membrane 14 near the control line 17, with the absorbent paper 15 overlapping the reaction membrane 14, and the overlap distance between the absorbent paper 15 and the reaction membrane 14 is within a preset distance range. In one embodiment, the preset distance range is 1 mm to 2 mm.

[0029] The prepared test strip is fixed inside the outer casing, thus becoming the serum amyloid A assay kit provided in this application. In one embodiment, the upper and lower covers of the serum amyloid A assay kit provided in this application can be connected via slots and pins. Alternatively, the test strip can be fixed inside the outer casing by adhesive, or a matching slot can be provided on the lower cover to fix the test strip in the slot of the lower cover.

[0030] It should be noted that the second serum amyloid A monoclonal antibody and the first serum amyloid A monoclonal antibody in this application bind to different sites with the serum amyloid A antigen. Therefore, the serum amyloid A antigen can bind to both the first serum amyloid A monoclonal antibody and the second serum amyloid A monoclonal antibody simultaneously. After the sample to be tested is added to the sample pad 12 through the sample well, the sample is drawn towards the absorbent paper 15 by capillary action and undergoes chromatography, passing sequentially through the binding pad 13, the reaction membrane 14, and the absorbent paper 15. When serum amyloid A antigen is present in the sample to be tested, the serum amyloid A antigen binds to the colloidal carbon labeled with the first serum amyloid A monoclonal antibody on the conjugate pad 13. The serum amyloid A antigen bound to the colloidal carbon labeled with the first serum amyloid A monoclonal antibody then binds to the second serum amyloid A monoclonal antibody on the test line 16, making the colloidal carbon on the test line 16 appear black. Since the control line 17 is coated with goat anti-mouse secondary antibody, the colloidal carbon labeled with the first serum amyloid A monoclonal antibody on the control line 17 continues to chromatographically binds to the goat anti-mouse secondary antibody, also appearing black. When the sample to be tested does not contain serum amyloid A antigen, without serum amyloid A antigen as an intermediate medium, the colloidal carbon labeled with the first serum amyloid A monoclonal antibody cannot bind to the second serum amyloid A monoclonal antibody on detection line 16, resulting in no color development on detection line 16. However, since goat anti-mouse secondary antibody is sprayed onto control line 17, the colloidal carbon labeled with the first serum amyloid A monoclonal antibody on control line 17 continues to precipitate and binds to the goat anti-mouse secondary antibody, appearing black. That is, when serum amyloid A antigen is present in the sample to be tested, both detection line 16 and control line 17 appear black; when serum amyloid A antigen is absent in the sample to be tested, detection line 16 does not develop color, and control line 17 appears black, thus enabling the detection of serum amyloid A antigen. It is understood that the distance between detection line 16 and control line 17 in this application needs to exceed the distance that can be distinguished by the naked eye.

[0031] In this application, the sample solution added to the sample pad from the sample well is chromatographically deposited towards the absorbent paper by capillary action, dragged along by the absorbent paper. By overlapping the various structures on the support plate by a certain distance, the chromatographic efficiency between the structures on the support plate is enhanced, thereby effectively improving the detection sensitivity. In commercially available serum amyloid A assay kits, the conjugate pad is coated with colloidal gold labeled with serum amyloid A monoclonal antibody, and the control and test lines appear red, resulting in a red-white contrast on the test strip. In this application, colloidal carbon labeled with a first serum amyloid A monoclonal antibody is sprayed onto the conjugate pad, resulting in a black control and test line, resulting in a black-and-white contrast on the test strip. Compared to red-and-white contrast, black-and-white contrast is more pronounced, indicating that colloidal carbon has a higher color intensity than colloidal gold. Furthermore, compared to colloidal gold, colloidal carbon has more binding sites with serum amyloid A antibodies, resulting in higher labeling efficiency and effectively improving the detection sensitivity of the serum amyloid A assay kit in this application. In addition, colloidal carbon is cheaper to produce than colloidal gold, thus effectively reducing the cost of serum amyloid A assay kits.

[0032] It should be understood that the specific embodiments described herein are merely illustrative of the application and not intended to limit it. All other embodiments derived by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0033] Obviously, the accompanying drawings are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar situations based on these drawings without any creative effort. Furthermore, it is understood that although the work done in this development process may be complex and lengthy, for those skilled in the art, certain design, manufacturing, or production modifications made based on the technical content disclosed in this application are merely conventional technical means and should not be considered as insufficient disclosure of this application.

[0034] The term "embodiment" in this application refers to a specific feature, structure, or characteristic described in connection with an embodiment that may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily imply the same embodiment, nor does it imply that it is mutually exclusive with or independent of other embodiments. It will be clearly or implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0035] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the appended claims.

Claims

1. A serum amyloid A assay kit, characterized by comprising: The kit comprises a shell and a test strip for detecting serum amyloid A; the test strip comprises a support plate, a sample pad, a binding pad, a reaction membrane and an absorbent paper; the shell comprises an upper cover and a lower cover, the upper cover is provided with a sample hole and an observation window; The binding pad is sprayed with colloidal carbon marked with a first serum amyloid A monoclonal antibody; The reaction membrane is provided with a detection line and a quality control line, the detection line is sprayed with a second serum amyloid A monoclonal antibody, and the quality control line is sprayed with a goat anti-mouse secondary antibody; The sample pad, the binding pad, the reaction membrane and the absorbent paper are fixed on the surface of the support plate in sequence; the binding pad overlaps with one end of the reaction membrane close to the detection line, and the overlapping distance of the binding pad and the reaction membrane is within a preset distance range; the sample pad overlaps with the other end of the binding pad, and the overlapping distance of the sample pad and the binding pad is within the preset distance range; the absorbent paper overlaps with one end of the reaction membrane close to the quality control line, and the overlapping distance of the absorbent paper and the reaction membrane is within the preset distance range; The test strip is fixed in the shell; the sample hole is located above the sample pad; the observation window is located above the reaction membrane, and the size of the observation window is sufficient to expose the detection line and the quality control line on the reaction membrane.

2. The serum amyloid A assay kit according to claim 1, characterized in that, The lower cover is provided with a clamping groove for fixing the test strip.

3. The serum amyloid A assay kit according to claim 1, characterized in that, The reaction membrane is a nitrocellulose membrane.

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