A rapid human interleukin-4 detection card based on a double antibody sandwich method

Abstract

This invention discloses a rapid human interleukin-4 test strip based on a double-antibody sandwich method. The test strip includes a sample pad, a latex pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The latex pad is made of latex microspheres labeled with CKIgY monoclonal antibody and red polystyrene latex microspheres. The conjugate pad is made of latex microspheres labeled with IL-mouse monoclonal antibody and red polystyrene latex microspheres. A detection line and a control line are located in the center of the nitrocellulose membrane. The detection line contains a protein conjugate for binding with the IL-mouse monoclonal antibody, and the control line contains an IgG monoclonal antibody. An upper shell is located on the outside of the test strip, and a matching lower shell is provided. This invention provides a rapid human interleukin-4 test strip based on a double-antibody sandwich method. This test strip has high sensitivity, low risk of cross-reaction, simple operation, and low cost.

Description

Technical Field

[0001] This utility model relates to an immunochromatographic reagent strip for human interleukin-4 based on a double-antibody sandwich method, which is applicable to the field of qualitative or semi-quantitative detection technology of interleukin-4 in samples such as serum, plasma, and cell culture supernatant. Specifically, it is a rapid detection card for human interleukin-4 based on a double-antibody sandwich method. Background Technology

[0002] Interleukin-4 is a key cytokine secreted by Th2 cells, involved in immune regulation, inflammatory responses, and the development of allergic diseases. Current methods for detecting interleukin-4 primarily use ELISA, which requires specialized equipment and takes 4-6 hours, failing to meet the needs of rapid clinical screening. While chemiluminescence immunoassays offer high sensitivity, the equipment is expensive and the operation is complex. Therefore, colloidal gold test strips have emerged, which can also perform detection and screening at a relatively lower cost. However, the aforementioned common detection devices still have shortcomings:

[0003] While traditional colloidal gold test strips are fast, they suffer from insufficient sensitivity and the risk of cross-reaction, which can easily lead to inaccurate test results, causing misunderstandings and harm to users. Furthermore, the traditional casing is usually installed by multiple clips, which requires aligning each clip to secure it, wasting time and affecting production efficiency. Utility Model Content

[0004] The purpose of this invention is to provide a rapid human interleukin-4 test strip based on a double-antibody sandwich method, in order to solve the problems mentioned in the background art. Although traditional colloidal gold test strips are fast, they have insufficient sensitivity, risk of cross-reaction, and are prone to inaccurate test results, which can lead to erroneous perceptions and harm to users. In addition, the traditional shell installation usually involves multiple clips that need to be aligned to be connected, which is time-consuming and affects production efficiency.

[0005] To achieve the above objectives, this utility model provides the following technical solution: a rapid human interleukin-4 detection card based on a double-antibody sandwich method, comprising a detection strip, wherein the detection strip includes a sample pad, a latex pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The latex pad is made of latex microspheres labeled with CKIgY monoclonal antibody and red polystyrene latex microspheres. The conjugate pad is made of latex microspheres labeled with IL-mouse monoclonal antibody and red polystyrene latex microspheres. A detection line and a control line are disposed in the middle of the nitrocellulose membrane. A protein conjugate for binding with the IL-mouse monoclonal antibody is disposed inside the detection line. An IgG monoclonal antibody is disposed inside the control line. An upper shell is disposed on the outside of the detection strip, and a lower shell is matched with the upper shell.

[0006] Preferably, one of the red polystyrene latex microspheres has a diameter of 300 nm, and the other red polystyrene latex microsphere has a diameter of 400 nm.

[0007] Preferably, both the upper and lower outer shells are rectangular in shape.

[0008] Preferably, the coating concentration of the detection line is 1.0 mg / mL.

[0009] Preferably, the coating concentration of the control line is 0.8 μL / cm.

[0010] Preferably, the surface of the upper shell is provided with a sample application hole, and an observation port is provided on one side of the sample application hole where it connects to the upper shell, which is beneficial for observing the test results.

[0011] Preferably, positioning blocks are fixedly connected to both ends of one side of the upper outer shell, anti-detachment blocks are fixedly connected to both sides of the two positioning blocks, and a connecting block is fixedly connected to one end of the surface of the upper outer shell.

[0012] Preferably, positioning grooves are provided at both ends on one side of the lower outer shell, and the interior of the positioning grooves is slidably connected to the surface of the positioning block.

[0013] Preferably, both positioning slots have anti-detachment grooves inside, and the inner wall of the anti-detachment groove is slidably connected to the surface of the anti-detachment block, which is beneficial to the stability of the connection.

[0014] Preferably, a connecting groove is provided at one end of the surface of the lower outer shell, and a pressure-sensitive adhesive layer is applied between the connecting groove and the connecting block, which facilitates the quick connection of the upper and lower outer shells.

[0015] Compared with the prior art, the beneficial effects of this utility model are: placing the test strip in the outer shell enhances the stability and protection of the test strip, reduces the potential influence of external factors on the test strip, and the sequential arrangement of the sample pad, latex pad, conjugate pad, nitrocellulose membrane and absorbent pad can effectively control the flow direction of the sample and improve uniform distribution. The absorbent pad helps the sample pad to separate the test sample that has been flowing over, preventing the backflow of the sample after the reaction, which could cause false positives when the interpretation is too late.

[0016] Meanwhile, this type of test strip has high sensitivity, low risk of cross-reaction, simple operation and low cost. In addition, it uses an adhesive method, eliminating the need for alignment of various clips required by traditional snap-fit, thus improving assembly efficiency and greatly increasing production efficiency. Attached Figure Description

[0017] Figure 1 This is a structural diagram of the rapid detection card of this utility model;

[0018] Figure 2 This is a structural diagram of the rapid detection card housing assembly of this utility model;

[0019] Figure 3 This is a structural diagram of the detection strip of the rapid detection card of this utility model;

[0020] Figure 4 This is a structural diagram of the positive display of the rapid detection card of this utility model;

[0021] Figure 5 This is a structural diagram of the negative display of the rapid test card of this utility model;

[0022] Figure 6 This is a structural diagram of the first type of invalid display on the rapid detection card of this utility model;

[0023] Figure 7 This is a structural diagram of the second type of invalid display of the rapid detection card of this utility model.

[0024] In the diagram: 1. Upper outer shell; 2. Lower outer shell; 3. Positioning groove; 4. Anti-detachment groove; 5. Positioning block; 6. Anti-detachment block; 7. Connecting groove; 8. Connecting block; 9. Detection strip; 10. Sample pad; 11. Latex pad; 12. Binding pad; 13. Nitrocellulose membrane; 14. Absorbent pad; 15. Detection line; 16. Control line; 17. Sample dispensing hole; 18. Observation port. Detailed Implementation

[0025] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of the present invention.

[0026] Please see Figure 1-7 This invention provides a rapid human interleukin-4 test strip based on a double-antibody sandwich method, comprising a test strip 9, which includes a sample pad 10, a latex pad 11, a conjugate pad 12, a nitrocellulose membrane 13, and an absorbent pad 14. The latex pad 11 is made of latex microsphere-labeled CKIgY monoclonal antibody and red polystyrene latex microspheres. The conjugate pad 12 is made of latex microsphere-labeled IL-4 mouse monoclonal antibody and red polystyrene latex microspheres. A test line 15 and a control line 16 are disposed in the middle of the nitrocellulose membrane 13. The interior of the test line 15 contains a protein conjugate for binding with the IL-4 mouse monoclonal antibody, and the interior of the control line 16 contains an IgG monoclonal antibody. An upper shell 1 is disposed on the outside of the test strip 9, and a lower shell 2 is matched to the upper shell 1.

[0027] See Figure 1 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7Furthermore, one of the red polystyrene latex microspheres has a diameter of 300 nm, and the other red polystyrene latex microsphere has a diameter of 400 nm. The coating concentration of the detection line 15 is 1.0 mg / mL, the coating concentration of the control line 16 is 0.8 μL / cm, and the surface of the upper shell 1 is provided with a sample application hole 17. An observation port 18 is provided on one side of the connection between the sample application hole 17 and the upper shell 1.

[0028] In use, the test strip 9 has a PVC base plate as its base, and sequentially adhered sample pad 10, latex pad 11, conjugate pad 12, nitrocellulose membrane 13, and absorbent pad 14. The test strip 9 is positioned inside the upper shell 1 and lower shell 2 for greater stability during use. The conjugate pad 12 contains antibodies labeled with latex microspheres or other markers, which bind to the analyte in the sample to form a complex. Additionally, the nitrocellulose membrane 13, serving as the middle section of the test strip 9, provides an ideal solid-phase carrier for the test line 15 and control line 16. Simultaneously, the absorbent pad 14 helps to remove the sample flowing from the sample pad 10, preventing backflow of the reacted sample. This backflow effect can lead to false positives when test results are interpreted too late. The latex pad 11 is made of glass fiber membrane and latex microspheres labeled with CK. IgY antibody was prepared, labeled, and sprayed onto a glass cellulose membrane. Binding pad 12 consisted of a glass cellulose membrane and latex microspheres labeled with the primary IL-4 antibody M031201M, and then sprayed onto the glass cellulose membrane. By labeling the primary antibody IL-4 M031201M and CKIgY separately onto the latex microsphere particles, the visibility and stability of the label can be increased, thereby improving the sensitivity and specificity of the detection. The latex microspheres and the antibody IL-4 M031201M or CKIgY... IgY binding is primarily achieved through covalent bonding. A chemical cross-linking agent activates the carboxyl groups on the surface of the latex microspheres, forming stable covalent bonds with the amino groups on the antibody. This enhances the antibody's binding stability in complex environments, reducing non-specific binding and improving detection sensitivity. The controllable hydrophilic / hydrophobic surface of the latex microspheres further reduces the impact of non-specific binding and improves detection specificity. The nitrocellulose membrane 13 is equipped with a detection line 15 and a control line 16. The detection line 15 is located on the side of the nitrocellulose membrane 13 closer to the binding pad 12, and the control line 16 is located on the side of the nitrocellulose membrane 13 closer to the absorbent pad 14. The detection line 15, being closer to the binding pad 12, allows for faster capture of antibodies bound to the latex microsphere markers (i.e., the latex microsphere particles), thus improving detection speed and efficiency. The control line 16, located on the nitrocellulose membrane 13 closer to the absorbent pad 14, further enhances detection speed and efficiency. On one side, the nitrocellulose membrane 13 is used to verify the flowability and reagent performance of the test strip, ensuring the effectiveness of the detection process. Additionally, the nitrocellulose membrane 13 has a high protein adsorption capacity, effectively immobilizing the antibodies of the detection line 15 and control line 16 on the membrane while maintaining capillary flow of the aqueous sample. This helps improve the sensitivity of the detection. The sample pad 10 is made of glass fiber and is a key component of the immunochromatographic test strip. Its main function is to absorb the sample and transfer it to the conjugation pad 12 via capillary force. Made of glass fiber, it has good absorbency and transport properties, helping to control the flow of the sample on the test strip and preventing inaccurate results due to excessively rapid sample flow. The absorbent pad 14's main function is to absorb the sample flowing through the chromatography membrane to balance the pressure difference across the membrane, encouraging more sample to flow laterally on the membrane.Adding samples through the dedicated sample well 17 reduces contact between the sample and the external environment, lowering the risk of cross-contamination.

[0029] See Figure 1 and Figure 2 Furthermore, positioning blocks 5 are fixedly connected to both ends of one side of the upper outer shell 1, and anti-detachment blocks 6 are fixedly connected to both sides of the two positioning blocks 5. A connecting block 8 is fixedly connected to one end of the surface of the upper outer shell 1. Positioning grooves 3 are provided at both ends of one side of the lower outer shell 2. Anti-detachment grooves 4 are provided inside the two positioning grooves 3. The inside of the positioning grooves 3 is slidably connected to the surface of the positioning blocks 5. A connecting groove 7 is provided at one end of the surface of the lower outer shell 2. A pressure-sensitive adhesive layer is applied between the connecting groove 7 and the connecting block 8. The upper outer shell 1 and the lower outer shell 2 are both rectangular in shape.

[0030] In use, first apply a layer of pressure-sensitive adhesive to the side of the connecting block 8 that contacts the inner wall of the connecting groove 7 using a suitable brush. Then, holding the lower outer shell 2, align the positioning block 5 on the upper outer shell 1 with the positioning groove 3 and push the upper outer shell 1 to one side until one end of the connecting block 8 abuts against the connecting groove 7. At this point, the outer shell is installed in place, and the connecting block 8 with the pressure-sensitive adhesive layer also abuts against the inner wall of the connecting groove 7. Press down firmly, and the upper outer shell 1 and the lower outer shell 2 will be bonded together. This method is quick and convenient, and the connection is relatively stable and strong.

[0031] In this embodiment, the following steps are taken: Serum, plasma, cell supernatant, culture medium, and other samples are added to the sample pad 10 corresponding to the sample well 17. Through capillary action, the samples migrate towards the binding pad 12 and bind to the monoclonal antibody M031201M therein, forming a complex. This complex continues to migrate to the nitrocellulose membrane 13 and is captured by the M031201M monoclonal antibody coated on the detection line 15. As the captured complex gradually accumulates, a red band appears on the detection line 15. When the complex migrates to the control line 16, the latex microspheres CK IgY labeled in the latex pad 11 are captured by the GT Anti-CK IgY polyclonal antibody, resulting in a red band on the control line 16. Two red bands appear on the detection line 9, namely the detection line 15 and the control line 16, indicating a positive result. If the control line 16 shows color but the detection line 15 does not, the result is negative. If the control line 16 does not show color, but the detection line 15 does, and neither the control line 16 nor the detection line 15 shows color, the test is invalid.

[0032] Although the present invention 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 invention should be included within the protection scope of the present invention.

Claims

1. A rapid human interleukin-4 detection card based on a double-antibody sandwich method, comprising a detection strip (9), characterized in that: The test strip (9) includes a sample pad (10), a latex pad (11), a conjugate pad (12), a nitrocellulose membrane (13), and an absorbent pad (14). The latex pad (11) is made of latex microsphere-labeled CKIgY monoclonal antibody and red polystyrene latex microspheres. The conjugate pad (12) is made of latex microsphere-labeled IL-4 mouse monoclonal antibody and red polystyrene latex microspheres. The nitrocellulose membrane (13) has a detection line (15) and a control line (16) in the middle. The detection line (15) has a protein conjugate for binding with the IL-4 mouse monoclonal antibody inside. The control line (16) has an IgG monoclonal antibody inside. The test strip (9) has an upper shell (1) on the outside. The upper shell (1) has a matching lower shell (2).

2. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: One of the red polystyrene latex microspheres has a diameter of 300 nm, and the other has a diameter of 400 nm.

3. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: Both the upper outer shell (1) and the lower outer shell (2) are rectangular in shape.

4. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: The coating concentration of the detection line (15) is 1.0 mg / mL.

5. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: The coating concentration of the control line (16) is 0.8 μL / cm.

6. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: The surface of the upper outer shell (1) is provided with a sample feeding hole (17), and an observation port (18) is provided on one side of the connection between the sample feeding hole (17) and the upper outer shell (1).

7. The rapid human interleukin-4 detection card based on the double-antibody sandwich method according to claim 1, characterized in that: Positioning blocks (5) are fixedly connected to both ends of one side of the upper outer shell (1), and anti-detachment blocks (6) are fixedly connected to both sides of the two positioning blocks (5). A connecting block (8) is fixedly connected to one end of the surface of the upper outer shell (1).

8. A rapid human interleukin-4 detection card based on a double-antibody sandwich method according to claim 7, characterized in that: The lower outer shell (2) has positioning grooves (3) at both ends on one side, and the interior of the positioning groove (3) is slidably connected to the surface of the positioning block (5).

9. A rapid human interleukin-4 detection card based on a double-antibody sandwich method according to claim 8, characterized in that: Both positioning grooves (3) have anti-detachment grooves (4) inside, and the inner wall of the anti-detachment groove (4) is slidably connected to the surface of the anti-detachment block (6).

10. A rapid human interleukin-4 detection card based on a double-antibody sandwich method according to claim 7, characterized in that: A connecting groove (7) is provided at one end of the surface of the lower outer shell (2), and a pressure-sensitive adhesive layer is applied between the connecting groove (7) and the connecting block (8).

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