Solution suitable for interference problem of immunochromatography test strip sample

Abstract

The invention discloses a solution suitable for solving the problem of sample interference of an immunochromatography test strip, the test strip comprises a sample pretreatment area, the sample pretreatment area is coated with a nano-material coupled basic protein, the nano-material is selected from magnetic microspheres, polystyrene microspheres and silicon dioxide microspheres, and the nano-material is selected from magnetic microspheres, polystyrene microspheres and silicon dioxide microspheres. The basic protein is coupled with the nano material through a chemical bond, and the basic protein is selected from arginine, histidine, lysine and protamine. According to the method, the solid-phase porous matrix is selected in the sample pretreatment area to coat the nanometer material to be coupled with the basic protein, strongly acidic heparin is neutralized through the basic protein, interference of a heparin medicine sample is reduced, meanwhile, the basic protein is effectively marked in the sample pretreatment area by utilizing the special particle size selection difference between the nanometer material and the solid-phase porous matrix, and the detection accuracy is improved. The coating efficiency is improved, the problem that a special sample interferes with a chromatographic detection result is solved, and the detection accuracy is effectively improved.

Description

Technical Field

[0001] The invention relates to the field of medical testing, and in particular to a solution to the problem of sample interference in immunochromatographic test strips. Background Art

[0002] Since the end of 2022, the in vitro diagnostic IVD market has seen a large number of complaints and feedback, with the feedback focusing on invalid detection issues with POCT immunochromatographic test strips. According to statistics, most of them are myocardial and inflammatory detection items, such as: cTnI, NT-proBNP, H-FABP, D-Dimer, PCT, hs-CRP, CRP and other detection kits.

[0003] After a large number of test statistics, the detection rate of invalid cards with test abnormalities is about 20%. Invalid cards generally have two abnormal phenomena during the test process: ① After adding serum, plasma, and whole blood samples, the tracer particles aggregate or adhere to the NC membrane, resulting in the inability to chromatograph the samples normally, affecting the detection of C and T lines; ② After adding serum, plasma, and whole blood samples, the tracer particles can chromatograph normally, but the reaction of the quality control C line is suppressed by interference and becomes lower. At the same time, we reviewed the samples retained in 2021 and before, and the reagent card test did not have the above abnormal problems, and all the retained sample reagent cards were tested with abnormal samples, and interference problems occurred.

[0004] According to the literature, the novel coronavirus (COVID-19) is spreading globally, the number of infected people has surged, and the number of severe and critical patients has continued to increase. Professor Zhang Shuyang and Professor Li Yongzhe are the corresponding authors of the study "Coagulopathy and Antiphospholipid Antibodies in Patients with COVID-19" published online in the New England Journal of Medicine. It is the first time that the clinical phenomenon of multiple high-titer antiphospholipid antibodies in patients with novel coronavirus has been reported internationally, indicating that autoimmune disorders are closely related to abnormal coagulation and thrombotic events in patients with novel coronavirus. Based on this, we speculate that there is a special interference phenomenon in blood samples after COVID-19 positive.

[0005] References:

[0006] 1.YanZhang,Meng Xiao,et al.Coagulopathy andAntiphospholipidAntibodiesinPatientswithCovid-19.NewEnglandJournal ofMedicine.April 8,2020 Summary of the invention

[0007] In order to solve the problem of the above-mentioned sample interference affecting the immunochromatographic detection results, the present application particularly relates to a novel sample pretreatment area suitable for immunochromatographic detection reagent cards. By selecting a solid-phase porous matrix coated nanomaterial to couple alkaline protein and fibrin polymerization inhibitor, the alkaline protein is used to neutralize the strongly acidic heparin, thereby reducing the interference of heparin drug samples, and utilizing the complementary combination of fibrin polymerization inhibitor and fibrinogen D to reduce the interference of the sample's hypercoagulable state. Ultimately, the problem of interference of special samples on the test results is effectively overcome, and the detection accuracy of the reagent card is significantly improved.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical means: a solution suitable for the problem of sample interference in immunochromatographic test strips, characterized in that the solution is to coat the sample pretreatment area of ​​the test strip with nanomaterials coupled with basic proteins.

[0009] Preferably, the nanomaterial is selected from at least one of magnetic microspheres, polystyrene microspheres, and silica microspheres.

[0010] Preferably, the basic protein is coupled to the nanomaterial via a chemical bond, and the basic protein is selected from at least one of arginine, histidine, lysine, and protamine.

[0011] Preferably, the sample pre-treatment area is also coated with a nanomaterial-coupled fibrin polymerization inhibitor, and the fibrin polymerization inhibitor is preferably GPRP.

[0012] Preferably, the sample pre-treatment area is a solid phase porous matrix, selected from at least one of a glass cellulose membrane and a rigid mesh membrane.

[0013] Preferably, the particle size of the nanomaterial is greater than or equal to the pore size of the solid porous matrix, preferably 1.0-2.0 um.

[0014] An immunochromatographic test strip comprises the sample pre-treatment area as described above.

[0015] Preferably, the test strip is suitable for detecting at least one of 3in1, cTnI, cTnT, and PCT.

[0016] Preferably, the test strip is a colloidal gold, fluorescent microsphere, or colored microsphere immunochromatographic test strip.

[0017] A method for preparing the immunochromatographic test strip as described above, characterized in that the preparation scheme of the sample pretreatment area is as follows:

[0018] a. Coupling of nanomaterials:

[0019] The basic protein and the fibrin polymerization inhibitor are respectively coupled to the nanomaterial by covalent coupling, and then the nanomaterial coupled with the basic protein and the nanomaterial coupled with the fibrin polymerization inhibitor are mixed to obtain a nanomaterial conjugate;

[0020] b. Preparation of sample area treatment solution:

[0021] Mixing a buffer, a surfactant, a protein and a nanomaterial conjugate to prepare a sample area treatment solution;

[0022] c. Sample pre-treatment area treatment:

[0023] The sample area treatment liquid is treated on the solid phase porous matrix by spraying or immersing, and then dried to form a sample pre-treatment area.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention uses a solid-phase porous matrix to coat nanomaterials and couple alkaline proteins in the sample pretreatment area. By differentially selecting the specific particle sizes of the nanomaterials and the solid-phase porous matrix, the alkaline proteins are effectively labeled in the sample pretreatment area, and the alkaline proteins are used to neutralize the highly acidic heparin, thereby reducing the interference problem of heparin drug samples.

[0026] (2) The present invention coats the nanomaterial coupled fibrin polymerization inhibitor in the sample pretreatment area, and utilizes the complementary binding between the nanomaterial and fibrinogen D to effectively reduce the interference problem from the hypercoagulable state sample.

[0027] (3) The present invention effectively improves the ability of the test strip to resist sample interference by setting a special sample pretreatment area, simplifies the user operation process while ensuring the detection performance, and the corresponding test strip preparation process of the present invention is simple and suitable for high-throughput industrial batch preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a structural diagram of the test strip prepared in Example 1 of the present invention (1-base, 2-sample pretreatment area; 3-binding pad, 4-nitrocellulose membrane, 5-sample pad, 6-T1 test line, 7-T2 test line, 8-T3 test line, 9-quality control line).

[0029] The following examples are included herein to more clearly and specifically illustrate the technical solutions of the present invention in an illustrative manner. It should be understood by those skilled in the art based on the disclosure herein that many changes can be made in the disclosed specific embodiments but still similar or similar results can be obtained without departing from the idea and scope of the present invention. The specific embodiments of the present invention are only used to explain the present invention and are not intended to limit the present invention in any way.

[0030] The embodiment of the present invention provides a solution to the sample interference problem of an immunochromatographic test strip, wherein the test strip comprises a special sample pretreatment area, a conjugation pad, an analysis pad, a water absorbent pad and a bottom liner. The sample pretreatment area is coated with nanomaterial-coupled alkaline protein and a fibrin polymerization inhibitor, the conjugation pad is coated with a tracer particle conjugate, the analysis pad is provided with a detection line and a quality control line, and the sample pretreatment area, the conjugation pad, the analysis pad and the water absorbent pad are sequentially arranged on the bottom liner.

[0031] The embodiment of the present invention provides another solution to the sample interference problem of an immunochromatographic test strip, wherein the test strip comprises a special sample pretreatment area, an analysis pad, a water absorbent pad and a bottom liner. The sample pretreatment area is coated with nanomaterial-coupled basic protein and nanomaterial-coupled fibrin polymerization inhibitor, the analysis pad is provided with a tracer particle conjugate line, a detection line and a quality control line, and the sample pretreatment area, the analysis pad and the water absorbent pad are sequentially arranged on the bottom liner.

[0032] Further, in some feasible embodiments, the nanomaterial can be selected from at least one of magnetic microspheres, polystyrene microspheres, and silica microspheres, and the basic protein can be selected from at least one of arginine, histidine, lysine, and protamine. Optionally, the nanomaterial and the basic protein are coupled via chemical bonds.

[0033] Furthermore, in some feasible embodiments, the material of the sample pre-treatment area is a solid phase porous matrix, which can be specifically selected from one of a glass cellulose membrane and a rigid mesh membrane.

[0034] Furthermore, the particle size of the nanomaterial needs to be greater than or equal to the pore size of the solid phase porous matrix, and the particle size of the nanomaterial is preferably 1.0-2.0 um. In this embodiment, by selecting a solid phase porous matrix to coat the nanomaterial and couple the basic protein in the sample pretreatment area, the basic protein is effectively labeled in the sample pretreatment area through the difference in the special particle size of the nanomaterial and the solid phase porous matrix, and the basic protein is used to neutralize the highly acidic heparin, thereby reducing the interference problem of the heparin drug sample.

[0035] Furthermore, in some feasible embodiments, the fibrin polymerization inhibitor is GPRP. This embodiment reduces the interference of the sample's hypercoagulable state through the complementary combination of the fibrin polymerization inhibitor and fibrinogen D, effectively overcomes the problem of interference of special samples on the test results, and significantly improves the detection accuracy of the reagent card.

[0036] Furthermore, in some feasible embodiments, the tracer particles can be selected from colloidal gold, fluorescent microspheres, and colored microspheres.

[0037] Furthermore, in some feasible embodiments, the solution described in the present application is applicable to test strips for 3in1, cTnI, cTnT, PCT and other projects.

[0038] The embodiment of the present invention provides a preparation scheme for a sample pre-treatment area applicable to an immunochromatographic test strip to solve the interference problem, comprising:

[0039] a. Coupling of nanomaterials:

[0040] The basic protein and the fibrin polymerization inhibitor are respectively coupled to the nanomaterial by covalent coupling, and then the nanomaterial coupled with the basic protein and the nanomaterial coupled with the fibrin polymerization inhibitor are mixed to obtain a nanomaterial conjugate;

[0041] b. Preparation of sample area treatment solution:

[0042] Mixing a buffer, a surfactant, a protein and a nanomaterial conjugate to prepare a sample area treatment solution;

[0043] c. Sample pre-treatment area treatment:

[0044] The sample area treatment liquid is treated on the solid phase porous matrix by spraying or soaking, and then dried to form a sample pre-treatment area.

[0045] Furthermore, the buffer can be selected from at least one of 10-100mM MOPSO buffer, HEPES buffer, Tris buffer or phosphate buffer; the protein can be selected from at least one of 0.1-1% bovine serum albumin or casein; the surfactant can be selected from at least one of 0.1-5% Tween20, Tween80, Triton or Brij; the nanomaterial can be selected from at least one of magnetic microspheres, polystyrene microspheres, and silica microspheres with a final concentration of 0.01-0.05mg / ml.

[0046] In view of the above feasible implementation scheme, this application particularly proposes the following embodiments:

[0047] Example 1 Preparation of 3in1 Test Strips

[0048] (1) Preparation of sample pretreatment area

[0049] a. Coupling of nanomaterials:

[0050] The basic amino acid polymer was mixed with the nano-microspheres at a ratio of 60 μg basic amino acid polymer / 300 μg polystyrene microspheres, and then 6 μg EDC was added. The mixture was stirred at room temperature for 120 minutes, 5% BSA was added, the mixture was closed and stirred for 60 minutes, and the mixture was centrifuged at 14000 r / min for 20 minutes. The supernatant was discarded, the precipitate was restored to its volume with preservation solution, and the mixture was stored at 4°C to obtain the nano-material coupled with the basic protein.

[0051] GPRP and nano-microspheres were mixed at a ratio of 60 μg of fibrin polymerization inhibitor GPRP / 300 μg of polystyrene microspheres, and then 6 μg of EDC was added. The mixture was stirred at room temperature for 120 minutes, 5% BSA was added, and the mixture was closed and stirred for 60 minutes. The mixture was centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, the precipitate was restored to its volume with preservation solution, and the mixture was stored at 4°C to obtain the nano-material-coupled fibrin inhibitor.

[0052] Then, the nanomaterial-coupled basic protein and the nanomaterial-coupled fibrin inhibitor are mixed in a volume ratio of 1:1 to obtain a nanomaterial conjugate.

[0053] b. Sample treatment solution preparation:

[0054] Prepare according to the following formula, stir thoroughly and store at 2-8℃.

[0055] MOPSO buffer 100 mM

[0056] Bovine serum albumin 1%

[0057] Tween20 5%

[0058] Nanomaterial conjugate 0.05g / l

[0059] The remaining solvent is water

[0060] c. Sample pre-treatment area treatment:

[0061] The sample treatment liquid is treated on the solid phase porous matrix by spraying or soaking, and then dried to form a sample pre-treatment pad.

[0062] (2) Assembly of test strips

[0063] a. Preparation of nitrocellulose membrane

[0064] 1.0 mg / mL cTnI monoclonal antibody, 1.0 mg / mL L-Myo monoclonal antibody, 1.0 mg / mL CK-MB monoclonal antibody, and 1.0 mg / mL goat anti-mouse IgG antibody coating solutions were streaked onto designated positions on the nitrocellulose membrane and dried at room temperature for 5 hours.

[0065] On the nitrocellulose membrane, the T1 detection line is coated with cTnI monoclonal antibody, the T2 detection line is coated with Myo monoclonal antibody, and the T3 detection line is coated with CK-MB monoclonal antibody.

[0066] b. Preparation of conjugate pad

[0067] The monoclonal antibody cTnI / Myo / CK-MB was slowly added to the colloidal gold sol to form a colloidal antibody mixed marker with the colloidal gold and the antibody. The colloidal gold antibody mixed marker was then coated on the conjugate pad and dried at room temperature for 5 hours.

[0068] c.Test strip assembly

[0069] Assemble the sample pretreatment pad, conjugate pad, NC membrane, and absorbent paper on the PVC backing in sequence, and then cut the test strips into corresponding widths as needed and assemble them in the card shell.

[0070] Example 23 Preparation of 1-in-1 Test Strips (Traditional Test Strips)

[0071] The test strip preparation method of this embodiment differs from that of Embodiment 1 only in the preparation of the sample pretreatment area, and the rest of the preparation process is the same.

[0072] (1) Preparation of sample pretreatment area

[0073] a. Sample treatment solution preparation:

[0074] Prepare according to the following formula, stir thoroughly and store at 2-8℃.

[0075] MOPSO buffer 100 mM

[0076] Bovine serum albumin 1%

[0077] Tween20 5%

[0078] The remaining solvent is water

[0079] b. Sample pre-treatment area treatment:

[0080] The sample treatment liquid is treated on the solid phase porous matrix by spraying or soaking, and then dried to form a sample pre-treatment pad.

[0081] (2) Assembly of test strips

[0082] a. Preparation of nitrocellulose membrane

[0083] 1.0 mg / mL cTnI monoclonal antibody, 1.0 mg / mL L-Myo monoclonal antibody, 1.0 mg / mL CK-MB monoclonal antibody, and 1.0 mg / mL goat anti-mouse IgG antibody coating solutions were streaked onto designated positions on the nitrocellulose membrane and dried at room temperature for 5 hours.

[0084] b. Preparation of conjugate pad

[0085] The monoclonal antibody cTnI / Myo / CK-MB was slowly added to the colloidal gold sol to form a colloidal antibody mixed marker with the colloidal gold and the antibody. The colloidal gold antibody mixed marker was then coated on the conjugate pad and dried at room temperature for 5 hours.

[0086] c.Test strip assembly

[0087] Assemble the sample pretreatment pad, conjugate pad, NC membrane, and absorbent paper on the PVC backing in sequence, and then cut the test strips into corresponding widths as needed and assemble them in the card shell.

[0088] Example 3 Test Strip Performance Verification

[0089] In order to verify the detection performance of the test strips described in Example 1 of the present invention, a total of 4 groups of test strips were set for verification:

[0090] Group A: the test strip prepared in Example 1 of the present invention;

[0091] Group B: the test strips prepared in Example 2 of the present invention;

[0092] Group C: commercially available test strips;

[0093] (1) Performance verification

[0094] Three groups of test strips were used to test the calibrators for clinical interference samples, and the signals were read by an immunoquantitative analyzer (Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., Q20). The test results are shown in the following table:

[0095] Table 1

[0096]

[0097]

[0098] From the above experimental results, it can be seen that coating the sample pretreatment area with nanomaterial basic protein and nanomaterial coupled fibrin polymerization inhibitor can effectively eliminate the interference of the sample on the C line.

[0099] Example 4 Effect of Nanomaterial Particle Size on Detection Performance

[0100] (1) Selection of nanomaterials

[0101] In order to verify the effect of the selection of nanomaterial particle size on the detection performance of the test strip, four groups of tests were set up for verification:

[0102] Group A Group B Group C Group D Particle size 100nm 300nm 1000nm 2000nm

[0103] *Note: The difference between the test strips in groups A, B, C, and D lies only in the nanomaterials and their particle sizes used in the sample pretreatment area. The rest of the preparation process is exactly the same.

[0104] (2) Performance Verification

[0105] Four groups of test strips were used to test the calibrators for clinical interference samples. The signals were read by an immunoquantitative analyzer (Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., Q20). The test results are shown in the following table:

[0106] Table 2

[0107]

[0108]

[0109] From the above experimental results, it can be seen that the particle size of nanomaterials is between 1000-2000nm, and its effect on eliminating sample interference is most obvious and effective.

[0110] Example 5 Effect of basic protein on detection performance

[0111] (1) Selection of basic protein

[0112] In order to verify the influence of the selection of basic protein on the detection performance of the test strip, 4 groups of experiments were set up for verification:

[0113]

[0114] *Note: The only difference between the test strips in groups A, B, C and D is whether the sample pretreatment area is coated with nanomaterial-coupled alkaline protein and the specific type of coated alkaline protein. The rest of the preparation process is exactly the same.

[0115] (2) Performance Verification

[0116] Four groups of test strips were used to test the calibrators for clinical interference samples. The signals were read by an immunoquantitative analyzer (Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., Q20). The test results are shown in the following table:

[0117] Table 3

[0118]

[0119] From the above experimental results, it can be seen that the effect of coating the nanomaterial coupled with histidine in the sample pretreatment area on eliminating sample interference is the most obvious and effective.

[0120] Example 6 Effect of Fibrin Polymerization Inhibitors on Detection Performance

[0121] (1) Selection of fibrin polymerization inhibitors

[0122] In order to verify the effect of the selection of fibrin polymerization inhibitor on the detection performance of the test strip, two groups of experiments were set up for verification:

[0123]

[0124] *Note: The difference between Group A test strips and Group B test strips is only whether the sample pretreatment area is coated with nanomaterials coupled with GPRP. The rest of the preparation process is exactly the same.

[0125] (2) Performance Verification

[0126] Two sets of test strips were used to test the calibrators for clinical interference samples, and the signals were read by an immunoquantitative analyzer (Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., Q20). The test results are shown in the following table:

[0127] Table 4

[0128]

[0129] From the above experimental results, it can be seen that the nanomaterial-coupled fibrin polymerization inhibitor GPRP coated in the sample pretreatment area is significantly effective in eliminating sample interference.

[0130] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and deform the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A solution to the problem of sample interference in immunochromatographic test strips, characterized in that: The scheme is to coat the sample pre-treatment area of ​​the test strip with nanomaterials coupled with alkaline proteins.

2. The solution according to claim 1, characterized in that: The nanomaterial is selected from at least one of magnetic microspheres, polystyrene microspheres and silica microspheres.

3. The solution according to claim 1, characterized in that: The basic protein is coupled to the nanomaterial via chemical bonds, and the basic protein is selected from at least one of arginine, histidine, lysine, and protamine.

4. The solution according to claim 1, characterized in that The sample pre-treatment area is also coated with a nanomaterial-coupled fibrin polymerization inhibitor, and the fibrin polymerization inhibitor is preferably GPRP.

5. The solution according to claim 2, characterized in that: The sample pre-treatment area is a solid phase porous matrix, which is selected from at least one of a glass cellulose membrane and a rigid mesh membrane.

6. The solution according to any one of claims 1 to 5, characterized in that: The particle size of the nanomaterial is greater than or equal to the pore size of the solid phase porous matrix, preferably 1.0-2.0 um.

7. An immunochromatographic test strip, characterized in that: The test strip comprises the sample pre-treatment zone as described in any one of claims 1-6.

8. The test strip according to claim 7, characterized in that: The test strip is suitable for detecting at least one of 3in1, cTnI, cTnT, and PCT.

9. The test strip according to claim 7, characterized in that: The test strip is a colloidal gold, fluorescent microsphere, or colored microsphere immunochromatographic test strip.

10. A method for preparing the immunochromatographic test strip according to any one of claims 7 to 9, characterized in that: The preparation scheme of the sample pretreatment area is as follows: a. Coupling of nanomaterials: The basic protein and the fibrin polymerization inhibitor are respectively coupled to the nanomaterial by covalent coupling, and then the nanomaterial coupled with the basic protein and the nanomaterial coupled with the fibrin polymerization inhibitor are mixed to obtain a nanomaterial conjugate; b. Preparation of sample area treatment solution: Mixing a buffer, a surfactant, a protein and a nanomaterial conjugate to prepare a sample area treatment solution; c. Sample pre-treatment area treatment: The sample area treatment liquid is treated on the solid phase porous matrix by spraying or soaking, and then dried to form a sample pre-treatment area.

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