Fluorescence immunochromatography test strip and kit

Abstract

The invention provides a fluorescence immunochromatography test strip and a kit. The test strip comprises a sample combination pad and a coating film, a first fluorescent microsphere marker is adsorbed on the sample combination pad, the first fluorescent microsphere marker is formed by coupling a labeled antibody on a first fluorescent microsphere, and the labeled antibody can be combined with a first site of an antigen to be detected; a first detection line and a second detection line are sequentially arranged on the coating film from front to back according to the flowing direction of a sample, the first detection line is coated with a first capture antibody, and the second detection line is coated with a first compound; the first compound is formed by specifically binding the capture antigen and the first capture antibody; the first capture antibody can be combined with a second site of the to-be-detected antigen, and the capture antigen and the to-be-detected antigen can be competitively combined with the labeled antibody.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing technology and relates to a fluorescent immunochromatographic test strip and a test kit. Background Art

[0002] In fluorescent immunochromatography, the double antibody sandwich method is often used for the detection of large molecules such as proteins. In the double antibody sandwich reaction, a coated antibody-analyte-labeled antibody complex is formed under normal circumstances. However, when the analyte concentration is very high, the number of antibodies is relatively small, and the area around the antibodies is filled with analytes. Both the coated antibody and the labeled antibody are saturated with the analyte. Therefore, the number of coated antibody-analyte-labeled antibody complexes formed will be reduced, resulting in a false negative or weak positive phenomenon.

[0003] In order to avoid the HOOK effect caused by excessive sample analyte concentration in the double antibody sandwich method of fluorescent immunochromatography, existing methods, such as the Chinese patent with publication number CN110596380A, set a detection H line between the detection T line and the quality control C line of the immunochromatographic test card, and the detection H line is coated with the analyte to be detected. When the sample analyte concentration is too high, the color of the detection H line becomes light or negative, and when the sample analyte concentration is small or even zero, the detection H line is positive, so that it is very good to find out whether the sample test has a HOOK effect.

[0004] However, when the molecular weight of the sample analyte is less than 40KDa, such as N-terminal B-type natriuretic peptide (NT-proBNP), the NT-proBNP with a smaller molecular weight is fixed on the detection line T2 as a capture antigen, which is prone to cause loose solidification. During the test, the capture antigen contaminates the detection line T1, resulting in affected detection accuracy. Summary of the invention

[0005] The present disclosure provides a fluorescent immunochromatographic test strip and a kit, which can effectively solve the above problems.

[0006] The present disclosure is implemented as follows:

[0007] In a first aspect, the present disclosure provides a fluorescent immunochromatographic test strip, the test strip comprising: a sample binding pad and a coating membrane;

[0008] The sample binding pad is adsorbed with a first fluorescent microsphere marker, which is formed by coupling a labeled antibody to the first fluorescent microsphere, and the labeled antibody can bind to the first site of the antigen to be detected;

[0009] The coating membrane is provided with a first detection line and a second detection line in sequence from front to back in the sample flow direction, the first detection line is coated with a first capture antibody, and the second detection line is coated with a first complex; the first complex is formed by the specific combination of the capture antigen and the first capture antibody;

[0010] The first capture antibody can bind to the second site of the antigen to be detected, and the capture antigen can compete with the antigen to be detected for binding to the labeled antibody.

[0011] In a second aspect, the present disclosure provides a test card, comprising the test strip described in the first aspect and a card housing.

[0012] In a third aspect, the present disclosure provides a kit, the kit comprising the test strip described in the first aspect or the test card described in the second aspect;

[0013] The kit also includes a sample buffer;

[0014] The sample buffer comprises at least one of an anti-erythrocyte antibody and a blocking agent, wherein the blocking agent is a HAMA blocking agent and / or an HBR blocking agent.

[0015] In a fourth aspect, the present disclosure provides a method for using the kit described in the third aspect, wherein the sample and the sample buffer are evenly mixed and then added to the test strip for reaction, and then, based on the detection value of the fluorescent signal enriched by the second detection line, it is determined whether the concentration of the antigen to be tested in the sample is within the linear detection range of the test strip.

[0016] The beneficial effects of the present disclosure are:

[0017] The fluorescent immunochromatographic test strip and kit provided by the present invention adopt a double antibody sandwich method and a competitive method to collaboratively detect the antigen to be tested, and determine whether the concentration of the antigen to be tested is excessive by the detection value of the fluorescent signal enriched in the second detection line, which can avoid the HOOK effect caused by the excessive concentration of the antigen to be tested, wherein the second detection line is coated with a complex of the capture antigen and the first capture antibody, which can improve the solidification effect of the capture antigen on the second detection line, and avoid its separation from the second detection line to interfere with the fluorescent signal of the first detection line, resulting in inaccurate detection value of the first detection line.

[0018] Furthermore, the present invention adopts fluorescent immunochromatography for detection, which can effectively eliminate the interference of non-specific fluorescence and greatly improve the analysis sensitivity.

[0019] Furthermore, the kit provided by the present disclosure includes a sample buffer comprising an anti-RBC antibody, which can bind to the red blood cells in the whole blood sample to form macromolecules, and can retain the red blood cells on the sample pad when adding the sample, thereby preventing the red blood cells from being chromatographed on the coating membrane and interfering with the fluorescent signal of the detection line, thereby realizing the detection of multiple sample types. In addition, the sample buffer also includes a blocking agent, which can greatly reduce the non-specificity of the sample, thereby avoiding the false positive problem of the traditional method. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 It is a schematic diagram of the structure of the test strip and the detection card provided in the embodiment of the present disclosure.

[0022] Figure 2 This is a trend graph of the standard curve of N-terminal B-type natriuretic peptide provided in the embodiments of the present disclosure.

[0023] Figure 3 This is a standard curve fitting diagram within the linear detection range of N-terminal B-type natriuretic peptide provided in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. Therefore, the following detailed description of the embodiments of the present disclosure provided in the drawings is not intended to limit the scope of the present disclosure for protection, but merely represents the selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0025] Abbreviation Description:

[0026] DNP: 2,4-Dinitrophenol.

[0027] BSA: Bovine serum albumin.

[0028] HAMA: Human anti-mouse antibody, Human Anti-mouse Antibody.

[0029] HBR: Heterophilic Blocking Reagent.

[0030] The time-resolved fluorescent microspheres used in the embodiments of the present disclosure were purchased from Changsha Meiniu Biotechnology Co., Ltd., with the item number MF03;

[0031] The labeled antibody NT-proBNP antibody 1 was purchased from Chongqing Tansheng Technology Co., Ltd., with the catalog number FAB-H001-T9;

[0032] The first capture antibody NT-proBNP antibody 2 was purchased from Chongqing Tansheng Technology Co., Ltd. with the catalog number FAB-H001-T6;

[0033] The capture antigen NT-proBNP was purchased from Chongqing Tansheng Technology Co., Ltd. with the catalog number FAP-HE001.

[0034] DNP-BSA was purchased from Xiamen Tongrenxin Biotechnology Co., Ltd. with the catalog number ZKC11;

[0035] Anti-DNP antibody was purchased from Xiamen Tongrenxin Biotechnology Co., Ltd., catalog number ZKC21;

[0036] HBR blocker was purchased from Jiangsu Fanbo Biological Products Co., Ltd., with the catalog number HBR-5;

[0037] Anti-RBC antibody was purchased from Jiangsu Fanbo Biological Products Co., Ltd. with the catalog number M7910.

[0038] The disclosed embodiment provides a fluorescent immunochromatographic test strip, the test strip comprising: a sample binding pad and a coating membrane;

[0039] The sample binding pad is adsorbed with a first fluorescent microsphere marker, which is formed by coupling a labeled antibody to the first fluorescent microsphere, and the labeled antibody can bind to the first site of the antigen to be detected;

[0040] The coating membrane is provided with a first detection line and a second detection line in sequence from front to back in the sample flow direction, the first detection line is coated with a first capture antibody, and the second detection line is coated with a first complex; the first complex is formed by the specific combination of the capture antigen and the first capture antibody;

[0041] The first capture antibody can bind to the second site of the antigen to be detected, and the capture antigen can compete with the antigen to be detected for binding to the labeled antibody.

[0042] The double antibody sandwich method and the competitive method are used to collaboratively detect the antigen with a smaller molecular weight. If the capture antigen used is also of a smaller molecular weight, or the capture antigen and the antigen to be detected are the same protein, the solidification effect of the capture antigen on the detection line T2 is poorer than that of the large molecule antibody, and it is easy to detach during the test strip preparation / detection process and contaminate the detection line T1.

[0043] When the detection line T1 is contaminated, the detection value will be high.

[0044] The disclosed embodiment specifically combines the capture antigen with the first capture antibody to form a first complex, wherein the first capture antibody is fixed on the detection line T2 as a fixed end to improve the overall solidification effect, and the capture antigen therein can specifically react with the labeled antibody NT-proBNP antibody 1 chromatographed to the detection line T2, thereby ensuring the accuracy of the detection.

[0045] Please refer to the attached Figure 1 In some embodiments, the reagent strip includes a base plate, a sample binding pad, a coating film and absorbent paper, and the sample binding pad, the coating film and the absorbent paper are sequentially adhered to the base plate.

[0046] In some embodiments, the bottom plate is a PVC plate with double-sided tape.

[0047] In some embodiments, the sample conjugation pad is a polyester membrane sample conjugation pad or a glass cellulose membrane sample conjugation pad.

[0048] In some embodiments, the coating membrane is a nitrocellulose membrane.

[0049] In some embodiments, the antigen to be detected is NT-proBNP protein.

[0050] In some embodiments, the labeled antibody is NT-proBNP antibody 1. The first capture antibody is NT-proBNP antibody 2. The capture antigen is NT-proBNP protein.

[0051] The labeled antibody NT-proBNP antibody 1 and the first capture antibody NT-proBNP antibody 2 are paired with different binding sites on the antigen NT-proBNP protein to be detected, and together form a double antibody sandwich complex. Therefore, the first capture antibody NT-proBNP antibody 2 can fix the complex formed by the first fluorescent microsphere marker and the antigen to be detected on the first detection line.

[0052] The capture antigen and the antigen to be tested are both NT-proBNP proteins. The two compete with the labeled antibody NT-proBNP antibody 1 for specific reaction. This is a double antigen competition reaction. The capture antigen in the first complex can fix the first fluorescent microsphere marker that is not bound to the antigen to be tested on the second detection line, thereby determining the relative amount between the antigen to be tested and the first fluorescent microsphere marker.

[0053] If the concentration of the antigen to be detected exceeds the limit, the number of the first fluorescent microsphere markers that have not been bound is close to zero, and it will be difficult for the second detection line to enrich the fluorescent signal.

[0054] In some embodiments, the capture antigen and the first capture antibody are mixed and then specifically reacted to obtain the first complex, wherein the capture antigen and the first capture antibody are mixed at a mass ratio of 1:0.1-0.5.

[0055] The Fab end of the antibody is the location that specifically reacts with the antigenic determinant. One IgG antibody has two Fab ends that can connect to two antigens. If the proportion of the first capture antibody in the first complex is too high, then the first complex and the first capture antibody will exist on the second detection line, causing part of the second complex that is not captured by the first detection line to sandwich react with the first capture antibody on the second detection line and enrich the fluorescence.

[0056] The second complex is a complex formed by the combination of the antigen to be detected and the first fluorescent microsphere marker.

[0057] In some embodiments, the test strip further includes a quality control line.

[0058] The quality control line is coated with a second capture antibody.

[0059] In some embodiments, the coating film is provided with a first detection line, a second detection line, and a quality control line in sequence from front to back in the sample flow direction. In other embodiments, the coating film is provided with a first detection line, a quality control line, and a second detection line in sequence from front to back in the sample flow direction. In other embodiments, the coating film is provided with a quality control line, a first detection line, and a second detection line in sequence from front to back in the sample flow direction.

[0060] In some embodiments, a second fluorescent microsphere marker is also adsorbed on the sample binding pad. The second fluorescent microsphere marker is formed by coupling a labeled protein to the second fluorescent microsphere. The labeled protein can bind to the second capture antibody.

[0061] In some embodiments, the second capture antibody is an anti-DNP antibody.

[0062] In some embodiments, the marker protein is a DNP protein conjugate.

[0063] In some embodiments, the marker protein is DNP-BSA.

[0064] The second capture antibody pairs with the labeled protein to form a complex, thereby fixing the second fluorescent microsphere marker on the quality control line.

[0065] In some embodiments, the mass ratio of the first fluorescent microsphere to the labeled antibody is 1:0.03-0.3.

[0066] In some embodiments, the preparation method of the first fluorescent microsphere marker and the second fluorescent microsphere marker comprises:

[0067] (1) Preparation of fluorescent markers:

[0068] a. Washing the fluorescent microspheres: Wash the fluorescent microspheres with MES buffer at pH 4.5 to 7.0, and remove the supernatant by centrifugation. The fluorescent microspheres include the first fluorescent microspheres or the second fluorescent microspheres.

[0069] b. Activate fluorescent microspheres: Dissolve NHS and EDC in purified water to 10 mg / mL, respectively, and add the NHS and EDC aqueous solutions to the fluorescent microspheres. Vortex to mix, rotate to mix for 0.5-2 h, and remove the supernatant by centrifugation.

[0070] c. Conjugated antibody: Add NT-proBNP antibody 1 to the activated fluorescent microspheres at a mass ratio of labeled protein to fluorescent microspheres of 0.03 to 0.3:1, oscillate to mix, rotate to mix for 0.5 to 4 hours, and remove the supernatant by centrifugation.

[0071] According to the mass ratio of the labeled protein to the fluorescent microspheres of 0.03 to 0.3:1, DNP-BSA was added to the activated fluorescent microspheres, and after oscillation and mixing, the mixture was rotated for 0.5 to 4 hours, and the supernatant was removed by centrifugation.

[0072] d. Blocking microspheres: Add bovine serum albumin and glycine to the coupled fluorescent microspheres, shake and mix, and rotate and mix for 0.5 to 18 hours to obtain a microsphere solution.

[0073] e. Washing and preservation: After the microsphere solution is centrifuged at 2-8°C, 15000 rpm for 15 min, the supernatant is removed and the microsphere solution is stored in 50 mM pH 8.0 Hepes buffer to obtain a storage solution.

[0074] The storage solution includes a storage solution containing a labeled first fluorescent microsphere marker and a storage solution containing a second fluorescent microsphere marker.

[0075] In some embodiments, the method for adsorbing the first fluorescent microsphere marker onto the sample binding pad includes:

[0076] f. Preparation of spray solution: dilute the preservation solution in the dilution buffer at a volume ratio of 1:5 to 100 to obtain a spray solution.

[0077] g. Preparation of sample binding pad: spray a line evenly on the sample binding pad at a rate of 2 to 4 μL / cm of the sample binding pad with the spray liquid, and then dry at 37°C to 55°C for 2 to 24 hours.

[0078] In some embodiments, the dilution buffer is a 50 mM Tris buffer at pH 8.0 containing 0.85% NaCl, 0.1% PEG2000, 0.1% BSA, 0.2% Tween-20, and 5.0% trehalose by mass fraction.

[0079] The preservation solution containing the labeled first fluorescent microsphere marker and the preservation solution containing the second fluorescent microsphere marker are diluted together in a dilution buffer to obtain a spraying solution.

[0080] The volume ratio of the preservation solution containing the labeled first fluorescent microsphere marker, the preservation solution containing the second fluorescent microsphere marker and the dilution buffer solution is 1:1:5-100.

[0081] In some embodiments, the first detection line is coated with the first capture antibody by: drawing the first capture antibody with a concentration of 0.3 to 1.5 mg / mL as the first detection line on the coating membrane for coating, and the coating amount is 0.8 to 2 μL / cm of membrane;

[0082] In some embodiments, the coating method of the second detection line with the first complex is: the first complex is drawn on the coating membrane as the second detection line for coating, and the coating amount is 0.8-2 μL / cm membrane, wherein the concentration of the captured antigen in the first complex is 0.3-1.5 mg / mL.

[0083] In some embodiments, the coating method of the quality control line with anti-DNP antibody is: the anti-DNP antibody with a concentration of 0.3-1.5 mg / mL is drawn on the coating membrane as the quality control line, and the coating amount is 0.8-2 μL / cm membrane.

[0084] In some embodiments, the method for preparing the coating solution comprises:

[0085] a. First test line coating solution: dilute NT-proBNP antibody 2 to a concentration of 0.3-1.5 mg / mL with 10 mM PB or PBS buffer at pH 7.2-8.0.

[0086] b. Second detection line coating solution: dilute the NT-proBNP protein to a concentration of 0.3-1.5 mg / mL with 10 mM PB or PBS buffer at pH 7.2-8.0, then add NT-proBNP antibody 2 at a mass ratio of 1:0.1-0.5, mix and react specifically to obtain the first complex.

[0087] c. Quality control line coating solution: dilute the anti-DNP antibody to a concentration of 0.3-1.5 mg / mL with 10 mM PB or PBS buffer at pH 7.2-8.0.

[0088] In some embodiments, the 10 mM PB or PBS buffer at pH 7.2-8.0 contains 2.0% sucrose by mass.

[0089] In some embodiments, the coating method of coating the coating membrane with the coating solution comprises:

[0090] The three coating solutions were coated on the coating membrane at a coating amount of 0.8 to 2 μL / cm membrane, and then dried at 37° C. to 55° C. for 2 to 24 hours.

[0091] In some embodiments, the coating film is provided with a first detection line, a second detection line, and a quality control line in order from front to back in the sample flow direction. The line spacing between the first detection line and the second detection line is greater than or equal to 4 mm. The line spacing between the second detection line and the quality control line is greater than or equal to 4 mm.

[0092] In some embodiments, the coating film is provided with a first detection line, a quality control line, and a second detection line in order from front to back in the sample flow direction. The line spacing between the first detection line and the quality control line is greater than or equal to 4 mm. The line spacing between the quality control line and the second detection line is greater than or equal to 4 mm.

[0093] In some embodiments, the coating film is provided with a quality control line, a first detection line, and a second detection line in order from front to back in the sample flow direction. The line spacing between the quality control line and the first detection line is greater than or equal to 4 mm. The line spacing between the first detection line and the second detection line is greater than or equal to 4 mm.

[0094] In some embodiments, the first fluorescent microsphere is one of fluorescein, time-resolved fluorescent microspheres, phycoerythrin, rhodamine red, and 2-methoxyfluorescein.

[0095] In some embodiments, the second fluorescent microsphere is one of fluorescein, time-resolved fluorescent microspheres, phycoerythrin, rhodamine red, and 2-methoxyfluorescein.

[0096] Principle of time-resolved fluorescence immunochromatography (TRFILF) double antibody sandwich method

[0097] When the sample containing the antigen to be tested is dropped on the sample addition area, the antigen in the sample to be tested combines with the antibody labeled with the fluorescent nano-microspheres in the sample binding pad and chromatographs forward through capillary action. When it reaches the detection area, it combines with the antibody fixed on the detection line to form a particle-antibody-antigen-antibody sandwich complex and is fixed on the detection line. After the reaction is completed, the detection area is scanned and detected with an ultraviolet light source (365nm). The fluorescent nano-microspheres on the detection line emit high-intensity fluorescence (615nm) and the decay time is also relatively long. By delaying the measurement time, the specific fluorescence of the rare earth element is measured after the naturally occurring short-lived fluorescence (1 to 10ns) in the sample matrix has completely decayed, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the analyte in the sample can be analyzed by the intensity of the fluorescence intensity of the detection line.

[0098] The test strip determines whether the concentration of the antigen to be tested exceeds the limit through the second detection line, thereby avoiding the HOOK effect caused by the excess and thus misjudging the concentration.

[0099] Detection principle: When the concentration of the antigen to be tested in the sample is not excessive, as the concentration of the antigen to be tested increases, the concentration of the double antibody sandwich complex first fluorescent microsphere marker-antigen to be tested-NT-proBNP antibody 2 enriched in the first detection line increases accordingly.

[0100] When the concentration of the antigen to be tested in the sample exceeds the limit, the double antibody sandwich complex enriched in the first detection line decreases as the concentration of the antigen to be tested increases, while the captured antigen in the second detection line competes with the antigen to be tested, and the first fluorescent microsphere marker enriched in the second detection line decreases as the concentration of the antigen to be tested increases until it is close to zero.

[0101] Please refer to the attached Figure 1 The disclosed embodiment also provides a fluorescent immunochromatographic detection card, which includes the above-mentioned test strip and a card shell.

[0102] The card shell includes an upper card shell and a lower card shell that are locked to each other. The inner surface of the lower card shell is provided with a card slot for placing the test strip. The upper card shell is provided with a sample addition port corresponding to the sample binding pad and an observation port corresponding to the coating membrane. The first detection line, the second detection line and the quality control line are all exposed at the observation port.

[0103] The card shell not only protects the test strip from damage and contamination, but also plays a role in fixing the test strip, making it less likely to slide and affect the measurement. The upper and lower card shells can fix and press the test strip, and the labeled antibody, labeled protein and sample diluent on the sample binding pad can run synchronously to ensure uniform liquid flow rate, thereby further reducing the CV coefficient and improving precision and accuracy.

[0104] In some embodiments, the test strip is cut into a width greater than or equal to 2.5 mm and then loaded into the cartridge.

[0105] The disclosed embodiments also provide a fluorescent immunochromatographic detection kit, which includes the above-mentioned test strip or the above-mentioned detection card.

[0106] The kit also includes a sample diluent;

[0107] The sample buffer comprises at least one of an anti-red blood cell antibody (anti-RBC antibody) and a blocking agent, wherein the blocking agent is a HAMA blocking agent and / or an HBR blocking agent.

[0108] Anti-RBC antibodies can bind to red blood cells and agglutinate into macromolecules. When adding samples, red blood cells can be retained on the sample binding pad to prevent red blood cells from chromatographing on the coating membrane and interfering with the fluorescent signal of the detection line, thereby realizing the detection of various types of samples.

[0109] For patients with autoimmune diseases and underlying diseases, autoimmune antibodies in the human body (such as rheumatoid factor, anti-mitochondrial antibodies, HAMA, etc.) often interfere with the test results, produce non-specific immune reactions, and produce false positives, leading to inaccurate test results. Blockers can greatly reduce non-specific reactions in samples, thereby avoiding the false positive problem of traditional methods.

[0110] The sample buffer has a diluting effect on the sample, which can reduce the viscosity of the sample and more effectively release the fluorescent markers coated on the sample binding pad, thereby improving the precision of the detection.

[0111] In some embodiments, the blocker is an active blocker and / or a passive blocker.

[0112] In some embodiments, the sample buffer comprises: 10-50 mM Tris solution, and the following mass fractions: 0.001-0.01% blocking agent, 0.001-0.01% anti-RBC antibody, 0.05-0.5% casein sodium (Casein-Na), 0.4-0.9% NaCl, 0.01-0.2% Tween-20, and 0.05-0.2% PC-300.

[0113] The disclosed embodiment also provides a method for using the above-mentioned kit, after the sample and the sample buffer are evenly mixed, they are added to the test strip for reaction, and then the fluorescence intensity on the first test line, the second test line and the quality control line are detected respectively by excitation through a fluorescent immunoassay analyzer, and according to the detection value of the fluorescent signal enriched by the second test line, it is judged whether the concentration of the antigen to be tested in the sample is within the linear detection range of the test strip.

[0114] In some embodiments, the ratio of the detection value of the second detection line to the detection value of the quality control line is taken as the first ratio.

[0115] If the first ratio is less than the first ratio of the linear upper limit of the standard curve when testing the sample, it is determined that the concentration of the antigen to be tested in the sample exceeds the linear detection range. If not less than, the concentration of the antigen to be tested in the sample is calculated using the standard curve.

[0116] In some embodiments, the sample is a whole blood sample.

[0117] Example 1

[0118] Preparation of fluorescent immunochromatographic kit for N-terminal B-type natriuretic peptide

[0119] 1. Preparation of sample conjugate pad

[0120] (1) Preparation of the first fluorescent microsphere marker:

[0121] a. Wash the fluorescent microspheres: Take 1 mg of fluorescent microspheres and add them to 400 μL of MES buffer at pH 6.0. After ultrasonic mixing for 1 min, centrifuge at 4°C, 15,000 rpm for 15 min, remove the supernatant, repeat twice, and finally suspend the precipitate in 400 μL of the above MES buffer and ultrasonically mix for 1 min.

[0122] b. Activate fluorescent microspheres: Dissolve NHS in purified water to a concentration of 10 mg / mL. Dissolve EDC in purified water to a concentration of 10 mg / mL.

[0123] 10 μL of NHS aqueous solution and 10 μL of EDC aqueous solution were added to the washed fluorescent microspheres in sequence, and the mixture was shaken for 30 seconds and then rotated for 1 hour.

[0124] After activation, the liquid was centrifuged at 4°C, 15000 rpm for 15 min, the supernatant was removed, and 400 μL of the above-mentioned MES buffer was added, and ultrasonic mixing was performed for 1 min, and this was repeated twice.

[0125] c. Conjugated antibody: dilute NT-proBNP antibody 1 with the above-mentioned MES buffer to a concentration of 1 mg / mL, take 100 μL of the diluted protein solution and put it into the activated fluorescent microspheres, shake and mix for 30 seconds, and rotate and mix for 1 hour.

[0126] d. Blocking microspheres: Add 50 μL of 5 mg / mL bovine serum albumin aqueous solution and 50 μL of 5 mg / mL glycine aqueous solution to the coupled fluorescent microspheres, shake and mix for 30 seconds, and rotate and mix for 2 hours.

[0127] e. Washing and storage: The blocked microspheres were centrifuged at 4°C, 15,000 rpm for 15 min. After removing the supernatant, 50 mM pH 8.0 Hepes buffer containing 0.5% Tween-20 was added and washed by centrifugation twice. Finally, the precipitate was stored in 100 μL of 50 mM pH 8.0 Hepes buffer.

[0128] (2) Preparation of the second fluorescent microsphere marker:

[0129] Steps a, b, d and e are the same as those for preparing the first fluorescent microsphere marker.

[0130] c. Conjugated antibody: dilute DNP-BSA with the above-mentioned MES buffer to a concentration of 1 mg / mL, take 100 μL of the diluted protein solution and put it into the activated fluorescent microspheres, shake and mix for 30 seconds, and rotate and mix for 1 hour.

[0131] (3) Preparation of spraying solution: dilute the preservation solution containing the labeled first fluorescent microsphere marker and the preservation solution containing the second fluorescent microsphere marker in a dilution buffer to obtain a spraying solution.

[0132] The volume ratio of the preservation solution containing the labeled first fluorescent microsphere marker, the preservation solution containing the second fluorescent microsphere marker, and the dilution buffer solution is 1:1:20.

[0133] The dilution buffer is a 50 mM Tris buffer at pH 8.0 containing 0.85% NaCl, 0.1% PEG2000, 0.1% BSA, 0.2% Tween-20, and 5.0% trehalose by mass fraction.

[0134] (4) Preparation of sample binding pad: Spray the spray solution onto the glass cellulose membrane sample binding pad at a rate of 3 μL / cm of sample binding pad, and then dry at 45° C. for 4 h.

[0135] 2. Coating membrane preparation

[0136] (1) Preparation of coating solution

[0137] a. Detection line 1 coating solution: dilute NT-proBNP antibody 2 to a concentration of 0.8 mg / mL using 10 mM pH 7.4 PB buffer containing 2.0% sucrose by mass.

[0138] b. Detection line 2 coating solution: dilute the NT-proBNP protein to a concentration of 0.8 mg / mL using PB buffer containing 2.0% sucrose by mass, then add NT-proBNP antibody 2 at a mass ratio of 1:0.5, mix and react specifically to obtain the first complex.

[0139] c. Quality control line coating solution: dilute the anti-DNP antibody to a concentration of 1.0 mg / mL using PB buffer containing 2.0% sucrose.

[0140] (2) Preparation of coating membrane

[0141] The three coating solutions were coated on the nitrocellulose membrane at a coating volume of 1.0 μL / cm membrane. According to the flow direction of the sample on the nitrocellulose membrane, the test line 1, test line 2 and quality control line were coated in sequence, and the lines were drawn parallel on the nitrocellulose membrane for coating, with the line spacing greater than or equal to 4 mm. Then dried at 45°C for 4 hours.

[0142] 3. Test card assembly:

[0143] Glue the sample conjugate pad, nitrocellulose membrane and absorbent paper onto the PVC plate with double-sided tape in sequence. Cut into 4mm width and put into the card case, then press the card case to tighten.

[0144] 4. Preparation of sample buffer:

[0145] The sample buffer includes: 10 mM pH 8.0 Tris solution, and the following mass fractions: 0.005% HBR blocking agent, 0.005% anti-RBC antibody, 0.1% Casein-Na, 0.85% NaCl, 0.2% Tween-20, and 0.05% PC-300.

[0146] Comparative Example

[0147] The difference between the comparative example and implementation case 1 is that:

[0148] 2. Preparation of coating membrane (1) Preparation of coating solution b. Detection line 2 coating solution:

[0149] The NT-proBNP protein was diluted to a concentration of 0.8 mg / mL using 10 mM pH 7.4 PB buffer containing 2.0% sucrose by mass.

[0150] That is, detection line 2 is only coated with NT-proBNP protein.

[0151] Test Example 1

[0152] Determination of standard curve

[0153] The NT-proBNP protein standard with a concentration of 1,000,000 ng / mL was diluted with a 0.01 M pH 7.4 PBS solution containing 1% BSA by mass to prepare standard solutions with gradient concentrations as shown in Table 1.

[0154] Take 20 μL of the standard solution and add it to 180 μL of the sample buffer in Example 1. After mixing, take 80 μL of the mixed solution and add it to the sample injection port of the test card of the kit in Example 1.

[0155] Place the test card into the fluorescent immunoassay instrument, and read the fluorescence intensity of test line 1, test line 2, and quality control line after 15 minutes of reaction.

[0156] The test data are shown in Table 1. With T1 / C as x and the logarithm of the analyte concentration as y, the standard curve trend diagram is drawn as follows: Figure 2 After analysis, the standard curve fitting diagram within the linear detection range is shown in Figure 3 As shown. The linear regression line obtained by fitting: y = 0.0443e 0.7036x , correlation coefficient R = 0.999.

[0157] The linear detection range of the kit is 20-35000 ng / mL. When the analyte concentration reaches above 35000 ng / mL, the corresponding T1 / C value does not show an obvious upward trend with increasing concentration, or begins to show a downward trend with increasing concentration, without a linear relationship, indicating that the kit has produced a HOOK effect at this analyte concentration.

[0158] Table 1 Standard curve data

[0159]

[0160] Sample testing:

[0161] (1) Take 20 μL of plasma sample or 30 μL of whole blood sample and add it to 180 μL of sample buffer and mix well to obtain a mixed solution.

[0162] (2) Take 80 μL of the mixed solution and add it to the sample injection port of the test card in Example 1.

[0163] (3) Place the test card into the fluorescence immunoassay instrument and read the fluorescence intensity of test line 1, test line 2, and quality control line after 15 minutes of reaction.

[0164] First, the ratio of the detection value of the fluorescent signal of the detection line 2 to the detection value of the fluorescent signal of the quality control line is predicted. If the ratio is lower than the ratio corresponding to the linear upper limit concentration of NT-proBNP of 35000 ng / mL, it is determined that the concentration of NT-proBNP in the sample is greater than 35000 ng / mL, which exceeds the linear detection range.

[0165] If the ratio is higher than the ratio corresponding to the linear upper limit concentration of NT-proBNP of 35000 ng / mL, the ratio of the detection value of the fluorescence signal of the detection line 1 to the detection value of the fluorescence signal of the quality control line is substituted into the standard curve to calculate the concentration of NT-proBNP in the sample.

[0166] Test Example 2

[0167] Linearity test of the kit

[0168] Please refer to Table 2, and use NT-proBNP standards with concentrations of 20 ng / mL and 35000 ng / mL to prepare detection solutions with gradient concentrations according to the mixing ratios shown in Table 2.

[0169] Each concentration of the test solution was tested three times using the kit in Example 1 to obtain the concentration test value. The mean of the test value and the theoretical concentration were subjected to linear regression and correlation coefficient analysis. The test data are shown in Table 2.

[0170] Table 2 Linear test data

[0171]

[0172] The linear regression line obtained by analysis is: y=0.9862x+49.934, and the correlation coefficient R=0.999.

[0173] The linear regression slope is within 1.0±0.1, and the correlation coefficient R>0.99, which indicates that the linear test of the kit provided in the embodiment of the present disclosure has a high linear correlation.

[0174] Precision test of the kit

[0175] The kit in Example 1 was tested with clinical whole blood and plasma samples, and each sample was tested 10 times. The mean and standard deviation were calculated, and the coefficient of variation CV = mean / standard deviation × 100%. The test results are shown in Table 3.

[0176] Table 3 Precision test data

[0177]

[0178] The CV values ​​of the plasma sample and the whole blood sample tests were both less than 11%, indicating that the kit provided in the embodiments of the present disclosure has good precision.

[0179] Test kit HOOK effect

[0180] The standard with a concentration of 500000 ng / mL was diluted to a concentration of 50000 ng / mL, 80000 ng / mL, 150000 ng / mL, and 200000 ng / mL, respectively. The standard and the above dilutions were detected using the kit in Example 1. The test results are shown in Table 4.

[0181] Table 4 Test data of HOOK effect

[0182]

[0183] It can be seen that the test results all determined that the concentration was greater than 35000 ng / mL, indicating that the analyte concentration exceeded the linear detection range, and the kit provided in the embodiments of the present disclosure can accurately determine the excess, and the test results did not show false negative or weak positive misjudgment.

[0184] Blank limit test

[0185] The kits of Example 1 and the comparative example were tested with the reference sample diluent, each tested 20 times, and the mean and standard deviation (SD) were calculated. The blank limit was the mean + 2SD. The test results are shown in Table 5.

[0186] Table 5 Blank limit test data

[0187]

[0188] It can be seen that the blank limit of the reference dilution in Example 1 is 13.27 ng / mL, which is lower than the linear lower limit of 20 ng / mL, while the concentration detection value of the reference dilution in the comparative test has large fluctuations, and there are multiple concentration detection values ​​greater than 300 ng / mL, that is, false positive test. This indicates that the test line T1 was contaminated during the comparative test.

[0189] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A fluorescent immunochromatographic test strip, characterized in that: The test strip comprises: a sample binding pad and a coating membrane; The sample binding pad is adsorbed with a first fluorescent microsphere marker, which is formed by coupling a labeled antibody to the first fluorescent microsphere, and the labeled antibody can bind to the first site of the antigen to be detected; The coating membrane is provided with a first detection line and a second detection line in sequence from front to back in the sample flow direction, the first detection line is coated with a first capture antibody, and the second detection line is coated with a first complex; the first complex is formed by the specific combination of the capture antigen and the first capture antibody; The first capture antibody can bind to the second site of the antigen to be detected, and the capture antigen can compete with the antigen to be detected for binding to the labeled antibody.

2. The test strip according to claim 1, characterized in that: The capture antigen and the first capture antibody are mixed and specifically reacted to obtain the first complex, wherein the capture antigen and the first capture antibody are mixed at a mass ratio of 1:0.1-0.

5.

3. The test strip according to claim 1, characterized in that: The test strip also includes a quality control line.

4. The test strip according to claim 1, characterized in that: The antigen to be detected is NT-proBNP.

5. The test strip according to claim 1, characterized in that: The first fluorescent microsphere is one of fluorescein, time-resolved fluorescent microspheres, phycoerythrin, rhodamine red, and 2-methoxyfluorescein.

6. The test strip according to claim 1, characterized in that: The mass ratio of the first fluorescent microsphere to the labeled antibody is 1:0.03-0.

3.

7. The test strip according to claim 1, characterized in that: The coating method of the first detection line with the first capture antibody is: the first capture antibody with a concentration of 0.3-1.5 mg / mL is drawn on the coating membrane as the first detection line for coating, and the coating amount is 0.8-2 μL / cm membrane.

8. The test strip according to claim 2, characterized in that: The coating method of the second detection line with the first complex is: the first complex is drawn on the coating membrane as the second detection line for coating, and the coating amount is 0.8-2 μL / cm membrane, wherein the concentration of the captured antigen in the first complex is 0.3-1.5 mg / mL.

9. A kit, characterized in that: The kit comprises the test strip according to any one of claims 1 to 8; The kit also includes a sample buffer; The sample buffer comprises at least one of an anti-erythrocyte antibody and a blocking agent, wherein the blocking agent is a HAMA blocking agent and / or an HBR blocking agent.

10. A method for using the kit according to claim 9, characterized in that: After the sample and the sample buffer are evenly mixed, they are added to the test strip for reaction, and then based on the detection value of the fluorescent signal enriched by the second detection line, it is determined whether the concentration of the antigen to be tested in the sample is within the linear detection range of the test strip.

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