Homocysteine competition method immunochromatography test paper based on antigen-antibody complex specific antibody, kit and application thereof

Abstract

The invention belongs to the technical field of preparation of in-vitro diagnostic products, and particularly relates to homocysteine competition method immunochromatography test paper based on an antigen-antibody complex specific antibody, a kit and application of the homocysteine competition method immunochromatography test paper and the kit. The homocysteine competition method immunochromatography test paper comprises a quality control line coated with an antibody and a detection line coated with an S-(5 '-adenosine)-L-cysteine and bovine serum albumin coupling antigen, wherein the antibody is a compound antibody of a solid phase carrier marked by an (S-(5 '-adenosine)-L-cysteine monoclonal antibody and an (S-(5'-adenosine)-L-cysteine monoclonal antibody, and the (S-(5 '-adenosine)-L-cysteine monoclonal antibody and the (S-( The compound antibody is obtained by compounding S-(5 '-adenosine)-L-cysteine serving as an antigen and a solid-phase carrier labeled by an S-(5'-adenosine)-L-cysteine monoclonal antibody, immunizing and purifying. According to the homocysteine competition method immunochromatography test paper provided by the invention, negative correlation linkage of the C line signal intensity and the T line is realized, the dynamic range of the T / C ratio is widened, and the homocysteine detection precision is improved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic product preparation, specifically relating to a homocysteine ​​competitive immunochromatographic test strip, kit, and its application based on antigen-antibody complex-specific antibodies. Background Technology

[0002] Homocysteine ​​(HCY) is a key biomarker for cardiovascular and cerebrovascular diseases, and its detection is of vital importance in clinical diagnosis.

[0003] Competitive immunochromatography is a rapid immunoassay technique based on the principle of antigen-antibody competitive binding. It is primarily used to detect small molecules containing only a single antigenic epitope (such as toxins, drug residues, hormones, etc.). Its core mechanism involves the target antigen in the sample competing with the fixed antigen (or analogue) on the detection line (T line) for binding to a limited number of gold-labeled antibodies, resulting in a negative correlation between the colorimetric signal and the concentration of the target antigen. Due to its ease of operation and rapid detection, competitive immunochromatography has been widely used in the field of HCY detection.

[0004] In existing technologies, the control line (C line) of immunochromatographic test strips used for HCY detection is typically designed using goat anti-mouse secondary antibody, rabbit IgG (immunoglobulin G) / goat anti-rabbit IgG, or chicken IgY (immunoglobulin Y) / goat anti-chicken antibody. These antibodies are mainly used to provide quality control signals, but their correlation with the detection line (T line) is weak. This results in a limited dynamic range for the T / C ratio, especially when the HCY concentration is in extreme ranges (e.g., <5 μmol / L or >30 μmol / L). The signal differences between the T line and the C line are difficult to distinguish effectively, directly affecting the precision of the detection results and making the reliability of the results insufficient. Summary of the Invention

[0005] The existing technology has the following problem: Although it uses a common secondary antibody as the C-line design, it fails to effectively solve the linkage between the T / C ratio and antigen concentration. In actual clinical testing, the T / C signal multiple within the linear dynamic range cannot reach the ideal state, making it difficult to meet the clinical needs for accurate detection of extreme value samples, thus limiting the application value of competitive immunochromatography in HCY detection.

[0006] To address the problems existing in the prior art, the present invention provides a homocysteine ​​competitive immunochromatographic test strip, a reagent kit, and their applications based on antigen-antibody complex-specific antibodies. The specific technical solution is as follows: Technical Solution 1: A homocysteine ​​competitive immunochromatographic test strip, characterized in that it includes an antibody-coated control line and a detection line coated with S-(5'-adenosine)-L-cysteine ​​and bovine serum albumin conjugate antigen; The antibody is a complex antibody of a solid-phase carrier labeled with a monoclonal antibody of S-(5'-adenosine)-L-cysteine ​​and S-(5'-adenosine)-L-cysteine. The complex antibody is prepared by a method comprising the following steps: conjugating the solid-phase carrier labeled with a monoclonal antibody of S-(5'-adenosine)-L-cysteine ​​as an antigen with the complex, and purifying the complex after immunization.

[0007] Technical Solution 2: The immunochromatographic test strip according to Technical Solution 1, characterized in that, in the complex, the mass ratio of S-(5'-adenosine)-L-cysteine ​​as the antigen to the solid-phase carrier labeled with S-(5'-adenosine)-L-cysteine ​​monoclonal antibody is 1:1-10:1. Preferably, the solid-phase carrier labeled with the S-(5'-adenosine)-L-cysteine ​​monoclonal antibody is prepared by using the S-(5'-adenosine)-L-cysteine ​​monoclonal antibody and the solid-phase carrier, wherein the mass ratio of the S-(5'-adenosine)-L-cysteine ​​monoclonal antibody to the solid-phase carrier is 0.5-1.5:9.9-10.1. The S-(5'-adenosine)-L-cysteine-bovine serum albumin conjugate antigen was prepared by mixing S-(5'-adenosine)-L-cysteine ​​and bovine serum albumin, wherein the mass ratio of S-(5'-adenosine)-L-cysteine ​​to bovine serum albumin was 1:5-10.

[0008] Technical Solution 3: The immunochromatographic test strip according to Technical Solution 1 or 2 is characterized in that the mass concentration of antibody protein in the complex antibody is ≥2 mg / mL, preferably, the mass concentration of antibody protein in the complex antibody is 2-5 mg / mL, and more preferably, the antibody protein includes one of mouse immunoglobulin G or rabbit immunoglobulin G.

[0009] Technical Solution 4: The immunochromatographic test strip according to any one of technical solutions 1-3 is characterized in that the solid phase support comprises one of colloidal gold, colored latex microspheres or time-resolved fluorescent latex microspheres, preferably, the particle size range of the solid phase support is 20-400 nm.

[0010] Technical Solution 5: The immunochromatographic test strip according to any one of Technical Solutions 1-4, characterized in that the solid-phase support is time-resolved fluorescent latex microspheres. Preferably, the labeled fluorescein in the time-resolved fluorescent latex microspheres includes a fluorescent substance with an excitation wavelength of 355-375 nm and an emission wavelength of 600-620 nm. More preferably, the fluorescent substance is a compound containing rare earth elements. More preferably, the rare earth element is europium.

[0011] Technical Solution 6: The immunochromatographic test strip according to any one of Technical Solutions 1-5, characterized in that the content of bovine serum albumin as a carrier protein in the S-(5'-adenosine)-L-cysteine-bovine serum albumin conjugate antigen of the detection line is 0.5-2 mg / mL.

[0012] Technical Solution 7: An immunochromatographic test strip according to any one of technical solutions 1-6, characterized in that the immunochromatographic test strip includes a backing plate and a sample pad, an enzyme conjugate pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially laid on the backing plate, wherein the nitrocellulose membrane is sequentially provided with a detection line strip and a control line strip.

[0013] Technical Solution 8: The immunochromatographic test strip according to any one of Technical Solutions 1-7, characterized in that the sample pad is glass fiber coated with a reducing agent to reduce bound homocysteine ​​to free homocysteine; preferably, the reducing agent is dithiothreitol, more preferably, a 5-50 mM dithiothreitol solution. Technical Solution 9: The immunochromatographic test strip according to any one of Technical Solutions 1-8, characterized in that the enzyme-conjugating pad is polyester cellulose coated with S-(5'-adenosine)-L-cysteine ​​tool enzyme and adenosine, wherein the S-(5'-adenosine)-L-cysteine ​​tool enzyme is an enzyme capable of catalyzing the reaction of free homocysteine ​​with adenosine to generate S-(5'-adenosine)-L-cysteine, preferably, the S-(5'-adenosine)-L-cysteine ​​tool enzyme includes recombinant enzymes or naturally extracted enzymes, more preferably, the S-(5'-adenosine)-L-cysteine ​​tool enzyme is an S-adenosine homocysteine ​​hydrolase. And / or the binding pad is polyester cellulose coated with a solid-phase carrier labeled with the S-(5'-adenosine)-L-cysteine ​​monoclonal antibody. And / or the material of the absorbent pad is plant fiber.

[0014] Technical Solution 10: The immunochromatographic test strip according to any one of technical solutions 1-9, characterized in that, during the chromatographic reaction, the molar ratio of S-(5'-adenosine)-L-cysteine ​​tool enzyme and adenosine in the enzyme conjugate pad is 1:5-50, preferably 1:45-50.

[0015] Technical Solution 11: The immunochromatographic test strip according to any one of technical solutions 1-10, characterized in that the binding pad contains a solid-phase carrier labeled with the S-(5'-adenosine)-L-cysteine ​​monoclonal antibody. And / or the purification is performed by chromatography using a Protein A affinity column or a Protein G affinity column.

[0016] Technical Solution 12: A homocysteine ​​competitive immunochromatographic assay kit, characterized in that it includes a detection unit and a reagent unit, wherein the detection unit is an immunochromatographic test strip as described in any one of Technical Solutions 1-11.

[0017] Technical Solution 13: The immunochromatographic reagent kit of Technical Solution 12, characterized in that the reagent unit is a diluent used to dilute the sample to be tested by 10-20 times. Preferably, each L of the diluent includes a Tris-HCl solution with a pH of 7.0±0.05 at a concentration of 10-100 mM, bovine serum albumin at a concentration of 0.1-0.5 w / v%, and Tween 20 at a concentration of 0.01-0.05 v / v.

[0018] Technical Solution 14: The immunochromatographic reagent kit described in Technical Solution 12 or 13, characterized in that the sample to be tested includes one of serum, plasma or whole blood.

[0019] Technical Solution 15: A competitive immunochromatographic detection method for homocysteine, characterized in that it uses the immunochromatographic reagent described in any one of Technical Solutions 12-14.

[0020] Technical Solution 16: The immunochromatographic detection method described in Technical Solution 15 is characterized by the following steps: (1) Pre-treatment of the sample: The sample is mixed with the sample diluent to obtain a diluted sample, wherein the dilution factor is 10-20 times; (2) Immunochromatographic reaction: 60-120 μL of the diluted sample obtained in step (1) is added to the sample pad for immunochromatographic reaction, preferably, the immunochromatographic reaction time is 10-15 min; (3) Reading signal values: After the immunochromatographic reaction is completed, the fluorescence signal values ​​or gray values ​​of the T line and C line are read to obtain the T value and C value. The T / C ratio is calculated based on the T value and C value to quantitatively determine the homocysteine ​​concentration, that is, the homocysteine ​​competitive immunochromatographic detection is completed.

[0021] Technical Solution 17: The immunochromatographic detection method described in Technical Solution 15 or 16 is characterized in that, in step (3), the quantitative judgment result is: if the homocysteine ​​concentration in the sample increases, the T line signal decreases, the C line signal increases, and the T / C ratio decreases significantly. When the concentration of homocysteine ​​in the sample decreases, the T-line signal increases, the C-line signal decreases, and the T / C ratio increases.

[0022] Technical Solution 18: The immunochromatographic test strip of any one of Technical Solutions 1-11 or the immunochromatographic reagent kit of any one of Technical Solutions 12-14, characterized in that it is used in the preparation of products for diagnosing or monitoring cardiovascular and cerebrovascular diseases.

[0023] Beneficial effects of this invention: This invention involves conjugating S-(5'-adenosine)-L-cysteine ​​(as an antigen) with a solid-phase carrier labeled with an S-(5'-adenosine)-L-cysteine ​​monoclonal antibody. The resulting complex is then purified after immunization to obtain an antibody conjugate of the S-(5'-adenosine)-L-cysteine ​​and S-(5'-adenosine)-L-cysteine ​​monoclonal antibody-labeled solid-phase carrier, which serves as a control line (C line). This antibody exhibits high specificity, accurately recognizing the complex as an immunogen and demonstrating a negative correlation between the C line signal intensity and the T line. This approach effectively broadens the dynamic range of the T / C ratio, significantly improving detection precision and meeting the urgent clinical need for high-precision HCY detection, providing stronger technical support for the early diagnosis and monitoring of cardiovascular and cerebrovascular diseases. Attached Figure Description

[0024] Figure 1 These are top and side views of the structure of the chromatography test strip; Figure 2 To compare the linear range of T / C values ​​in samples with HYC concentrations ranging from 0 to 51.92 μmol / mL; Figure 3 To detect HCY standard solutions at different dilution ratios using traditional detection methods, a linear regression equation was obtained, along with data on the theoretical concentration, measured concentration, and relative deviation of the HCY standard solutions. Figure 4 To utilize the detection method of this invention to detect HCY standard solutions at different dilution ratios, a linear regression equation was obtained, along with data on the theoretical concentration, measured concentration, and relative deviation of the HCY standard solutions. Detailed Implementation

[0025] To address the problems existing in the prior art, this invention innovatively selects a complex of S-(5'-adenosine)-L-cysteine ​​(hereinafter referred to as SAH) as the antigen and a solid-phase carrier labeled with S-(5'-adenosine)-L-cysteine ​​monoclonal antibody (hereinafter referred to as SAH monoclonal antibody-labeled solid-phase carrier). The antibody obtained after immunizing and purifying this complex (i.e., the complex antibody of SAH and SAH monoclonal antibody-labeled solid-phase carrier) is used as the coating antibody for the control line (C line). This complex antibody has high specificity and can accurately recognize the immunogen (i.e., the "SAH and SAH monoclonal antibody-labeled solid-phase carrier complex"). Through this unique design, a negative correlation between the C line signal and the T line signal is achieved. Specifically, when the HCY concentration in the sample increases, the T line signal decreases, the C line signal increases, and the T / C ratio decreases significantly; conversely, when the HCY concentration decreases, the T line signal increases, the C line signal decreases, and the T / C ratio increases. This signal linkage mechanism effectively improves the concentration gradient spacing within the linear range of 2-50 μmol / L, significantly enhancing detection precision with an intra-assay coefficient of variation (CV) of less than 5%. This invention successfully solves the problem of insufficient discrimination in traditional immunochromatography when detecting extreme value samples, enabling more precise differentiation of HCY samples at different concentrations. It provides a reliable technical means for high-precision clinical detection of HCY and has significant clinical application value.

[0026] The immunochromatographic test strip provided by this invention contains a T line and a C line. The T line is coated with an SAH / BSA (bovine serum albumin) conjugate antigen, and the C line is coated with a complex antibody of SAH and a solid-phase carrier labeled with SAH monoclonal antibody. When a sample containing HCY is added for reaction, the competitive immunochromatographic principle and signal linkage mechanism of this invention are as follows: The principle of competitive chromatography-immunoassay: During the detection process, the T line is coated with SAH and BSA conjugated antigen, which competes with SAH generated from HCY transformation in the sample for binding to the SAH monoclonal antibody-labeled solid-phase carrier, and can only bind to the SAH monoclonal antibody-labeled solid-phase carrier. The C line, on the other hand, is coated with anti-(SAH and SAH monoclonal antibody-labeled solid-phase carrier) antibody, which specifically binds to the complex formed by the combination of HCY-transformed SAH and the SAH monoclonal antibody-labeled solid-phase carrier in the sample.

[0027] The signal correlation mechanism is as follows: When the HCY concentration in the sample increases, the amount of SAH generated by transformation increases accordingly, leading to an increase in the number of SAH-SAH monoclonal antibody-labeled solid-phase carrier complexes and a decrease in the number of free SAH monoclonal antibody-labeled solid-phase carriers. This series of changes ultimately results in a weakening of the T-line signal, an enhancement of the C-line signal, and a significant decrease in the T / C ratio. Conversely, when the HCY concentration in the sample (e.g., serum, plasma, calibrator, or quality control) decreases, the amount of transformed SAH decreases, the number of SAH-SAH monoclonal antibody-labeled solid-phase carrier complexes in the sample decreases, and the number of free SAH monoclonal antibody-labeled solid-phase carriers increases, resulting in an enhancement of the T-line signal, a weakening of the C-line signal, and a significant increase in the T / C ratio. Through this unique signal linkage mechanism, a non-linear amplification effect of the T / C ratio changing with HCY concentration is achieved, effectively expanding the T / C ratio difference between different concentrations within the linear range, greatly broadening the dynamic linear range between concentrations, and significantly improving the precision of detection.

[0028] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely explained below in conjunction with specific embodiments. It should be noted that the content of the specific embodiments is only a specific implementation and explanation of the technical solutions of the present invention, and should not be construed as a limitation on the scope of protection of the present invention.

[0029] In some specific embodiments, the present invention provides an HCY competitive immunochromatographic test strip, characterized in that it includes an antibody-coated control line and a detection line coated with SAH and BSA conjugate antigen. The antibody is a complex antibody of SAH and a solid-phase carrier labeled with SAH monoclonal antibody. The complex antibody is prepared by a method including the following steps: SAH as an antigen is compounded with a solid-phase carrier labeled with SAH monoclonal antibody to obtain a complex, and the complex is purified after immunization.

[0030] Preferably, the mass ratio of SAH as antigen to the solid-phase carrier labeled with SAH monoclonal antibody is 1:1 to 10:1. More preferably, the mass ratio of SAH as antigen to the solid-phase carrier labeled with SAH monoclonal antibody can be 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1 or 10:1, or any two of the above values ​​constitute the mass ratio of SAH as antigen to the solid-phase carrier labeled with SAH monoclonal antibody within the numerical range.

[0031] Preferably, the SAH monoclonal antibody-labeled solid-phase carrier is prepared by using SAH monoclonal antibody and a solid-phase carrier, wherein the mass ratio of SAH to solid-phase carrier is 0.5-1.5:9.9-10.1. More preferably, the mass ratio of SAH monoclonal antibody to solid-phase carrier can be 0.5:9.9, 0.55:9.9, 0.6:9.9, 0.65:9.9, 0.7:9.9, 0.75:9.9, 0.8:9.9, 0.85:9.9, 0.90:9.9, or 0.95:9. 0.9, 1.0:9.9, 1.05:9.9, 1.1:9.9, 1.15:9.9, 1.2:9.9, 1.25:9.9, 1.3:9.9, 1.35:9.9, 1.4:9.9, 1.45:9.9, 1.5:9.9, 0.5:10.0, 0.55:10.0, 0.6:10.0, 0.65:10.0, 0.7:10.0, 0.75:10.0, 0.8:10.0, 0.85:10.0, 0.90:10.0 , 0.95:10.0, 1.0:10.0, 1.05:10.0, 1.1:10.0, 1.15:10.0, 1.2:10.0, 1.25:10.0, 1.3:10.0, 1.35:10.0, 1.4:10.0, 1.45:10.0, 1.5:10.0, 0.5:10.1, 0.55:10.1, 0.6:10.1, 0.65:10.1, 0.7:10.1, 0.75:10.1, 0.8:10.1, 0.85:10.1, 0.90:10.1, 0.95:10.1, 1.0:10.1, 1.05:10.1, 1.1:10.1, 1.15:10.1, 1.2:10.1, 1.25:10.1, 1.3:10.1, 1.35:10.1, 1.4:10.1, 1.45:10.1 or 1.5:10.1, or any two of the above values ​​constitute the mass ratio of S-(5'-adenosine)-L-cysteine ​​monoclonal antibody to solid-phase carrier within the numerical range.

[0032] In some specific embodiments, the present invention provides a method for preparing a solid-phase carrier labeled with a SAH monoclonal antibody, as detailed below: (1) Activation of solid-phase carrier: Centrifuge 0.99-1.01 mL (i.e., 9.9-10.1 mg) of solid-phase carrier at 4℃ and 14000 rpm for 20 min and discard the supernatant. Then, reconstitute the solid-phase carrier with the original volume (1 mL) of activation buffer, mix well, and separate the solid and liquid. Discard the supernatant. Then, reconstitute the solid-phase carrier with the original volume of buffer, preferably 2-(N-morpholino)ethanesulfonic acid (MES) solution, mix well, and sonicate to obtain a mixed solution. Then, mix the mixed solution with 100 μL of buffer with a concentration of 2-4 mg / mL, preferably carbodiimide (EDC) solution and 100 μL of buffer with a concentration of 3-6 mg / mL, preferably N-hydroxysuccinimide (NHS) solution in the dark to obtain the activated solid-phase carrier. The solid-liquid separation is performed by centrifugation, with a centrifugation temperature of 2-8℃, and / or a centrifugation speed of 10000-14000 rpm, and / or a centrifugation time of 20-30 min; and / or the mixing speed in the dark is 15-100 r / min, and / or the mixing time in the dark is 25-35 min.

[0033] (2) Solid-phase carrier conjugation of SAH monoclonal antibody: After solid-liquid separation of the activated microspheres obtained in step (1), the supernatant is discarded. Then, the microspheres are reconstituted in the original volume using a buffer solution, preferably 2-(N-morpholino)ethanesulfonic acid (MES) solution. After mixing, 0.5-1.5 mg of SAH monoclonal antibody is added and mixed in the dark. This mixture is then used to complete the conjugation of the solid-phase carrier and SAH monoclonal antibody, resulting in a conjugate of the solid-phase carrier and SAH monoclonal antibody. The solid-liquid separation is performed by centrifugation, with a centrifugation temperature of 2-8℃, and / or a centrifugation speed of 10000-14000 rpm, and / or a centrifugation time of 20-30 min, and / or a light-protected mixing speed of 15-100 r / min, and / or a light-protected mixing time of 25-35 min.

[0034] (3) Blocking: In the conjugate obtained in step (2), the fluorescently labeled antibody microsphere complex is mixed with a bovine serum albumin (BSA) solution with a concentration of 15-20 g / mL (containing 15-20 g of BSA per 100 mL of solution) in the dark to obtain a mixed solution; after solid-liquid separation of the mixed solution, the supernatant is discarded, the solution is reconstituted and subjected to ultrasonic disruption to obtain a solid-phase carrier labeled with SAH monoclonal antibody; The solid-liquid separation is performed by centrifugation, with a centrifugation temperature of 2-8℃, and / or a centrifugation speed of 10000-14000 rpm, and / or a centrifugation time of 20-30 min, and / or a light-protected mixing speed of 15-100 r / min, and / or a light-protected mixing time of 25-35 min.

[0035] In some specific embodiments, the present invention provides a method for preparing a complex antibody of a solid-phase carrier labeled with a monoclonal antibody of S-(5'-adenosine)-L-cysteine ​​and S-(5'-adenosine)-L-cysteine, as detailed below: (1) Preparation of immunogen: SAH and SAH monoclonal antibody-labeled solid-phase carrier are mixed in a mass ratio of SAH:SAH monoclonal antibody-labeled solid-phase carrier = 1:1-10:1. The mixture is placed in a constant temperature environment of 37±1℃ and incubated for 2-4 hours to allow it to react fully, so as to obtain a complex of SAH and SAH monoclonal antibody-labeled solid-phase carrier, i.e., immunogen.

[0036] (2) Immunization procedure: Inject the immunogen obtained in step (1) into the skin of the experimental animal at a dose of 0.5-1 mg / animal and perform booster immunization. After booster immunization, collect the blood of the experimental animal and centrifuge it at 2-8℃ and 2000-3000 rpm for 10-20 min to obtain serum containing immunoglobulin G. Preferably, the experimental animal is a mouse or a rabbit.

[0037] (3) Antibody purification: Protein A affinity chromatography column or Protein G affinity chromatography column were used for chromatographic purification of the experimental animal serum containing antibody proteins. After purification, the purity of antibody proteins in the experimental animal serum reached ≥95% (based on the relative content of the target protein, representing the purity of the protein), and the antibody titer of the experimental animal serum was ≥1×10⁻⁶. -6 The cross-reactivity rate of the antibody in the experimental animal with free SAH is <5%, the cross-reactivity rate of the antibody in the experimental animal with SAH monoclonal antibody is <5%, and the binding affinity (KD) of the antibody in the serum of the experimental animal with the complex is ≤1×10⁻⁶. - 6 M, to obtain anti-(SAH and SAH monoclonal antibody labeled solid-phase carrier) antibody with an antibody protein mass concentration of 2-5 mg / mL, preferably, the antibody protein is immunoglobulin G.

[0038] In some specific embodiments, the solid support comprises one of colloidal gold, colored latex microspheres, and a solid support. More preferably, the particle size range of the solid support is 20-400 nm.

[0039] Preferably, the solid support is time-resolved fluorescent latex microspheres. Preferably, the labeled fluorophore in the time-resolved fluorescent latex microspheres comprises a fluorescent substance with an excitation wavelength of 355-375 nm and an emission wavelength of 600-620 nm. More preferably, the fluorescent substance is a compound containing rare earth elements. Even more preferably, the rare earth element is europium. To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise stated, all raw materials / reagents / instruments used in the embodiments of the present invention are conventional commercially available products. The sources of information on the experimental raw materials used in the present invention are shown in Table 1.

[0040]

[0041] The specific method for preparing the solution used in this invention is as follows: (1) Preparation method of 2-(N-morpholino)ethanesulfonic acid (MES) solution with pH 6.0±0.1 and concentration of 20mM: Weigh 4.265g MES and dissolve it in 500mL purified water. Adjust the pH to 6.0±0.1 with 0.1mol / L NaOH. Make up the volume of purified water to 1L. After sterilization by 0.22μm filter membrane, store at 2-8℃.

[0042] (3) Preparation method of 2-(N-morpholino)ethanesulfonic acid (MES) solution with pH 6.5±0.1 and concentration of 20mM: Weigh 4.265g MES and dissolve it in 500mL purified water. Adjust the pH to 6.5±0.1 with 0.1mol / L NaOH. Make up the volume of purified water to 1L. After sterilization by 0.22μm filter membrane, store at 2-8℃.

[0043] (4) Preparation method of carbodiimide (EDC) solution with a concentration of 4 mg / mL: Weigh 4 mg EDC (accurate to 0.1 mg), dissolve it in purified water and make up to 1 mL, and use it immediately.

[0044] (5) Preparation method of N-hydroxysuccinimide (NHS) solution with a concentration of 6 mg / mL: Weigh 6 mg NHS (accurate to 0.1 mg), dissolve in purified water and make up to 1 mL, and use immediately.

[0045] (6) Preparation method of carbonate buffer with pH 9.6±0.1 and concentration of 50mM: Weigh 5.3g Na2CO3 and 4.2g NaHCO3 and dissolve them in 500mL purified water. Adjust the pH to 9.6±0.1 and then make up the volume of purified water to 1L. After sterilization by 0.22μm filter membrane, store at 2-8℃.

[0046] (7) Blocking solution: First, prepare 10mM PBS (the preparation method of 10mM PBS is: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 1.07g, NaH2PO4·2H2O 0.156g and purified water, then make up to 1L with purified water and adjust the pH to 7.4±0.05). Add 50g sucrose and 5g casein to 10mM PBS and stir to dissolve. After sterilization and filtration through a 0.22μm filter membrane, dispense and freeze.

[0047] (8) Storage solution: Weigh 2.4228g Tris (molecular weight 121.14) and dissolve it in 800mL of purified water. Adjust the pH to 8.0±0.1 with 1M HCl. Add 10g sucrose and 5g BSA to dissolve. Add 1mL Proclin300 and mix well. After sterilization by 0.22μm filter membrane, store at 2-8℃.

[0048] (9) Antibody dilution solution: First, prepare 10mM PBS (the preparation method of 10mM PBS is: 8.0g NaCl, 0.2g KCl, 1.07g Na2HPO4·12H2O, 0.156g NaH2PO4·2H2O and purified water, then make up to 1L with purified water and adjust the pH to 7.4±0.05). Add 10g sucrose and 10g casein to 10mM PBS and stir to dissolve. Add 1mL Proclin300 and mix well. After sterilization by 0.22μm filter membrane, store at 2-8℃.

[0049] (10) Washing solution: First, prepare 10mM PBS (the preparation method of 10mM PBS is: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 1.07g, NaH2PO4·2H2O 0.156g and purified water, then make up to 1L with purified water and adjust the pH to 7.4±0.05). Add 0.1mL of Tween20 to the 10mM PBS and mix well. Store in a sealed container at room temperature.

[0050] The preparation method of SAH monoclonal antibody-labeled fluorescent latex microspheres (i.e., SAH monoclonal antibody-labeled solid-phase carriers) used in the embodiments of the present invention is as follows: (1) Activation of fluorescent latex microspheres: Take a 2mL EP tube, pipette 1mL of fluorescent latex microspheres (i.e., 10mg of fluorescent latex microspheres) into the EP tube, and centrifuge at 4℃ and 14000rpm for 20min; then pipette the supernatant, reconstitute with 20mM MES solution (pH 6.0±0.1), shake thoroughly with a micro vortex mixer, and centrifuge at 4℃ and 14000rpm for 20min; then pipette the supernatant, reconstitute with 20mM MES solution (pH 6.0±0.1), and centrifuge at 4℃ and 14000rpm for 20min. The original volume of 20 mM MES solution was reconstituted and thoroughly mixed by shaking. The mixture was then sonicated (using a 5-second interval between sonication sessions, for a total duration of 2 minutes at 10% power). Next, 100 μL of 4 mg / mL EDC solution was added and mixed, followed by 100 μL of 6 mg / mL NHS solution. The mixture was then placed on a DNA mixer and mixed at 15 rpm in the dark for 30 minutes to obtain activated fluorescent latex microspheres, which were then used to activate the fluorescent latex microspheres. (2) Fluorescent latex microspheres conjugated with antibody: After centrifuging the activated fluorescent latex microspheres obtained in step (1) at 14000 rpm for 20 min, the supernatant was discarded. Then, MES solution with a pH of 6.5±0.1 and a concentration of 20 mM was added and reconstituted in the original volume (200 uL). The mixture was then thoroughly shaken and mixed using a micro vortex mixer. Then, 1 mg of S-(5'-adenosine)-L-cysteine ​​(SAH) monoclonal antibody was added and shaken by hand for 2 min. The mixture was then placed on a DNA mixer and mixed at 15 r / min in the dark for 120 min to complete the conjugation of fluorescent latex microspheres with SAH monoclonal antibody, thus obtaining the conjugate of fluorescent latex microspheres and SAH monoclonal antibody. (3) Blocking: Add 100 μL of BSA solution with a concentration of 20 g / 100 mL (20 g BSA is diluted to 100 mL with purified water) to the conjugate obtained in step (2), place it on a DNA mixer and mix it at 15 r / min in the dark for 30 min to obtain a mixed solution; centrifuge the mixed solution at 14000 rpm for 20 min and discard the supernatant, then add the storage solution to reconstitute and sonicate it with an ultrasonic disruptor to obtain SAH monoclonal antibody-labeled fluorescent latex microspheres.

[0051] (4) Storage: Store the fluorescent latex microspheres labeled with SAH monoclonal antibody at 4°C and sonicate before each use.

[0052] Example 1: Preparation of a complex antibody of SAH and SAH monoclonal antibody labeled on a solid-phase carrier (1) Preparation of immunogen: S-(5'-adenosine)-L-cysteine ​​(hereinafter referred to as SAH) and fluorescent latex microspheres labeled with SAH monoclonal antibody (fluorescent latex microspheres as solid phase carrier) are mixed at a mass ratio of SAH:SAH monoclonal antibody-labeled fluorescent latex microspheres = 5:1. The mixed substance is placed in a constant temperature environment of 37°C for 2 hours to allow it to react fully and obtain a complex of SAH and SAH monoclonal antibody-labeled solid phase carrier, i.e., immunogen.

[0053] (2) Immunization program: Healthy 6-week-old New Zealand white rabbits were selected and injected intradermally into their backs with the immunogen obtained in step (1), at a dose of 1 mg / rabbit. Thereafter, booster immunizations were performed every 2 weeks for a total of 3 booster immunizations. On the 7th day after the last booster immunization, blood was collected from the rabbits and centrifuged at 4°C and 2500 rpm for 15 minutes to obtain serum containing immunoglobulin G (IgG).

[0054] (3) Antibody purification: Rabbit serum containing IgG was purified using a Protein A affinity chromatography column. After purification, the purity of IgG in the rabbit serum reached ≥95% (based on the relative content of the target protein, representing protein purity). The antibody titer of the rabbit serum was then determined by indirect ELISA to be ≥1×10⁻. 6 The cross-reactivity of the antibody in this rabbit serum with free S-(5'-adenosine)-L-cysteine ​​is <5%, the cross-reactivity of the antibody in this rabbit serum with S-(5'-adenosine)-L-cysteine ​​monoclonal antibody is <5%, and the binding affinity (KD) of the antibody in this rabbit serum to the complex is ≤1×10⁻⁶. -6 M, a complex antibody of SAH and SAH monoclonal antibody labeled on a solid-phase carrier with a mass concentration of 2 mg / mL of antibody protein IgG was obtained.

[0055] The specific steps for the indirect ELISA method are as follows: 1. Coating: The SAH and SAH monoclonal antibody-labeled solid-phase carrier were diluted to a solution of 1 μg / mL with 50 mM carbonate buffer at pH 9.6 ± 0.1. The solution was added to a well plate (100 μL / well) and incubated overnight at 4°C. 2. Blocking: After washing the well plate once with physiological saline (0.9% (w / v) NaCl solution), add the blocking solution to the well plate (200 μL / well) and incubate at 37°C for 2 hours; 3. Add primary antibody: Dilute the antibody from the rabbit serum to a concentration of 10 mM PBS to a concentration of 10 μg / mL, add the solution to a well plate (100 μL / well), and incubate at 37°C for 0.5 h; 4. Add enzyme-labeled secondary antibody: Dilute anti-rabbit-HRP at a ratio of 1:5000 using antibody dilution buffer, add the diluted solution to the well plate (100 μL / well), and incubate at 37°C for 0.5 h; 5. Color development: After washing 5 times with the washing solution, add the color developing solution (this color developing solution is TMB (3,3',5,5'-tetramethylbenzidine). This color developing solution is oxidized under the action of HRP and H2O2, first forming a blue intermediate product, which turns yellow after adding the stop solution). The time is determined according to the color development speed. This color development takes about 2 minutes. 6. Stop reading: Add 100 μL of stop solution (1 M HCl) to each well and read the value using a microplate reader at 450 nm.

[0056] Example 2: Preparation of Chromatographic Test Strips (1) Sample pad preparation: Prepare a Tris-HCl buffer solution containing 50 mM DTT (50 mM, pH 7.4). This is the sample pad buffer solution. Immerse the glass fiber material completely in the buffer solution for 20 min to ensure that the material fully absorbs the components in the buffer solution. After immersion, dry the glass fiber material at 37°C for 4 hours to achieve a completely dry state, thus completing the sample pad preparation. The specific steps for preparing a Tris-HCl buffer solution containing 50 mM DTT (50 mM, pH 7.4) are as follows (taking 100 mL as an example): Weigh 0.7713 g of DTT (Note: DTT is hygroscopic and needs to be weighed quickly; you can pre-weigh the paper beforehand); Add 0.7713 g of DTT to a 50 mM Tris-HCl buffer solution (100 mL system) and stir magnetically until completely dissolved. Filter aseptically through a 0.22 μm filter membrane to obtain 100 mL of Tris-HCl buffer solution containing 50 mM DTT. The specific method for preparing the 50mM Tris-HCl buffer solution with pH 7.4 is as follows (taking 100 mL as an example): Weigh 0.6057 g of Tris base using an electronic balance, pour it into a 100 mL beaker, add about 80 mL of purified water, stir magnetically until completely dissolved, and adjust the pH to 7.4 with hydrochloric acid. Then transfer the solution to a 100 mL volumetric flask, dilute to the mark with purified water, shake well, and store at 4℃ to obtain a 50mM Tris-HCl buffer solution with pH 7.4.

[0057] (2) Preparation of enzyme conjugate pad: Prepare a Tris-HCl (50mM, pH 8.0) buffer containing 1mg / mL SAH tool enzyme and 1mM adenosine. This is the enzyme pad buffer. Use a gold sprayer to evenly spray the enzyme pad buffer onto the polyester cellulose material, and then dry it at 37℃ for 4 hours to complete the preparation of the enzyme conjugate pad. This is to ensure that the molar ratio of adenosine to SAH tool enzyme is about 49:1 during the reaction, so that adenosine and SAH tool enzyme are stably bound on the polyester cellulose material.

[0058] The specific preparation method of Tris-HCl (50mM, pH 8.0) buffer containing 1 mg / mL SAH enzyme and 1 mM adenosine is as follows: a. Add approximately 80 mL of ultrapure water to a 100 mL beaker, then add 0.6057 g of Tris base (50 mM final concentration) and 26.7 mg of adenosine (1 mM final concentration) sequentially. Stir magnetically at room temperature for 10 minutes until completely dissolved. Adjust the pH to 8.0 with hydrochloric acid, then transfer the solution to a 100 mL volumetric flask and dilute to the mark with ultrapure water. Shake well to obtain a 1 mM adenosine Tris-HCl (50 mM, pH 8.0) buffer solution (i.e., 50 mM Tris-HCl buffer solution (containing 1 mM adenosine)).

[0059] b. Weigh 100 mg of SAH enzyme powder and slowly add it in 3-5 portions to the Tris-HCl (50 mM, pH 8.0) buffer containing 1 mM adenosine obtained in step 1, which is placed in an ice bath. Stir for 2 minutes after each addition until completely dissolved (to avoid excessive local concentration that could cause enzyme protein aggregation). Continue stirring for 10 minutes to ensure complete enzyme dissolution, resulting in a mixed solution (each mL of this mixed solution contains 1 mg of SAH enzyme). Then filter the solution through a 0.22 μm sterile filter to obtain a Tris-HCl (50 mM, pH 8.0) buffer containing 1 mg / mL SAH enzyme and 1 mM adenosine.

[0060] (3) Preparation of conjugate pad: SAH monoclonal antibody-labeled fluorescent latex microspheres, sucrose, and proclin300 were mixed to obtain a mixed solution. The concentration of SAH monoclonal antibody-labeled fluorescent latex microspheres in the mixed solution was 1 mg / mL, the concentration of sucrose was 5 wt%, and the concentration of proclin300 was 0.1 vol. The mixed solution was uniformly sprayed onto polyester cellulose material using a gold sprayer and dried at 37°C for 4 hours to complete the preparation of the conjugate pad.

[0061] (4) Preparation of detection line (T line) and control line (C line) on nitrocellulose membrane (NC membrane): Preparation of T line: SAH-BSA conjugate antigen containing 0.5 mg / mL carrier protein BSA is uniformly sprayed onto nitrocellulose membrane using a spraying device. Then, the nitrocellulose membrane is placed in a dry environment at 37°C for 2 hours to dry so that SAH-BSA conjugate antigen is firmly attached to the surface of NC membrane, thus completing the preparation of T line.

[0062] Preparation of C-line: The complex antibody of SAH and SAH monoclonal antibody-labeled solid-phase carrier obtained in step (3) of Example 1 with a mass concentration of 2 mg / mL of antibody protein IgG was sprayed onto the NC membrane using a spraying machine, and then dried at 37°C for 2 hours to complete the preparation of C-line.

[0063] The specific method for preparing SAH-BSA conjugate antigen containing a carrier protein BSA at a concentration of 0.5 mg / mL is as follows: 1. SAH activation (carboxyl activation): Weigh 1 mg SAH and dissolve it in 5 mL of coupling buffer (10 mM MES, pH 5.5) to obtain SAH solution. Then, add 10 μL of EDC solution (20 mg / mL) and 10 μL of NHS solution (40 mg / mL) to the SAH solution in sequence. Mix and react at room temperature in the dark for 2 hours to obtain activated SAH.

[0064] 2. SAH-to-BSA Coupling: Weigh 10 mg of BSA and dissolve it in 1 mL of coupling buffer (final concentration 10 mg / mL) to obtain a BSA solution; then, slowly add the activated SAH solution obtained in step 1 to the BSA solution (stirring while adding), and react at room temperature in the dark for 4 hours to carry out the coupling reaction; then add 50 μL of termination buffer (1 M Tris-HCl, pH 7.5) to terminate the coupling reaction, and continue stirring for 30 minutes to obtain the solution after the coupling reaction has been terminated.

[0065] 3. Ultrafiltration concentration: Transfer the solution (approximately 6 mL) obtained after the coupling reaction in step 2 to an ultrafiltration concentration tube (10 kDa cutoff), and centrifuge at 4°C and 3000×g for 30 minutes (the remaining volume can be observed multiple times during this period) until the solution volume is concentrated to approximately 0.5 mL (the concentration factor is approximately 12 times the original volume), obtaining the concentrated sample; then add 5 mL of PBS buffer (10 mM, pH 7.4) to the concentrated sample, and centrifuge at 4°C and 3000×g for 30 minutes, discarding the filtrate; repeat the above steps for 2-3 times to ensure that small molecule impurities are fully removed. After the last solution change, invert the ultrafiltration tube and centrifuge at 4°C and 1000×g for 2 minutes, collecting the retained sample into a new centrifuge tube to obtain SAH-BSA conjugate antigen containing a concentration of 0.5 mg / mL carrier protein BSA.

[0066] (6) Assembling the chromatography strip: From the sample application end to the absorbent end, the chromatography strip consists of: a sample pad, an enzyme conjugate pad, a conjugate pad, a nitrocellulose membrane (with integrated control and detection lines), and an absorbent pad (made of plant fiber). The absorbent pad drives the sample migration during chromatography. These are then laminated onto a PVC base plate. After lamination, the strips are cut into 4mm wide reagent strips using specialized equipment and assembled into reagent cards. The strips are then labeled and ready for use. See the top and side views of the chromatography strip structure for details. Figure 1 As shown.

[0067] (7) Preparation of sample diluent (1L): Weigh 1.2114g Tris and dissolve it in 800mL of purified water. Adjust the pH to 7.0 with 1M HCl. Add 5.0g BSA and 0.5mL Tween 20 and stir until completely dissolved. Make up the volume with purified water to 1L. After sterilization by 0.22μm filter membrane as needed, store at 4℃ in the dark to obtain a sample diluent with pH 7.0 containing 10mM Tris-HCl, 0.5% (w / v) BSA and 0.05% (v / v) Tween 20.

[0068] Example 3 Detection Method Sample pretreatment: Mix 10 μL of sample with 90 μL of the sample diluent to obtain the diluent.

[0069] Sample addition reaction: Accurately pipette 100 μL of diluent and slowly add it to the sample well of the chromatography strip in step (6) of Example 2 for chromatography reaction for 15 minutes.

[0070] The principle of this chromatographic reaction is as follows: The HCY sample first flows through a sample pad pre-coated with dithiothreitol (DTT) solution. Under the action of the DTT solution, bound HCY in the sample is reduced to free HCY. During this process, the pH is maintained at 7.0-8.5, and the reaction time is 1-5 minutes. Next, the free HCY in the sample flows through an enzyme-binding pad pre-coated with SAH enzyme and adenosine. Under the catalysis of the SAH enzyme, the free HCY and adenosine in the sample react to generate SAH. During this process, the reaction temperature is controlled at 20-40℃, and the reaction time is 2-8 minutes. The SAH enzyme is an enzyme capable of catalyzing the reaction of free HCY and adenosine in the sample to generate SAH, and the reaction formula is as follows:

[0071] Signal reading: After the chromatography reaction is completed, the signals of the T line and C line on the chromatography test strip are accurately read using a fluorescence detector to obtain the T value and C value, and the T / C ratio is calculated to achieve quantitative determination of the HCY concentration in the HCY sample.

[0072] Technical effect evaluation: 1. Evaluation of Technical Effect 1: It should be noted that the sample solutions used in Evaluation of Technical Effect 1 are DL-homocysteine, which are diluted with 10mM PBS to obtain sample solutions containing different concentrations of DL-homocysteine, as shown in Table 2 below.

[0073] (1) Detection method of the present invention: Detection is performed using the method described in Example 3 of the present invention. (2) Traditional detection method: The difference between the traditional method and the detection method of the present invention is that the C line used is different. The C line used in the traditional method is: goat anti-mouse IgG polyclonal antibody.

[0074] (3) Linear range discrimination test and linear range test: The values ​​obtained in steps (1) and (2) were compared for linear range discrimination, and the results are shown in Table 2 below; The linear range of T / C values ​​was compared under the condition that the concentration of DL-homocysteine ​​in the sample solution was 0-51.92 μmol / mL, and the results are shown in Table 2 below. Figure 2 As shown.

[0075]

[0076] As shown in Table 2, when the concentration of DL-homocysteine ​​in the sample solution was 5.01 μmol / L, the T / C ratio was 5.0; when the concentration of DL-homocysteine ​​in the sample solution was 51.92 μmol / L, the T / C ratio dropped to 0.4. The variation range of the T / C ratio (i.e., the ratio of the T / C value when the concentration of DL-homocysteine ​​in the sample solution was 51.92 μmol / mL to the T / C value when the concentration of DL-homocysteine ​​in the sample solution was 5.01 μmol / mL) was as high as 12.5. In contrast, the variation range of the T / C ratio of the traditional detection method within the same concentration range was only 2.9. This result fully demonstrates that the linear range discrimination of the present invention is wider than that of the traditional method, that is, the linear range discrimination of the present invention is better, and it can more effectively distinguish HCY samples of different concentrations, significantly improving the accuracy and reliability of detection.

[0077] like Figure 2 As shown, Figure 2 The figures show the T / C ratio of the detection method of this invention and the conventional detection method as a function of the concentration of DL-homocysteine ​​in the sample solution. The results show that the curve of the detection method of this invention maintains a good linear relationship over a wider concentration range, exhibiting a broader linear range. This further demonstrates the advantages of the detection method of this invention in detecting HCY samples of different concentrations.

[0078] 2. Evaluation of Technical Effect II: It should be noted that the preparation method of Sample 1 used in Evaluation of Technical Effect II is as follows: DL-homocysteine ​​is diluted with 10mM PBS to obtain a sample solution containing 10μmol / mL DL-homocysteine; the preparation method of Sample 2 used is as follows: DL-homocysteine ​​is diluted with 10mM PBS to obtain a sample solution containing 25μmol / mL DL-homocysteine.

[0079] (1) Detection method of the present invention: Detection is performed using the method described in Example 3 of the present invention. (2) Traditional detection method: The difference between the traditional method and the detection method of the present invention is that the C line used is different. The C line used in the traditional method is: goat anti-mouse IgG polyclonal antibody.

[0080] (3) Take sample 1 and sample 2 respectively, set 10 parallel groups for each sample, and use the detection methods in steps (1) and (2) respectively to detect them, and obtain the T value, C value and T / C ratio to evaluate the precision of the traditional detection method and the detection method of the present invention. The results are shown in Tables 3 and 4.

[0081]

[0082]

[0083] As shown in Tables 3 and 4, the results indicate that the T-values ​​obtained by the method of this invention are 9011-178273, C-values ​​are 4162-109732, and T / C values ​​are 0.09-3.2. Furthermore, the SD and CV of the method of this invention for T-values, C-values, T / C, and sample concentrations (especially concentration, as the final detection target) are significantly lower than those of the traditional method. Specifically, the concentration CV is controlled within 4% (3.56% for sample 1 and 3.86% for sample 2), while the traditional method is above 6% (7.63% for sample 1 and 6.34% for sample 2), clearly demonstrating a significant advantage in precision compared to the traditional method. Simultaneously, the deviation of the mean concentration (AVG) obtained by the two methods is <5% (1.15% for sample 1 and 4.87% for sample 2), indicating that the present invention improves precision without sacrificing detection accuracy. The detection method of this invention is more reliable.

[0084] 3. Technical Effect Evaluation Three: Linear Range Test. DL-homocysteine ​​standard solutions at different dilution ratios were tested using traditional detection methods to obtain linear regression equations. The theoretical concentration (i.e., the concentration of the DL-homocysteine ​​standard solution after dilution at the corresponding factor), measured concentration, and relative deviation for each sample were recorded in detail (the formula for calculating the relative deviation is: (measured concentration / theoretical concentration) - 1). The results are as follows: Figure 3 As shown, the detection method of this invention was used to detect HCY standard solutions at different dilution ratios, and a linear regression equation was obtained. The theoretical concentration (i.e., the concentration of DL-homocysteine ​​standard solution after dilution at the corresponding factor), measured concentration, and relative deviation of each sample were recorded in detail. The results are as follows: Figure 4 As shown.

[0085] like Figure 3 and Figure 4 As shown, the results indicate that under the conditions of the traditional detection method, the correlation coefficient r = 0.9971. However, under the conditions of the detection method of the present invention, the correlation coefficient r = 0.9997. At the same time, the measured concentration of the present invention at different dilution factors is closer to the theoretical concentration, and the relative deviation is smaller, indicating that its linear range is better and it can detect HCY samples of different concentrations more accurately.

[0086] 4. Evaluation of Technical Effects (Part Four): (1) Detection method of the present invention: Detection is performed using the method described in Example 3 of the present invention. (2) Traditional detection method: The difference between the traditional method and the detection method of the present invention is that the C line used is different. The C line used in the traditional method is: goat anti-mouse IgG polyclonal antibody.

[0087] (3) Serum sample testing: 42 serum samples were taken for comparative testing. The concentration of L-homocysteine ​​in serum samples under the traditional detection method and the concentration of L-homocysteine ​​in serum samples under the detection method of the present invention were recorded. The relative deviations of the traditional detection method, the detection method of the present invention and the reference reagent concentration of L-homocysteine ​​in serum samples were also recorded. The concentration of the reference reagent was detected using a homocysteine ​​(HCY) enzymatic kit. The results are shown in Table 5.

[0088]

[0089] The results show that the detection method of the present invention is closer to the concentration of the reference reagent and has a smaller relative deviation, which fully demonstrates that the present invention has higher accuracy in clinical sample detection and can provide a more reliable basis for clinical diagnosis.

[0090] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not intended to limit the present invention. Any improvements made by those skilled in the art on this basis that do not highlight substantive features or make significant progress should fall within the protection scope of the present invention.

Claims

1. A homogeneous cysteine competition method immunochromatographic test strip, characterized by, The quality control line is coated with an antibody, and the detection line is coated with S-(5'-adenosine)-L-cysteine and bovine serum albumin conjugated antigen; The antibody is a complex antibody of S-(5'-adenosine)-L-cysteine and S-(5'-adenosine)-L-cysteine monoclonal antibody labeled solid phase carrier, wherein the complex antibody is prepared by a method comprising the following steps: complexing S-(5'-adenosine)-L-cysteine as an antigen with S-(5'-adenosine)-L-cysteine monoclonal antibody labeled solid phase carrier to obtain a complex, and purifying the complex after immunization to obtain the complex antibody.

2. The immunochromatographic test strip according to claim 1, characterized by In the complex, the mass ratio of S-(5'-adenosine)-L-cysteine as an antigen to S-(5'-adenosine)-L-cysteine monoclonal antibody labeled solid phase carrier is 1:1-10:

1.

3. The immunochromatographic test strip according to claim 1 or 2, characterized in that, The S-(5'-adenosine)-L-cysteine monoclonal antibody labeled solid phase carrier is prepared from S-(5'-adenosine)-L-cysteine monoclonal antibody and a solid phase carrier, wherein the mixed mass ratio of S-(5'-adenosine)-L-cysteine monoclonal antibody and the solid phase carrier is 0.5-1.5:9.9-10.1, The S-(5'-adenosine)-L-cysteine and bovine serum albumin conjugated antigen is prepared from S-(5'-adenosine)-L-cysteine and bovine serum albumin, wherein the mixed mass ratio of S-(5'-adenosine)-L-cysteine and bovine serum albumin is 1:5-10.

4. The immunochromatographic test strip according to any one of claims 1 to 3, characterized in that, The mass concentration of antibody protein in the complex antibody is ≥2 mg / mL.

5. The immunochromatographic test strip according to any one of claims 1 to 4, characterized in that, The mass concentration of antibody protein in the complex antibody is 2-5 mg / mL.

6. The immunochromatographic test strip according to any one of claims 1 to 5, characterized in that, The antibody protein comprises one of mouse immunoglobulin G or rabbit immunoglobulin G.

7. The immunochromatographic test strip according to any one of claims 1 to 6, characterized in that, The solid phase carrier comprises one of colloidal gold, colored latex microspheres or time-resolved fluorescent latex microspheres.

8. The immunochromatographic test strip according to any one of claims 1 to 7, characterized in that, The solid phase carrier is time-resolved fluorescent latex microspheres.

9. The immunochromatographic test strip according to any one of claims 1 to 8, characterized in that, The content of bovine serum albumin as a carrier protein in the S-(5'-adenosine)-L-cysteine and bovine serum albumin conjugated antigen is 0.5-2 mg / mL.

10. The immunochromatographic test strip according to any one of claims 1 to 9, characterized in that, The immunochromatography test paper comprises a back plate and a sample pad, an enzyme binding pad, a binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially laid on the back plate, wherein the nitrocellulose membrane is sequentially provided with a strip of the detection line and a strip of the quality control line.

11. The immunochromatographic test strip according to any one of claims 1 to 10, characterized in that, The sample pad is a glass fiber coated with a reducing agent, which is used to reduce and convert the bound homocysteine into free homocysteine, The enzyme binding pad is polyester cellulose coated with S-(5'-adenosine)-L-cysteine tool enzyme and adenosine, wherein the S-(5'-adenosine)-L-cysteine tool enzyme is an enzyme capable of catalyzing free homocysteine to generate S-(5'-adenosine)-L-cysteine, The binding pad is polyester cellulose coated with the S-(5'-adenosine)-L-cysteine monoclonal antibody labeled solid phase carrier, And / or the material of the water absorption pad is plant fiber.

12. The immunochromatographic test strip according to any one of claims 1 to 11, characterized in that, In the chromatographic reaction, the molar ratio of S-(5'-adenosine)-L-cysteine tool enzyme and adenosine in the enzyme binding pad is 1:5-50.

13. The immunochromatographic test strip according to any one of claims 1 to 12, characterized in that, The purification is chromatographic purification by a Protein A affinity chromatographic column or a Protein G affinity chromatographic column.

14. A homogeneous cysteine competition method immuno-chromatographic test kit, characterized by, The kit comprises a detection unit and a reagent unit, wherein the detection unit is the immunochromatographic test paper according to any one of claims 1-13.

15. The immunochromatographic test kit according to claim 14, characterized in that, The reagent unit is a diluent for diluting the sample to be detected by 10-20 times.

16. The immunochromatographic test kit according to claim 14 or 15, characterized in that, Each L of the diluent comprises 10-100 mM of Tris-Hcl solution with a pH of 7.0±0.05, 0.1-0.5 w / v% of bovine serum albumin, and 0.01-0.05 v / v% of Tween 20.

17. The immunochromatographic test kit according to any one of claims 14 to 16, characterized in that, The sample to be detected comprises one of serum, plasma or whole blood.

18. The immunochromatographic test strip of any one of claims 1 to 13 or the immunochromatographic test kit of any one of claims 14 to 17, characterized in that, The application in the preparation of products for diagnosing or monitoring cardiovascular and cerebrovascular diseases.

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