tat enzyme-labeled antibody stock solution

Abstract

The present application relates to the field of biotechnology, in particular to a TAT enzyme-labeled antibody storage solution, and provides application of proteins, saccharides and aromatic amino acids in preparation of a thrombin-antithrombin III complex (TAT) chemiluminescence assay kit, wherein the kit reduces non-specific binding of the antibody, reduces interference of other substances in the sample on the detection result, improves the calibration curve of the chemiluminescence detection, and improves the repeatability of antigen detection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to compositions and their applications. Background Technology

[0002] Thrombin is a multifunctional serine proteolytic enzyme containing two polypeptide chains, A and B, linked by an interchain disulfide bond. Thrombin acts directly on the final step of the blood coagulation process, causing soluble fibrinogen in plasma to convert into insoluble fibrin, thus achieving rapid hemostasis. Once activated, prothrombin rapidly covalently binds to antithrombin III to form the thrombin-antithrombin III complex (TAT). TAT serves as a molecular marker of coagulation system activation, indicating thrombin activation and thrombus formation. Elevated TAT levels are closely associated with diseases such as disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), pulmonary embolism (PE), and atrial fibrillation. Therefore, the measurement of thrombin-antithrombin III complex levels is of significant importance for the diagnosis and treatment of some diseases. Because thrombin has a very short half-life of only a few seconds, it is difficult to detect directly. In contrast, the half-life of thrombin-antithrombin III complex (TAT) is much longer, reaching tens of minutes. Therefore, existing methods primarily detect the content of TAT. Currently available methods for detecting TAT mainly include enzyme-linked immunosorbent assay (ELISA) and tube-based chemiluminescence immunoassay. ELISA is cumbersome, time-consuming, and produces unstable and poorly reproducible results, making it inconvenient for on-call testing and hospital emergencies. Tube-based chemiluminescence immunoassay also suffers from poor stability of enzyme-labeled antibodies and low reproducibility. Magnetic microparticle chemiluminescence immunoassay combines magnetic separation technology, immunoassay technology, and chemiluminescence technology to quantitatively determine specific antigens. Existing TAT magnetic microparticle chemiluminescence assay kits use streptavidin-biotin or radioactive element labeling, which can easily lead to interference from excessive labeling and poses a radioactive hazard. Summary of the Invention

[0003] In view of this, the composition and its application provided by the present invention, along with the kit, combine immunoassay and chemiluminescence technologies. Detection is performed by hydrolyzing the chemiluminescent substrate with alkaline phosphatase. The composition in the enzyme-labeled antibody can stabilize the stereostructure of the complex, reduce non-specific binding of the antibody, and improve the repeatability of chemiluminescence assays.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the use of carbohydrates in any of the following:

[0006] (I) Improve the repeatability of chemiluminescence assay kits;

[0007] (II) Used to prepare enzyme-labeled antibody storage buffer;

[0008] The sugars include one or more of trehalose, glucose, mannose, hyaluronic acid, or galactose;

[0009] The chemiluminescence assay kit includes a chemiluminescence assay kit for the thrombin-anthirombin III complex.

[0010] This invention also provides the use of aromatic amino acids in any of the following:

[0011] (I) Improve the calibration curve of the chemiluminescence assay kit;

[0012] (II) Used to prepare enzyme-labeled antibody storage buffer;

[0013] The aromatic amino acids include one or more of tryptophan, tyrosine, or phenylalanine.

[0014] The chemiluminescence assay kit includes a chemiluminescence assay kit for the thrombin-anthirombin III complex.

[0015] The present invention also provides compositions comprising proteins, carbohydrates, and aromatic amino acids;

[0016] The protein includes one or more of bovine serum albumin, human serum albumin, or rabbit serum albumin; the carbohydrates include one or more of trehalose, glucose, mannose, hyaluronic acid, or galactose.

[0017] The aromatic amino acids include one or more of tryptophan, tyrosine, or phenylalanine.

[0018] In some specific embodiments of the present invention, the above composition comprises:

[0019] The protein is bovine serum albumin;

[0020] The sugar is trehalose;

[0021] The aromatic amino acid is tyrosine.

[0022] The present invention also provides for the use of the above composition in any of the following:

[0023] (I) Improve the calibration curve of the chemiluminescence assay kit;

[0024] (II) Improve the repeatability of chemiluminescence assay kits;

[0025] (III) Preparation of enzyme-labeled antibody storage buffer;

[0026] The chemiluminescence assay includes a chemiluminescence assay for the thrombin-antithrombin III complex.

[0027] The present invention also provides an enzyme-labeled antibody storage buffer comprising the above-described composition, and:

[0028] (A) Buffer solution; and / or

[0029] (B) Preservatives; and / or

[0030] (C) Acceptable excipients or additives.

[0031] In some specific embodiments of the present invention, the concentration of sugars in the enzyme-labeled antibody storage buffer is 10 mg / mL.

[0032] In some specific embodiments of the present invention, the sugar in the enzyme-labeled antibody storage buffer is trehalose; and the aromatic amino acid is tyrosine.

[0033] In some specific embodiments of the present invention, the above-mentioned enzyme-labeled antibody storage buffer includes 50 mM Tris, 5 mg / mL BSA, 10 mg / mL trehalose, 2.5 mg / mL tyrosine and 1 mg / mL NaN3.

[0034] In some specific embodiments of the present invention, the pH value of the enzyme-labeled antibody storage buffer is 8.0.

[0035] The present invention also provides a kit comprising acceptable excipients or adjuvants, and:

[0036] (a) the above-mentioned composition; or

[0037] (b) Storage buffer for the enzyme-labeled antibody mentioned above.

[0038] In some specific embodiments of the present invention, the above-mentioned kit further includes enzyme-labeled antibodies;

[0039] The method for preparing the enzyme-labeled antibody includes: mixing the activated enzyme and the thiolized antibody at a mass ratio of 2:1 to obtain the enzyme-labeled antibody;

[0040] The enzyme includes alkaline phosphatase;

[0041] The antibodies include antithrombin III antibodies.

[0042] In some specific embodiments of the present invention, the above-mentioned kit also includes immunomagnetic beads;

[0043] The method for preparing the immunomagnetic beads includes: activating carboxyl magnetic beads with an activating agent, and then adding an antibody for conjugation after activation to obtain the immunomagnetic beads;

[0044] The antibodies include thrombin antibodies;

[0045] The ratio of the carboxyl magnetic beads to the antibody is 50:1 (w / w);

[0046] The feeding ratio of the carboxyl magnetic beads to the activator is 20:1 (w / w);

[0047] The activators are EDC and Sulfo-NHS.

[0048] The compositions of the present invention, their applications, and the kits thereof have the following effects:

[0049] The immunomagnetic beads described in this invention bind a certain amount of thrombin antibody to the surface of activated magnetic beads via chemical coupling. The magnetic beads used are superparamagnetic, generating a rapid magnetic response without hysteresis. The enzyme-labeled antibody is coupled to the Sulfo-SMCC-activated alkaline phosphatase and Traut's thiolized antithrombin III antibody using a heterobifunctional reagent cross-linking method. The storage buffer in this invention contains proteins, carbohydrates, and aromatic amino acids, which effectively reduces non-specific antibody binding, minimizes interference from other substances in the sample, and improves the repeatability of the test results. The thrombin antibody and antithrombin III antibody used in this invention exhibit high specificity and high affinity for thrombin and antithrombin III antigens, enabling rapid and accurate capture of the thrombin-antithrombin III complex in the sample. Quantitative detection is achieved by using the substrate solution in a fully automated immunoassay system containing chromogenic substrates such as AMPPD or APS-5. The kit of this invention exhibits good calibration curves and test repeatability. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 , Figure 2 Calibration curves for different storage buffer groups are shown;

[0052] Figure 3 This paper presents a consistency analysis of the test results between the reagent kit of this invention and the Sysmex reagent kit. Detailed Implementation

[0053] This invention discloses the composition and its application. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0054] This invention relates to the preparation and detection method of a thrombin-antithrombin III complex (TAT) chemiluminescence assay kit, specifically to an enzyme-labeled antibody storage buffer and a kit preparation method. The enzyme-labeled antibody storage buffer comprises an enzyme-labeled antibody storage buffer containing proteins, aromatic amino acids, preservatives, etc. This invention also relates to the preparation methods of magnetic microparticle suspensions (immunomagnetic beads) coated with thrombin antibodies and alkaline phosphatase-labeled antithrombin III antibodies (enzyme-labeled antibodies).

[0055] The kit of this invention is based on magnetic microparticle chemiluminescence immunoassay, which provides a simple, rapid, and accurate method for the preparation of thrombin-antithrombin III complexes. Magnetic nanoparticles have advantages such as large specific surface area, fast separation, good reproducibility, and the ability to be coupled with labels. The magnetic microparticle chemiluminescence immunoassay (CLIA) based on magnetic nanoparticles combines highly sensitive chemiluminescence assay technology with highly specific immunoreaction, featuring high sensitivity, wide linear range, high specificity, stable measurements, and high automation.

[0056] This invention utilizes alkaline phosphatase-labeled antithrombin III antibodies for quantitative detection, achieved by combining the antibody with substrate solutions from a fully automated immunoassay system containing chromogenic substrates such as AMPPD or APS-5. The antigen first binds to the thrombin antibody coated on the surface of magnetic beads, forming a stable antigen-thrombin antibody complex. The enzyme-labeled antibody then binds to other sites on the antigen, forming an antigen-thrombin antibody-antibody and antithrombin III antibody complex. The alkaline phosphatase labeled on the antibody catalyzes the hydrolysis of the luminescent substrate, emitting a light signal. The light signal value is positively correlated with the alkaline phosphatase concentration. Quantitative testing of the double-antibody sandwich complex (antigen) is achieved through chemiluminescence analysis. The enzyme-labeled antibody storage buffer contains proteins and aromatic amino acids, effectively reducing non-specific antibody binding, minimizing interference from other substances in the sample on alkaline phosphatase activity and instrumental results, and improving the repeatability of the detection. Specifically:

[0057] The enzyme-labeled antibody storage buffer of the kit of the present invention contains added proteins, sugars, and aromatic amino acids, which can not only reduce the non-specific binding of antibodies, but also reduce the interference of other substances in the sample on the detection results, thereby improving the repeatability of antigen detection.

[0058] This invention provides the following technical solution:

[0059] The TAT chemiluminescence assay kit provided by this invention uses a double-antibody sandwich method and an enzyme-catalyzed chemiluminescence method for detection. The kit includes a magnetic microparticle suspension (immunomagnetic beads) coated with thrombin antibody and an alkaline phosphatase-labeled antithrombin III antibody (enzyme-labeled antibody).

[0060] The magnetic microparticles are superparamagnetic, and the core material can be one or more of the following: iron oxide, zinc ferrite, or ferrous oxide. The surface should be modified with a uniform and sufficient amount of carboxyl groups.

[0061] The magnetic microparticles may have a particle size ranging from 0.5 μm to 5 μm, preferably 1.5 μm.

[0062] The antibody may be a monoclonal antibody, a modified antibody fragment with Fab activity, an antibody or antibody fragment polymer, etc., which can specifically bind to the antigenic determinant and may be derived from animals such as mice, rabbits, sheep, and dogs, preferably mouse monoclonal antibodies.

[0063] The immunomagnetic beads can be conjugated with antibodies using activators such as EDC, EDC and NHS, and EDC and Sulfo-NHS, with EDC and Sulfo-NHS being preferred. The conjugation steps are as follows:

[0064] a) Disperse the magnetic particles uniformly in the activation buffer solution;

[0065] b) Dissolve the activator in activation buffer, add it to the magnetic microparticle suspension and mix well. Incubate at room temperature (10-30℃) for 30 min to 2 h to obtain activated magnetic beads.

[0066] c) Remove residual activator by magnetic separation, and redisperse magnetic particles by adding cross-linking buffer;

[0067] d) Dilute the thrombin antibody with cross-linking buffer, add it to the activated magnetic microparticle suspension, mix well, and incubate at 4–30°C for 0.5–12 h to obtain the immunomagnetic bead suspension;

[0068] e) Dissolve the blocking agent in cross-linking buffer, add it to the immunomagnetic bead suspension after the reaction is complete, and incubate at 4–30°C for 0.5–3 h;

[0069] f) After magnetic separation, disperse the immunomagnetic beads using storage buffer and store for later use.

[0070] The ratio of magnetic beads to antibodies is 10:1 to 200:1 (w / w), preferably 50:1.

[0071] The feeding ratio of the magnetic beads to the activator is 0.1:1 to 20:1 (w / w), preferably 20:1.

[0072] The blocking agent may be one or more of the following substances containing free amino groups: ethanolamine, bovine serum albumin, casein, amino acids, etc.

[0073] The activation and cross-linking buffer can be one of the buffers that do not contain amino and carboxyl groups, such as MES buffer, borate buffer, PBS buffer, MOPS buffer, and HEPES buffer, with MES buffer being preferred.

[0074] The pH range of the activation and cross-linking buffer solution is 5.5 to 7.0, preferably 6.5.

[0075] The storage buffer system can be one of MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer, etc., preferably Tris-HCl buffer.

[0076] The storage buffer contains bovine serum albumin (BSA) and the preservative NaN3.

[0077] The storage buffer solution has a pH range of 6.0 to 9.0, preferably 7.5.

[0078] The enzyme-labeled antibody can be prepared by methods such as the glutaraldehyde method, the sodium periodate method, or the heterobifunctional reagent cross-linking method (Sulfo-SMCC), with the heterobifunctional reagent cross-linking method being preferred. The preparation process can utilize Sulfo-SMCC to activate alkaline phosphatase and thiothreitol (DTT) / 2-mercaptoethylamine (2-MEA) to thiolated antithrombin III antibody, or use Sulfo-SMCC to activate antithrombin III antibody and Traut's / DTT / 2-MEA reagent to thiolated alkaline phosphatase.

[0079] The preparation steps are as follows:

[0080] a) Dissolve / dilute alkaline phosphatase with enzyme-labeled buffer, add a certain amount of Sulfo-SMCC, and incubate gently at 4–30°C for 0.5–3 h. Desalt the product and set it aside for later use.

[0081] b) Dissolve / dilute the antithrombin III antibody with enzyme-labeled buffer, add a certain amount of Traut's reagent, incubate gently at 4-30℃ for 0.5-3h, then quench with 5-25mg / mL glycine solution for 5min, and desalt the product for later use;

[0082] c) After mixing the activated alkaline phosphatase with the thiolized antithrombin III antibody, gently stir and incubate at 4–30°C for 2–30 h, then add 5–25 mg / mL of cysteine ​​solution to block for 0.5–2 h.

[0083] d) After desalting, the product is stored in a storage buffer for later use.

[0084] The ratio of alkaline phosphatase / antibody to Sulfo-SMCC is 2 to 30:1 (w / w), preferably 15:1.

[0085] The alkaline phosphatase / antibody to Traut's feed ratio is 20-250:1 (w / w), preferably 20:1.

[0086] The ratio of alkaline phosphatase to antibody is 1 to 5:1 (w / w). The ratio of alkaline phosphatase to antibody affects the stereostructure of the enzyme-labeled antibody complex and the number of antibodies bound to the alkaline phosphatase, thereby affecting the formation of immunomagnetic beads, antigen, and enzyme-labeled antibody complex, causing differences in chemiluminescence signals and affecting the repeatability of the test results. A ratio of 2:1 is preferred.

[0087] The enzyme-labeled buffer can be one of the buffers that do not contain amino and carboxyl groups, such as MES buffer, borate buffer, or PBS buffer, with PBS buffer being preferred.

[0088] The pH of the enzyme-labeled buffer solution is 7.0 to 8.0, preferably 7.0.

[0089] The storage buffer solution mainly consists of a buffer system, stabilizers (proteins, sugars, aromatic amino acids, etc.), and preservatives.

[0090] The concentration of amino acids in the storage buffer is 1–3 mg / mL, preferably 2.5 mg / mL;

[0091] The sugars in the storage buffer can be trehalose, glucose, mannose, hyaluronic acid, galactose, etc., with trehalose being preferred; the concentration of the sugars can be 5-15 mg / mL, with 10 mg / mL being preferred;

[0092] The aromatic amino acid can be one or more of tryptophan, tyrosine, phenylalanine, etc., with tyrosine being preferred;

[0093] The storage buffer system can be one of MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer, etc., preferably Tris-HCl buffer.

[0094] The pH of the enzyme-labeled buffer solution is 7.0 to 8.0, preferably 8.0.

[0095] The storage buffer stabilizer can be one or more of the following: polyethylene glycol, bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, aromatic amino acids, cellulose (sodium carboxymethyl cellulose, ethyl cellulose, etc.), and amino polysaccharides (chondroitin sulfate, chitosan, etc.). The aromatic amino acids can be phenylalanine, tyrosine, and tryptophan. The stabilizer can reduce the non-specific binding of antibodies and can also bind to interfering substances in the sample, thereby improving the repeatability of the detection.

[0096] The preservative in the storage buffer solution can be one or more of NaN3, Proclin 300, and Proclin 600.

[0097] The TAT chemiluminescence assay kit can be used in chemiluminescence analyzers with an enzyme-catalyzed glow discharge chemiluminescence detection system.

[0098] The TAT chemiluminescence assay kit uses immunomagnetic beads with a test concentration of 0.1–1 mg / mL (based on bead concentration), preferably 0.5 mg / mL.

[0099] The TAT chemiluminescence assay kit uses an enzyme-labeled antibody concentration of 0.1–1 μg / mL (based on antibody concentration), preferably 0.5 μg / mL.

[0100] Furthermore, the linear range of the kit is 0.5–120 ng / mL.

[0101] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0102] The present invention will be further illustrated below with reference to the embodiments:

[0103] Preparation Example

[0104] 1. Preparation of immunomagnetic beads

[0105] Use a vortex mixer to thoroughly mix the carboxyl magnetic beads. Take 5 mg of the magnetic bead solution into a centrifuge tube, add activation buffer (0.1 M MES, pH 6.5) to a final volume of 1 mL, mix well, and then magnetically separate for 1 min. Discard the supernatant. Add another 1 mL of activation buffer, mix well, and then magnetically separate for 1 min. Discard the supernatant, add another 1 mL of activation buffer, and mix well before use.

[0106] Add 100 μL of activator (2.5 mg / mL EDC & 2.5 mg / mL Sulfo-NHS, freshly prepared) and mix well. Incubate at room temperature by rotating the mixture for 30 min.

[0107] After magnetic separation, the supernatant was removed, and 100 μg of thrombin antibody was taken and added to cross-linking buffer (0.1 M MES, pH 7.0) to make up to 1 mL. The mixture was then added to the activated magnetic beads, mixed evenly, and incubated at room temperature by rotation for 6 h.

[0108] After magnetic separation, the supernatant was removed, and 1 mL of cross-linking buffer containing 10 mg / mL BSA was added. The mixture was rotated and blocked at room temperature for 1 h. After magnetic separation, the supernatant was removed.

[0109] Disperse the conjugated immunomagnetic beads using 1 mL of storage buffer and store at 2–8°C. Dilute to 0.5 mg / mL with storage buffer before instrumental testing.

[0110] 2. Preparation of enzyme-labeled antibodies

[0111] Dissolve / dilute 2 mg of alkaline phosphatase to a 10 mg / mL alkaline phosphatase solution using 0.2 mL of enzyme-labeled buffer, add 33.4 μL of Sulfo-SMCC (4 mg / mL), and gently incubate at room temperature for 1 h. After desalting the product, disperse it using enzyme-labeled buffer for later use.

[0112] Dilute 2 mg of antithrombin antibody to 10 mg / mL using 0.2 mL of enzyme-labeled buffer, add 10 μL of Traut's (10 mg / mL), and incubate gently at room temperature for 1 h. Add 40 μL (5 mg / mL) of glycine solution, mix well, and continue incubation for 5 min. After desalting the product, disperse it with enzyme-labeled buffer for later use.

[0113] After mixing the activated alkaline phosphatase and the thiolized antithrombin antibody evenly, the mixture was gently incubated at 4°C for 24 hours. Then, 100 μL (10 mg / mL) of cysteine ​​was added and mixed well. The mixture was then incubated for another 30 minutes. After desalting the product, it was diluted with enzyme-labeled storage buffer and then diluted to 0.5 μg / mL with enzyme-labeled storage buffer before testing.

[0114] Example 1: Composition of storage buffer solution

[0115] 1. Trehalose concentration

[0116] Prepare enzyme-labeled antibody storage buffers (50mM Tris, 5mg / mL BSA, 1mg / mL NaN3, pH 8.0) containing 0mg / mL, 5mg / mL, 7.5mg / mL, 10mg / mL, 12.5mg / mL, and 15mg / mL trehalose, respectively. Dilute the enzyme-labeled antibody (preparation method as shown in the preparation example), and test plasma samples with immunomagnetic beads (preparation method as shown in the preparation example). Use a one-step method for testing. Add 50μL of immunomagnetic beads, 50μL of enzyme-labeled antibody, and 10μL of sample. After incubation for 30min, wash three times with washing buffer, add 200μL of substrate solution, incubate for 5min, and then detect the photon quantity. The test was repeated five times, and the coefficient of variation (CV) of the test results was calculated to examine the repeatability of the test results. The results are shown in Table 1. Compared with the 0 mg / mL trehalose group, the coefficient of variation of the test results decreased and the repeatability was improved after adding trehalose to the enzyme-labeled antibody storage buffer. Among them, the enzyme-labeled antibody storage buffer containing 10 mg / mL trehalose showed the lowest coefficient of variation and the best repeatability for samples 1 and 2. Therefore, the enzyme-labeled antibody storage buffer containing 10 mg / mL trehalose was selected to store the enzyme-labeled antibody.

[0117] Table 1: Analysis of Repeatability Test Results for Different Storage Buffer Groups

[0118]

[0119] 2. Ratio of alkaline phosphatase to antibody

[0120] Enzyme-labeled antibodies were prepared according to the method shown in the preparation example, with alkaline phosphatase to antibody mass ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. These antibodies were then used with immunomagnetic beads (prepared as shown in the preparation example) to test plasma samples. The tests were repeated five times, and the coefficient of variation (CV) was calculated to examine the repeatability of the test results. The results are shown in Table 2. When the ratio of alkaline phosphatase (AP) to antibody (lgG) was 2:1, the coefficient of variation for samples 1 and 2 was the lowest, indicating the best repeatability. This suggests that the stereostructure of the enzyme-labeled antibody prepared at this ratio, and the binding ratio of alkaline phosphatase and antibody in the complex, are more conducive to antigen detection and improve the stability of the complex, thereby improving the repeatability of the measured values. Therefore, an alkaline phosphatase to antibody ratio of 2:1 was chosen for preparing the enzyme-labeled antibody.

[0121] Table 2: Analysis of repeatability test results for different enzyme-labeled antibody groups

[0122]

[0123] 3. Calibration curve test

[0124] Prepare enzyme-labeled antibody storage buffers according to Table 3, dilute the enzyme-labeled antibodies (preparation method as shown in the preparation example), and test different concentrations of TAT antigen using immunomagnetic beads (preparation method as shown in the preparation example). Perform linear regression based on antigen concentration and measured photon intensity, and the results are as follows. Figure 1 and Figure 2 As shown in Table 4 (corresponding data), compared with the control group storage buffer 1 (without amino acids), the correlation coefficients of the standard curves of the other four groups were all increased, indicating that the calibration curves were improved after adding amino acids to the enzyme-labeled antibody storage buffer. Compared with storage buffer 2 (containing the non-aromatic amino acid glycine), the correlation coefficients of the standard curves of storage buffers 3-5 (containing aromatic amino acids) were further increased. Among them, the correlation coefficient γ of the standard curve of storage buffer 5 was 0.9976, showing good linearity and a high measured photon value. This indicates that adding the aromatic amino acid tyrosine to the storage buffer is more conducive to the stability of the enzyme-labeled antibody, maintaining the stereostructure of the enzyme-labeled antibody, thereby improving the calibration curve of the TM assay kit and increasing the repeatability of the test. Therefore, storage buffer 5 was selected to prepare the storage buffer for the enzyme-labeled antibody.

[0125] Table 3: Composition of Storage Buffer

[0126]

[0127] Table 4: Calibration Curve Test Results

[0128] Antigen concentration (ng / mL) Storage buffer 1 Storage Buffer 2 Storage Buffer 3 Storage Buffer 4 Storage buffer 5 0 27132 24158 21143 27131 18148 5 114157 129365 123435 153733 259075 10 1531726 1651357 842121 1132731 1251516 40 4521154 4649776 3205465 4630903 4849576 80 8012570 8656245 7423578 8548846 11453243 100 8424028 10629964 9463820 9071871 15629639 120 11876420 14829144 12973709 11511218 17829144

[0129] Example 2: Sample Test Comparison

[0130] One hundred blood samples were randomly collected from inpatients and outpatients at Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The blood was mixed with 0.109 mol / L sodium citrate anticoagulant at a ratio of 9:1, and centrifuged at 2500 rpm for 15 minutes to separate plasma. The same samples were tested using the kit of this invention (immunomagnetic beads 0.5 mg / mL, volume 50 μL; enzyme-labeled antibody 0.5 μg / mL, volume 50 μL, preparation method as shown in the preparation example, the storage buffer prepared according to the formula of storage buffer 5 described in effect example 1) and the Sysmex kit. The obtained values ​​were subjected to linear regression, and the correlation coefficient between the two was calculated. The results are as follows: Figure 3 As shown, the correlation coefficient γ = 0.996 between the two kits, and the linear regression equation is y = 0.932x + 0.6155. The detection data of the kit of this invention and the Sysmex kit have good consistency.

[0131] Example 3: Repeatability Test

[0132] The kit of this invention (0.5 mg / mL immunomagnetic beads, 50 μL; 0.5 μg / mL enzyme-labeled antibody, 50 μL, prepared as shown in the preparation example, wherein the storage buffer is prepared according to the formulation of storage buffer 5 described in effect example 1), the Sysmex kit, and the plasma sample were placed in the designated position of the chemiluminescence analyzer, and the following procedure was followed for testing. The test was repeated 10 times, and the average value and coefficient of variation (CV) of the test results were calculated:

[0133] a) Mix 50 μL of immunomagnetic beads, 50 μL of enzyme-labeled antibody and 10 μL of sample evenly, and incubate at 37°C for 30 min;

[0134] b) Magnetic separation, using chemiluminescence analyzer cleaning solution to clean the complex;

[0135] c) Add 200 μL of substrate solution for fully automated immunoassay system to the cleaned magnetic bead complex and incubate at 37°C in the dark for 5 min;

[0136] d) Use a photoelectrochemical reactor to measure the amount of photons in the substrate solution after the reaction;

[0137] e) Calculate the sample concentration using the fitted calibration curve.

[0138] Table 5: Repeatability Test Results

[0139]

[0140] The test results are shown in Table 5. The coefficient of variation of the test results of the kit of the present invention is small, indicating that the test repeatability is good.

[0141] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of tyrosine in enzyme-labeled antibody storage buffer for preparing a reagent kit for chemiluminescence assay of thrombin-antithrombin III complex, wherein the enzyme-labeled antibody storage buffer is composed of buffer, BSA, trehalose, tyrosine and preservative.

2. An enzyme label antibody storage buffer for a thrombin-antithrombin III complex chemiluminescent assay kit, characterized in that, The enzyme-labeled antibody storage buffer is composed of 50 mM Tris, 5 mg / mL BSA, 10 mg / mL trehalose, 2.5 mg / mL tyrosine and 1 mg / mL NaN3; and the pH value of the enzyme-labeled antibody storage buffer is 8.

0.

3. A kit for the chemiluminescent assay of the thrombin-antithrombin III complex, characterized in that, The enzyme-labeled antibody storage buffer of claim 2 further comprises acceptable adjuvants or auxiliaries.

4. The kit of claim 3, wherein The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody.

5. The method of claim 4, wherein the kit is prepared by, The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody. The enzyme-labeled antibody storage buffer of claim 2 further comprises enzyme-labeled antibody.

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