Antigen preserving fluid as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to antigen preserving fluid and a preparation method and application thereof. The invention provides an antigen preservation solution and a preparation method thereof, the antigen preservation solution adopts a sodium tetraborate buffer solution, and potassium chloride, glycerol, bovine serum albumin, triton, ADP, ProClin300 and methylisothiazolinone are added as an inorganic salt, a stabilizer, an excipient, a surfactant, a protein protective agent and a preservative respectively. A stable preservation environment is provided for the biotinylated U1RNP antigen, the problems that the biotinylated U1RNP antigen is prone to dissociation and degradation, consequently, the biotinylated U1RNP antigen cannot be stably preserved, and inconvenience is brought to transportation and use are solved, and the problems that an anti-U1RNP antibody IgG detection kit is poor in carrier stability and real-time stability are further solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an antigen preservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Ribonucleoprotein (RNP) is one of the extractable nuclear antigens (ENA) and belongs to the small ribonucleoprotein (snRNP) family. The snRNP contains five particles, namely U1, U2, U4, U5, and U6. The so-called anti-RNP antibody can precipitate the U1 part therein, so it is also called anti-U1RNP antibody. High-titer U1RNP is a marker antibody for mixed connective tissue disease (MCTD), and the U1RNP particle also contains three specific proteins, U1-70K, U1A, and U1C, with relative molecular weights of 70,000 or 68,000, 32,000, and 17,500 respectively.

[0003] Due to the fact that the U1RNP antigen consists of multiple protein components, there are charge effects among the protein components, and the charges among the protein components will interact with each other. If the biotinylated U1RNP antigen is not properly stored, it is easily dissociated and degraded, which in turn leads to instability in the storage and use of the detection kit, affecting the robustness of in vitro diagnostic tests and ensuring the accuracy of test results. Currently, no relevant patents have mentioned how to solve the stability problems of the storage and application of autoantigens containing component proteins. Patent CN106596919 A proposes a kit (chemiluminescence method) for detecting anti-ribonucleoprotein 70 antibody IgG and its preparation method. This method is for the detection method of ribonucleoprotein 70 antibody, and the antigen coated is only the RNP70K protein, without mentioning the application of UIA and UIC; Patent CN102621310 B proposes a colloidal gold immunoassay strip for detecting anti-U1-RNP antibody and its preparation method. This method 1 is the colloidal gold method and does not involve the application of the magnetic particle chemiluminescence platform; Patent CN102360016A proposes the preparation of a kit for detecting Sm-RNP antibody in SLE by flow cytometry microspheres. This method is the flow cytometry microsphere method, a chemiluminescence platform, and the antigen coated is Sm-RNP, without mentioning how to improve the stability of the Sm-RNP antigen; Patent CN 105300965A proposes a kit for quantitatively detecting anti-nRNP / Sm antibody IgG using magnetic particle chemiluminescence and its preparation method and detection method. This method is the magnetic particle chemiluminescence method, and the antigen coated is nRNP / Sm. The coating method is to directly coat the nRNP / Sm antigen onto the magnetic particle reagent, but it does not mention how to improve the stability of the nRNP / Sm antigen; Patent CN117110601 A proposes a preparation method of an immunomagnetic bead preservation solution. This preservation solution is only used for the preservation of magnetic beads and cannot be used for antigen solutions in the biotin-avidin system. Currently, there is no solution to the problem that biotinylated U1RNP antigen is not easily stored. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an antigen preservation solution, its preparation method and application. The antigen preservation solution provided by the present invention can achieve the long-term preservation of biotinylated U1RNP antigen, and the preparation and use costs are economical and affordable.

[0005] The present invention provides an antigen preservation solution, which includes a buffer solution with a pH of 8.0 - 11.0 and a concentration of 0.01 - 2M, 0.1 - 20g / L of inorganic salts, 1 - 100g / L of stabilizers, 1 - 100g / L of excipients, 1 - 50g / L of surfactants, 0.5 - 50g / L of protein protectants, and 0.5 - 50g / L of preservatives.

[0006] The U1RNP antigen subtype has many components. The connection between U1-70K and U1A is not only through the RNA connected thereto, but also through the direct connection between proteins. When the biotinylated U1RNP antigen is applied to the kit, the antigen is liable to dissociate and degrade in the conventional preservation solution, resulting in the unstable preservation of the kit, which brings inconvenience to the transportation and use of the kit. The antigen preservation solution provided by the present invention adopts a sodium tetraborate buffer solution, and provides a stable preservation environment for the biotinylated U1RNP antigen by adding a combination of excipients, protein protectants, and preservatives. It can improve the problems of poor on-board stability and real-time stability of the anti-U1RNP antibody IgG detection kit.

[0007] In some embodiments, the buffer solution includes at least one of boric acid-borax buffer solution, borax buffer solution, boric acid-potassium chloride buffer solution, and sodium tetraborate buffer solution;

[0008] In some embodiments, the inorganic salt includes at least one of potassium chloride, magnesium chloride, and sodium chloride;

[0009] In some embodiments, the stabilizer includes at least one of glycerol, mannitol, sorbitol, sucrose, and trehalose;

[0010] In some embodiments, the excipient includes at least one of bovine serum albumin, calf serum, and casein;

[0011] In some embodiments, the surfactant includes Triton and / or Tween;

[0012] In some embodiments, the protein protectant includes ADP and / or BB2;

[0013] In some embodiments, the preservative includes at least one of sodium azide, ProClin300, gentamicin, dichlorodiphenyltrichloroethane, and methylisothiazolinone.

[0014] In some specific embodiments, the buffer solution is a sodium tetraborate buffer solution;

[0015] The inorganic salt is potassium chloride;

[0016] The stabilizer is glycerol;

[0017] The excipient is bovine serum albumin;

[0018] The surfactant is Triton;

[0019] The protein protectant is ADP;

[0020] The preservative is ProClin300 and methylisothiazolinone.

[0021] Compared with the prior art, the inorganic salts, stabilizers, excipients, surfactants, protein protectants, and preservatives provided by the present invention cooperate with each other, have strong compatibility, and can synergistically enhance the effect, providing a stable storage environment for the biotinylated U1RNP antigen, thereby achieving a more accurate detection effect.

[0022] In some specific embodiments, the antigen preservation solution includes 0.1M sodium tetraborate buffer with a pH of 10.0 ± 0.05, 5 g / L potassium chloride, 20 g / L glycerol, 20 g / L bovine serum albumin, 1 g / L Triton, 1 g / L ADP, 1 g / L ProClin300, and 1 g / L methylisothiazolinone.

[0023] Verified by experiments, at the above concentrations, the inorganic salts, stabilizers, excipients, surfactants, protein protectants, and preservatives provided by the present invention have the best cooperation effect, the strongest compatibility, and can synergistically enhance the effect, providing the most stable storage environment for the biotinylated U1RNP antigen, thereby achieving a more accurate detection effect.

[0024] The present invention provides a method for preparing the antigen preservation solution, including the following steps:

[0025] Add buffer salts to water, mix well, and adjust the pH to 8.0 - 11.0;

[0026] Add inorganic salts, stabilizers, excipients, surfactants, protein protectants, and preservatives, and obtain the antigen preservation solution after mixing well.

[0027] The present invention provides a detection kit for anti-U1RNP antibody IgG, including the antigen preservation solution described above or the antigen preservation solution prepared by the preparation method described above, and biotinylated U1RNP antigen dispersed in the antigen preservation solution.

[0028] Compared with the prior art, the beneficial effects of the present invention include:

[0029] 1. The present invention provides an antigen preservation solution and a preparation method thereof. The antigen preservation solution uses sodium tetraborate buffer, and by adding potassium chloride, glycerol, bovine serum albumin, Triton, ADP, ProClin300, and methylisothiazolinone as inorganic salts, stabilizers, excipients, surfactants, protein protectants, and preservatives respectively, it provides a stable storage environment for the biotinylated U1RNP antigen, overcomes the problems that the biotinylated U1RNP antigen is prone to dissociation and degradation, resulting in inconvenient transportation and use, and further improves the problems of poor on-board stability and real-time stability of the anti-U1RNP antibody detection kit;

[0030] 2. The antigen preservation solution can be applied to the preservation of biotinylated U1RNP antigen and the detection of anti-U1RNP antibody IgG, which can improve the carrier stability of biotinylated U1RNP antigen. The signal value variation within 35 days of the carrier can be reduced to within 10%, and it can be stored at 2-8°C for 1 year. After 7 days of thermal acceleration test at 37°C and then detection, the variation is within 10%. According to the Arrhenius formula, the validity period is calculated to be 1 year. Detailed implementation mode

[0031] The present invention provides an antigen preservation solution, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] Ribonucleoprotein (RNP), which is one of the extractable nuclear antigens (ENA), belongs to the small nuclear ribonucleoprotein (snRNP) family. SnRNP contains five particles: U1, U2, U4, U5, and U6. The so-called anti-RNP antibody can precipitate the U1 part among them, so it is also called anti-U1RNP antibody. High-titer U1RNP is a marker antibody for mixed connective tissue disease (MCTD). The U1RNP particle also contains three specific proteins, U1-70K, U1A, and U1C, with relative molecular masses of 70,000 or 68,000, 32,000, and 17,500 respectively. The U1RNP antigen is mainly composed of these three proteins. The connection between U1-70K and U1A is not only through the connected RNA but also through direct protein-protein connection. U1-70K has a constant structural region at the N-terminus, called the RNA binding domain (RBD), RNA recognition motif (RRM), or RNP motif (within 104-184 aa). The RBD and the amino acid sequences of the N- and C-terminal side branches are necessary for binding to U1 snRNA. Its tail has two arginine-serine-rich regions (within 241-303 aa and 348-389 aa). The U1A protein has an RBD at each end. The N-terminal RBD participates in the connection with U1 snRNA, and the C-terminal RBD has no affinity for RNA. The U1A protein contains a proline- and methionine-rich region (within 140-202 aa). The U1C protein has a zinc finger-like structure at the C-terminus, which is necessary for the binding of U1C protein to U1 snRNP. Its C-terminus also has a proline- and methionine-rich region (within 62-159 aa).

[0033] Since U1RNP contains three proteins and RNA, and the three proteins are connected by RNA and amino acids between proteins, U1RNP in a liquid environment requires the presence of an RNA enzyme inhibitor, a protease inhibitor, and a chemical substance that can strengthen amino acid connection to ensure its long-term stability. The borate contained in the preservation solution in the present invention can not only act as a protease inhibitor but also serve as a bridge for the connection between serine in U1RNP and RNA. At the same time, the protein protectant and stabilizer in the preservation solution can also play a role in protecting the U1RNP antigen. Stability is an important indicator for the robustness of in vitro diagnostic test kits and ensuring the accuracy of test results. Therefore, in view of the problem that biotinylated U1RNP antigen is not easily preserved, the present invention proposes a solution.

[0034] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with the examples.

[0035] Example 1

[0036] The preparation process of a biotinylated U1RNP antigen preservation solution of the present invention is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0037] (1) Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0038] (2) Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0039] (3) Accurately weigh 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), 1 g of ProClin300, and add them into the buffer solution with adjusted pH in (2), stir evenly, and place it at 2 - 8 °C for standby;

[0040] Example 2

[0041] The preparation process of comparative preservation solution 1 is as follows (the buffer solution is PBS buffer solution with pH 7.4):

[0042] (1) Add 1000 ml of purified water into a container, and then add 14.2 g of accurately weighed sodium dihydrogen phosphate (NaH2PO4), 81 g of disodium hydrogen phosphate (Na2HPO4), and 8.5 g of sodium chloride (NaCl), and stir well to dissolve;

[0043] (2) Adjust the pH value of the solution to pH 7.4 ± 0.05 with 6M hydrochloric acid;

[0044] (3) Accurately weigh 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), 1 g of ProClin300, and add them into the buffer solution with adjusted pH in (2), stir evenly, and place it at 2 - 8 °C for standby.

[0045] Example 3

[0046] The preparation process of comparative preservation solution 2 is as follows (the buffer solution is Tris - Nacl buffer system with pH 7.4):

[0047] (1) Add 1000 ml of purified water into a container, and then add 12.114 g of accurately weighed Tris and 5.85 g of NaCl, and stir well to dissolve;

[0048] (2) Adjust the pH value of the solution to pH 7.4 ± 0.05 with 6M hydrochloric acid

[0049] (3) Weigh accurately 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin 300 that need to be added, and add them to the buffer solution with the pH adjusted in (2), stir evenly, and place it at 2 - 8 °C for standby.

[0050] Example 4

[0051] The preparation process of Comparative Preservation Solution 3 is as follows (the buffer solution is a MES buffer system with pH 6.3):

[0052] (1) Add 1000 ml of purified water to the container, and then add 19.525 g of MES weighed accurately, and stir well to dissolve.

[0053] (2) Adjust the pH value of the solution to pH 6.3 ± 0.05 with 6 M sodium hydroxide.

[0054] (3) Weigh accurately 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, `g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin 300 that need to be added, and add them to the buffer solution with the pH adjusted in (2), stir evenly, and place it at 2 - 8 °C for standby.

[0055] Example 5

[0056] The preparation process of Comparative Preservation Solution 4 is as follows (the buffer solution is a HEPES buffer system with pH 7.0):

[0057] (1) Add 1000 ml of purified water to the container, and then add 23.83 g of HEPES weighed accurately, and stir well to dissolve.

[0058] (2) Adjust the pH value of the solution to pH 7.0 ± 0.05 with 6 M sodium hydroxide.

[0059] (3) Weigh accurately 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 2 g of Triton, 100 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin 300 that need to be added, and add them to the buffer solution with the pH adjusted in (2), stir evenly, and place it at 2 - 8 °C for standby.

[0060] Example 6

[0061] The preparation process of Comparative Preservation Solution 5 is as follows (the buffer solution is a sodium borate buffer solution with pH 10.0):

[0062] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0063] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0064] Accurately weigh 5 g of potassium chloride, 10 g of glycerol, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2), stir evenly, and store at 2 - 8 °C for standby;

[0065] Example 7

[0066] The preparation process of Comparative Preservation Solution 6 is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0067] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0068] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0069] Accurately weigh 5 g of potassium chloride, 20 g of glycerol, 10 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2), stir evenly, and store at 2 - 8 °C for standby;

[0070] Example 8

[0071] The preparation process of Comparative Preservation Solution 7 is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0072] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0073] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0074] Accurately weigh 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 2 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2), stir evenly, and store at 2 - 8 °C for standby;

[0075] Example 9

[0076] The preparation process of Comparative Preservation Solution 8 is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0077] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0078] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0079] Accurately weigh 5 g of potassium chloride, 20 g of glycerol, 20 g of bovine serum albumin, 1 g of Triton, 2 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2), stir evenly, and place at 2 - 8°C for standby;

[0080] Example 10

[0081] The preparation process of Comparative Preservation Solution 9 is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0082] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0083] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0084] Accurately weigh 5 g of potassium chloride, 10 g of glycerol, 10 g of bovine serum albumin, 2 g of Triton, 2 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2), stir evenly, and place at 2 - 8°C for standby.

[0085] Example 11

[0086] The preparation process of Comparative Preservation Solution 9 is as follows (the buffer solution is sodium tetraborate buffer solution with pH 10.0):

[0087] Add 1000 ml of purified water into a container, and then add 37.8 g of accurately weighed sodium tetraborate, and stir well to dissolve;

[0088] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0089] Accurately weigh 5 g of magnesium chloride, 20 g of mannitol, 20 g of calf serum, 1 g of Tween, 1 g of BB2, 1 g of MIT (methylisothiazolinone), and 1 g of sodium azide, and add them to the buffer solution with adjusted pH in (2). Stir evenly and store at 2 - 8 °C for later use.

[0090] Example 12

[0091] The preparation process of Comparative Preservation Solution 9 is as follows (the buffer solution is sodium tetraborate buffer with pH 10.0):

[0092] Add 1000 ml of purified water to the container, and then add accurately weighed 37.8 g of sodium tetraborate, and stir well to dissolve.

[0093] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6 M sodium hydroxide.

[0094] Accurately weigh 5 g of sodium chloride, 20 g of sorbitol, 20 g of casein, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of gentamicin, and add them to the buffer solution with adjusted pH in (2). Stir evenly and store at 2 - 8 °C for later use.

[0095] Example 13

[0096] The preparation process of Comparative Preservation Solution 9 is as follows (the buffer solution is sodium tetraborate buffer with pH 10.0):

[0097] Add 1000 ml of purified water to the container, and then add accurately weighed 37.8 g of sodium tetraborate, and stir well to dissolve.

[0098] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6 M sodium hydroxide.

[0099] Accurately weigh 5 g of potassium chloride, 20 g of sucrose, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of dichlorodiphenyltrichloroethane, and add them to the buffer solution with adjusted pH in (2). Stir evenly and store at 2 - 8 °C for later use.

[0100] Example 14

[0101] The preparation process of Comparative Preservation Solution 9 is as follows (the buffer solution is sodium tetraborate buffer with pH 10.0):

[0102] Add 1000 ml of purified water to the container, and then add accurately weighed 37.8 g of sodium tetraborate, and stir well to dissolve.

[0103] Adjust the pH value of the solution to pH 10.0 ± 0.05 with 6M sodium hydroxide;

[0104] Accurately weigh 5 g of potassium chloride, 20 g of trehalose, 20 g of bovine serum albumin, 1 g of Triton, 1 g of ADP, 1 g of MIT (methylisothiazolinone), and 1 g of ProClin300 to be added, and add them to the buffer solution with adjusted pH in (2). Stir evenly and store at 2 - 8°C for later use.

[0105] Effect Example 1 Carrier Stability Assessment

[0106] Apply the 5 kinds of biotinylated U1RNP antigen preservation solutions prepared in Examples 1 - 5 to the dilution of biotinylated U1RNP antigen to prepare antigen reagents, and conduct carrier stability assessment on them. The biotinylated U1RNP antigen is sourced from Yimeinuo Company (product number AA410004). Detect the signal values at 7 days, 21 days, and 35 days of being on the carrier respectively, and compare them with the signal value at 0 day of being on the carrier (using Antu Bio A2000Plus instrument with the reagents supporting the project). The test steps are as follows:

[0107] After preparing the test samples (calibrators or samples to be tested) and placing them correctly, click the start button to perform the calibration program or

[0108] sample detection program, and the instrument will perform the following operations:

[0109] 1) Transfer the sample rack to the sampling position and load the reaction container to the reagent addition position.

[0110] 2) Dilute the sample to be tested at a ratio of 1:19 with the system diluent (mix 20 μL of the sample evenly with 380 μL of the system diluent).

[0111] Note: The calibrator has been pre - diluted and can be used directly.

[0112] 3) When performing the calibration program, dispense 100 μL of the calibrator and 20 μL of the magnetic particle suspension.

[0113] 4) When performing the sample detection program, dispense 10 μL of the sample to be tested, 20 μL of the magnetic particle suspension, and 90 μL of the sample diluent.

[0114] 5) Mix and incubate the reaction solution. The incubation temperature is 37°C and the incubation time is 15 minutes.

[0115] 6) After incubation, perform magnetic separation on the reaction solution and wash and separate it with the washing solution.

[0116] 7) Dispense 100 μL of the enzyme conjugate and mix the reaction solution.

[0117] 8) Mix and incubate the reaction solution at a temperature of 37 °C for 15 minutes.

[0118] 9) After incubation, wash and separate the reaction solution with the washing solution.

[0119] 10) Dispense 50 μL of Substrate A solution and 50 μL of Substrate B solution.

[0120] 11) Mix the reaction solution and measure the luminescence intensity.

[0121] For the relevant functions and operations of the instrument, refer to the operation instructions of the corresponding instrument system.

[0122] Results: For the antigen reagent carrier prepared with the preservation solution of Example 1 of the present invention for 7 days, 21 days, and 35 days, the bias compared with the carrier at day 0 is within ±10%. For the antigen reagents prepared with the antigen preservation solutions of Examples 2-5 for 7 days, 21 days, and 35 days, the bias compared with the carrier at day 0 exceeds ±10%. The detection effects are shown in Table 1.

[0123] Table 1

[0124]

[0125]

[0126] Effect Example 2 Thermal Accelerated Stability Assessment

[0127] Apply the 5 biotinylated U1RNP antigen preservation solutions prepared in Examples 1-5 to the dilution of biotinylated antigens to prepare antigen reagents. Place the antigen reagents in an oven at 37 °C for 7 days, and after taking them out, compare the detection results with those of the control at 4 °C (using the Antu Biotech A2000Plus instrument with the supporting reagents for the project). The test steps are the same as in Effect Example 1. Results: For the antigen reagent prepared with the preservation solution of Example 1 of the present invention, the bias of the detection results at 37 °C for 7 days compared with the detection results of the 4 °C control is within ±10%. For the antigen reagents prepared with the preservation solutions of Examples 2-5, the bias of the detection results at 37 °C for 7 days compared with the detection results of the 4 °C control exceeds ±20%. Calculated according to the Arrhenius formula, it shows that the antigen reagent prepared with the preservation solution of the present invention can be stored stably at 2-8 °C for 1 year. The detection effects are shown in Table 2.

[0128] Table 2

[0129]

[0130]

[0131] Effect Example 3 Thermal Accelerated Stability Assessment

[0132] Six biotinylated U1RNP antigen preservation solutions of Example 1 and Examples 6-10 were applied to the dilution of biotinylated antigen in parallel to prepare antigen reagents. The antigen reagents were placed in an oven at 37 °C for 7 days, and after taking them out, the test results were compared with those of the 4 °C control (using the Antu Bio A2000Plus instrument with the reagents supporting the project). The test steps were the same as those in Effect Example 1. Results: Compared with the test results of the 4 °C control, the stability of the antigen reagents prepared with the six preservation solutions after heat acceleration at 37 °C for 7 days showed that the stability of Example 1 was significantly better than the other five. Calculated according to the Arrhenius formula, it was shown that the antigen reagents prepared with the preservation solution of the present invention could be stored stably at 2-8 °C for 1 year. The test effects are shown in Table 3.

[0133] Table 3

[0134]

[0135]

[0136] Effect Example 4 Assessment of Thermal Acceleration Stability of Different Formulations

[0137] Five biotinylated U1RNP antigen preservation solutions of Example 1 and Examples 11-14 were applied to the dilution of biotinylated antigen in parallel to prepare antigen reagents. The antigen reagents were placed in an oven at 37 °C for 7 days, and after taking them out, the test results were compared with those of the 4 °C control (using the Antu Bio A2000Plus instrument with the reagents supporting the project). The test steps were the same as those in Effect Example 1. Results: Compared with the test results of the 4 °C control, the stability of the antigen reagents prepared with the five preservation solutions after heat acceleration at 37 °C for 7 days showed that the stability of Example 1 was significantly better than the other five, and the signal value of Example 1 was significantly higher than the other four examples. Calculated according to the Arrhenius formula, it was shown that the antigen reagents prepared with the preservation solution of the present invention could be stored stably at 2-8 °C for 1 year. The test effects are shown in Table 4.

[0138] Table 4

[0139]

[0140]

[0141] The U1RNP antigen has many components. It is easily dissociated and unstable in ordinary buffer solutions and is easily degraded by proteases. Sodium tetraborate buffer contains borate ions. The electrophilicity of borate ions can form covalent bonds, which can connect different subtypes again. At the same time, borate ions can also act as protease inhibitors to avoid the degradation of U1RNP antigen by proteases.

[0142] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An antigen storage solution, characterized in that: The invention comprises 0.01-2M buffer solution with a pH value of 8.0-11.0, 0.1-20 g / L inorganic salt, 1-100 g / L stabilizer, 1-100 g / L excipient, 1-50 g / L nonionic surfactant, 0.5-50 g / L protein protective agent and 0.5-50 g / L preservative.

2. The antigen preservation solution according to claim 1, characterized in that The buffer comprises at least one of a boric acid-borax buffer, a borax buffer, a boric acid-potassium chloride buffer and a sodium tetraborate buffer.

3. The antigen preservation solution according to claim 1, characterized in that The inorganic salt includes at least one of potassium chloride, magnesium chloride and sodium chloride; The stabilizer includes at least one of glycerol, mannitol, sorbitol, sucrose and trehalose.

4. The antigen preservation solution according to claim 1, characterized in that The excipient includes at least one of bovine serum albumin, calf serum and casein; The surfactant includes Triton and / or Tween.

5. The antigen preservation solution according to claim 1, characterized in that The protein protecting agent includes ADP and / or BB2.

6. The antigen preservation solution according to claim 1, characterized in that The preservative comprises at least one of sodium azide, ProClin300, gentamicin, bis(p-chlorophenyl)trichloroethane and methylisothiazolinone.

7. The antigen storage solution according to any one of claims 1 to 6, characterized in that The buffer is sodium tetraborate buffer; The inorganic salt is potassium chloride; The stabilizer is glycerol; The excipient is bovine serum albumin; The surfactant is Triton; The protein protective agent is ADP; The preservatives are ProClin 300 and methylisothiazolinone.

8. The antigen storage solution according to any one of claims 1 to 7, characterized in that It includes 0.1MPH 10.0±0.05 sodium tetraborate buffer, 5g / L potassium chloride, 20g / L glycerol, 20g / L bovine serum albumin, 1g / L Triton, 1g / LADP, 1g / L ProClin300 and 1g / L methylisothiazolinone.

9. The method for preparing the antigen storage solution according to any one of claims 1 to 8, characterized in that: The steps include: Add buffer salt to water, mix well, and adjust the pH to 8.0-11.0; Inorganic salts, stabilizers, excipients, surfactants, protein protective agents and preservatives are added and mixed to obtain the antigen preservation solution.

10. A detection reagent or kit for anti-U1RNP antibody IgG, characterized in that: The invention comprises the antigen preservation solution according to any one of claims 1 to 8 or the antigen preservation solution prepared by the preparation method according to claim 9, and the biotinylated U1RNP antigen dispersed in the antigen preservation solution.

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