A rubella virus-IgM antibody rapid detection kit

Abstract

This invention belongs to the field of medical testing technology, specifically relating to a rapid detection kit for rubella virus-IgM antibodies. The kit comprises independently stored first lyophilized microspheres, second lyophilized microspheres, and a sample processing solution. The first lyophilized microspheres contain a complex I formed by coupling and coating rubella virus antigen with epoxy-based magnetic beads. The second lyophilized microspheres contain a complex II formed by acrid ester and anti-human IgM antibody through a light-protected reaction. The sample processing solution includes a buffer, sodium chloride, a surfactant, and a preservative. Compared with existing IgM antibody detection kits, the rapid detection kit for rubella virus-IgM antibodies provided by this invention has advantages such as strong anti-interference ability, high detection accuracy and sensitivity, higher stability, ease of use, room temperature storage, and controllable cost, and has broad prospects for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a rapid test kit for rubella virus-IgM antibodies. Background Technology

[0002] Rubella virus IgM antibodies are specific antibodies produced early in the body after rubella virus infection. They typically appear 5-7 days after infection, peak at 2-3 weeks, and last for 6-8 weeks (sometimes extending to several months). Therefore, a positive IgM antibody test is a key indicator for determining recent or acute rubella virus infection. When pregnant women are infected with rubella virus, the virus can cross the placental barrier and infect the fetus, causing congenital rubella syndrome (CRS). This can lead to multiple systemic malformations in the fetus, including congenital heart disease, cataracts, hearing impairment, intellectual disability, and hepatosplenomegaly. The earlier the infection occurs, the higher the risk (>90% risk in early pregnancy, approximately 25% in mid-pregnancy), posing a significant threat to maternal and infant health. Furthermore, rubella virus IgM antibody testing provides important clinical evidence for the differentiation of rash-related diseases and the screening of susceptible populations.

[0003] Currently, chemiluminescence immunoassay (CLIA) technology is widely used for rubella virus IgM antibody detection due to its speed and convenience. However, existing detection technologies have two major drawbacks: (1) The problem of false positives is prominent: during the testing process, it is easily interfered with by impurities such as rheumatoid factor (RF), antinuclear antibody (ANA), and high concentration of albumin in the sample, which leads to non-specific binding and misdiagnosis. This not only increases the psychological burden on patients, but may also lead to unnecessary medical intervention. (2) Insufficient detection sensitivity: It is difficult to accurately capture low concentrations of rubella virus IgM antibodies, which can easily lead to missed diagnoses, delay the best treatment and intervention time for patients, and have an adverse impact on public health prevention and control.

[0004] Existing IgM detection kits mostly use carboxyl magnetic beads as the solid-phase carrier, which requires stepwise activation via EDC / NHS, a cumbersome process. Furthermore, the acidic conditions (pH=4.0~5.5) during activation can easily lead to denaturation of rubella virus antigens. Simultaneously, the negatively charged surface of carboxyl magnetic beads readily adsorbs interfering substances from the sample, further exacerbating false positives and insufficient sensitivity. In addition, traditional kits have complex reagent components, require refrigerated storage, and have a shortened shelf life after opening, increasing both operational difficulty and cost, and limiting their application in primary healthcare institutions and resource-scarce areas.

[0005] Therefore, developing a rapid rubella virus-IgM test kit that can solve the problems of false positives and low sensitivity, and that is convenient to store and use and cost-effective, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to address the problems of high false positive rate and low sensitivity in existing cost-controllable rubella virus-IgM detection kits, and to provide a stable and interference-resistant rapid detection kit for rubella virus-IgM antibodies.

[0007] Specifically, the present invention provides a rapid detection kit for rubella virus-IgM antibody, comprising independently stored first lyophilized microspheres, second lyophilized microspheres, and sample processing solution; the first lyophilized microspheres contain a complex I formed by coupling and coating rubella virus antigen with epoxy magnetic beads; the second lyophilized microspheres contain a complex II formed by acridine ester and anti-human IgM antibody through a light-protected reaction; the sample processing solution includes a buffer, sodium chloride, surfactant, and preservative.

[0008] In a preferred embodiment, the particle size of the first freeze-dried microspheres and the second freeze-dried microspheres are each independently 3.0~3.5 mm.

[0009] In a preferred embodiment, the water content of both the first and second freeze-dried microspheres is less than 3 wt%.

[0010] In a preferred embodiment, the mass ratio of rubella virus antigen to epoxy magnetic beads in the first freeze-dried microspheres is (0.1~0.3):10.

[0011] In a preferred embodiment, the ratio of acridine ester to anti-human IgM antibody in the second lyophilized microspheres is (1~3) μL: 0.1 mg.

[0012] In a preferred embodiment, the pH value of the sample processing solution is 6.0 to 7.0.

[0013] In a preferred embodiment, the buffer is selected from at least one of 2-(N-morpholino)ethanesulfonic acid, phosphate buffer, and tris(hydroxymethyl)aminomethane-hydrochloric acid buffer.

[0014] In a preferred embodiment, the surfactant is selected from at least one of Tween 80, Tween 60, Triton 405, and Triton 100.

[0015] In a preferred embodiment, with a sample processing solution volume of 1L, the content of the buffer is 8.0~15.0g, the content of the sodium chloride is 8.0~10.0g, the content of the surfactant is 0.5~3.0g, and the content of the preservative is 0.5~1.5g.

[0016] This invention also provides a method for preparing the above-mentioned rapid detection kit for rubella virus-IgM antibody. The preparation method includes: Preparation of the first lyophilized microspheres: S1. Rubella virus antigen, epoxy magnetic beads and first buffer solution are mixed and coupled and coated. The product of the coupling and coating reaction is magnetically separated and washed and then resuspended in reagent storage solution to obtain the first working solution; S2. The first working solution is frozen and spotted to form frozen microspheres, and then vacuum frozen to obtain the first lyophilized microspheres. Preparation of the second lyophilized microspheres: S3. Acridinium ester, anti-human IgM antibody, and second buffer solution are mixed and reacted in the dark. The resulting reaction product is filtered through a gel filter column and diluted with reagent storage solution to obtain the second working solution; S4. The second working solution is subjected to freeze spot treatment to form frozen microspheres, which are then subjected to vacuum freezing treatment to obtain the second lyophilized microspheres.

[0017] Preparation of sample processing solution: The sample processing solution is obtained by mixing and dissolving buffer, sodium chloride, surfactant, preservative and ultrapure water.

[0018] In a preferred embodiment, in step S1, the mass ratio of the rubella virus antigen to the epoxy magnetic beads is (0.1~0.3):10.

[0019] In a preferred embodiment, in step S1, the epoxy-based magnetic beads have a particle size of 2.4~3.0 μm, a surface epoxy density of 400~600 μmol / g, and a specific surface area of ​​15~25 m². 2 / g.

[0020] In a preferred embodiment, in step S1, the first buffer solution contains disodium hydrogen phosphate and sodium dihydrogen phosphate, and has a pH value of 7.5 to 8.5.

[0021] In a preferred embodiment, in step S1, the conditions for the coupling coating reaction include: a temperature of 20~30℃ and a time of 2~5h.

[0022] In a preferred embodiment, in step S1, the concentration of epoxy-based magnetic beads in the first working solution is 0.3~0.7 mg / mL.

[0023] In a preferred embodiment, in step S3, the ratio of acridine ester to anti-human IgM antibody is (1~3) μL: 0.1 mg.

[0024] In a preferred embodiment, in step S3, the acridine ester is selected from at least one of NSP-DMAE-NHS, DMAE-NHS, Me-DMAE-NHS, and NSP-SA-NHS.

[0025] In a preferred embodiment, in step S3, the second buffer solution contains sodium carbonate and sodium bicarbonate.

[0026] In a preferred embodiment, in step S3, the conditions for the light-protected reaction include: a temperature of 20~30℃ and a time of 1~3h.

[0027] In a preferred embodiment, in step S3, the concentration of anti-human IgM antibody in the second working solution is 0.001~0.003 mg / mL.

[0028] In a preferred embodiment, the reagent storage solution contains amino acids, mannitol, trehalose, bovine serum albumin, antioxidants, surfactants, gelatin, polyethylene glycol, sodium chloride, antibacterial agents, and buffers, with a pH value of 7.0 to 8.0.

[0029] In a preferred embodiment, the method of freezing and spotting includes: spotting the working solution at a rate of 15~25 μL / drop, with each drop forming an independent droplet, and then freezing it in liquid nitrogen until solidified, thereby forming frozen microspheres.

[0030] In a preferred embodiment, the first and second lyophilized microspheres are each individually packaged and sealed with nitrogen gas, and the sample processing solution is packaged in reaction flasks at 3.5 mL / parts and stored away from light.

[0031] Compared with existing kits, the rubella virus-IgM antibody rapid detection kit provided by this invention has the following advantages: (1) Strong anti-interference ability, high detection accuracy and sensitivity: The surface of epoxy magnetic beads is electrically neutral and has few hydrophobic regions, which can effectively reduce non-specific binding and reduce the probability of false positives; at the same time, epoxy magnetic beads have a high specific surface area and a large binding area, which can efficiently capture low concentrations of IgM antibodies. Combined with the characteristics of acridine ester high luminescence intensity and low background signal (≤500RLU), the detection sensitivity is significantly improved, and the minimum detection limit is no higher than 0.1AU / mL, avoiding missed diagnoses; (2) Simplified process and excellent stability: Epoxy magnetic beads do not require an activation step and can react directly with RV antigen in a near-neutral or weakly alkaline environment to complete the coating in one step, avoiding detection deviation caused by antigen denaturation; The lyophilized microspheres have low moisture content and can be stored at room temperature for 2 years when combined with nitrogen-sealed packaging, and the shelf life is not affected after opening, which is far superior to existing kits that require cold chain storage (2~8℃) and have a shelf life of only 1 year. (3) Easy to use and cost controllable: The kit contains only three components: the first lyophilized microspheres, the second lyophilized microspheres, and the sample processing solution, which are stored independently. The sample processing solution has both reconstitution and dilution functions, simplifying the operation steps and reducing human error. It also supports single-use packaging and use, avoiding contamination and waste caused by repeated use of reagents. Room temperature storage does not require cold chain equipment, reducing transportation and storage costs by more than 30%, making it suitable for primary medical institutions and resource-scarce areas. (4) Wide applicability and high clinical value: It can not only accurately detect rubella virus-IgM antibodies, but also be extended to TORCH multi-target detection (toxoplasmosis, cytomegalovirus, herpes simplex virus, etc.). That is, when coating epoxy magnetic beads, multi-target (such as TOX, CMV, etc. in TORCH multi-target detection) can be completed in one step, and the consistency and accuracy of multi-target detection can be guaranteed. It provides a reliable diagnostic basis for the prevention of congenital rubella syndrome in pregnant women, the identification of skin rash diseases, and the screening of susceptible populations, and has broad prospects for clinical promotion. Attached Figure Description

[0032] Figure 1 This is a physical packaging image of the lyophilized magnetic bead microspheres and acridine ester lyophilized microspheres prepared in Example 1 of this invention.

[0033] Figure 2 This is a graph showing the fitting results of the RV-IgM standard curve for the kit in Example 1.

[0034] Figure 3 This is a graph showing the fitting results of the RV-IgM standard curve for the kit in Example 2.

[0035] Figure 4 This is a graph showing the fitting results of the RV-IgM standard curve for the kit in Example 3.

[0036] Figure 5 This is a graph showing the fitting results of the RV-IgM standard curve for the kit in Example 4.

[0037] Figure 6 This is a graph showing the fitting results of the RV-IgM standard curve for the kit in Example 5.

[0038] Figure 7 This is a graph showing the fitting results of the RV-IgM standard curve for Comparative Example 1 kit.

[0039] Figure 8 This is a graph showing the fitting results of the RV-IgM standard curve for Comparative Example 2 kit. Detailed Implementation

[0040] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Furthermore, unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions, and such technical solutions should be considered to be included in the disclosure of the present invention.

[0041] The rubella virus-IgM antibody rapid detection kit provided by the present invention comprises a first lyophilized microsphere, a second lyophilized microsphere, and a sample processing solution, each stored independently. The first lyophilized microsphere contains a complex I formed by coupling and coating rubella virus antigen (RV antigen) with epoxy magnetic beads. The second lyophilized microsphere contains a complex II formed by acridinium ester and anti-human IgM antibody through a light-protected reaction. The sample processing solution includes a buffer, sodium chloride, a surfactant, and a preservative.

[0042] In this invention, the particle size of the first freeze-dried microsphere and the second freeze-dried microsphere is preferably 3.2~3.5 mm, such as 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm or any value between them.

[0043] In this invention, the water content of both the first and second freeze-dried microspheres is preferably below 3 wt%, which is beneficial for improving the room temperature storage stability of the freeze-dried microspheres and extending their shelf life.

[0044] In this invention, the preferred mass ratio of rubella virus antigen to epoxy magnetic beads in the first freeze-dried microspheres is (0.1~0.3):10, such as 0.1:10, 0.15:10, 0.2:10, 0.25:10, 0.3:10 or any ratio therebetween.

[0045] In this invention, the preferred ratio of acridine ester to anti-human IgM antibody in the second lyophilized microspheres is (1~3) μL:0.1 mg, such as 1 μL:0.1 mg, 1.5 μL:0.1 mg, 2 μL:0.1 mg, 2.5 μL:0.1 mg, 3 μL:0.1 mg or any ratio thereof.

[0046] In this invention, the pH value of the sample processing solution is preferably 6.0 to 7.0, such as 6.0, 6.2, 6.5, 6.8, 7.0, or any value between them. Controlling the pH of the sample processing solution within the above-mentioned preferred range is more conducive to improving the dilution stability of the sample and providing favorable environmental conditions for participating in the immune response.

[0047] In this invention, there are no particular limitations on the specific content and type of each component in the sample processing solution, as long as it enables the sample processing solution to have dilution and reconstitution capabilities. Specifically, the buffer can be a conventional choice in the art, and specific examples include, but are not limited to, at least one of: 2-(N-morpholino)ethanesulfonic acid, phosphate buffer (PBS), and tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl). The surfactant can be a conventional choice in the art, and specific examples include, but are not limited to, at least one of: Tween 80, Tween 60, Triton 405, and Triton 100.

[0048] In this invention, based on a sample processing solution volume of 1L, the content of the buffer is preferably 8.0~15.0g, such as 8.0g, 9.0g, 10.0g, 11.0g, 12.0g, 15.0g, or any value between them; the content of the sodium chloride is preferably 8.0~10.0g, such as 8.0g, 8.5g, 9.0g, 9.5g, 10.0g, or any value between them; the content of the surfactant is preferably 0.5~3.0g, such as 0.5g, 0.8g, 1.0g, 1.2g, 1.5g, 1.8g, 2.0g, 2.5g, 3.0g, or any value between them; the content of the preservative is preferably 0.5~1.5g, such as 0.5g, 0.8g, 1.0g, 1.2g, 1.5g, or any value between them. Within the above-mentioned preferred range, the resulting sample processing solution has a better ability to dilute samples and dissolve lyophilized microspheres during detection, which is beneficial for enabling the test kit to better achieve detection accuracy and sensitivity.

[0049] The preparation method of the rubella virus-IgM antibody rapid detection kit provided by the present invention includes: Preparation of the first lyophilized microspheres: S1. Rubella virus antigen, epoxy magnetic beads, and the first buffer solution are mixed and coupled for coating reaction. The resulting product of the coupling and coating reaction is magnetically separated and washed (complex I) and then resuspended in reagent storage solution to obtain the first working solution; S2. The first working solution is subjected to freeze spotting to form frozen microspheres, which are then subjected to vacuum freezing to obtain the first lyophilized microspheres; Preparation of the second lyophilized microspheres: S3. Acridinium ester, anti-human IgM antibody, and second buffer solution are mixed and subjected to a light-protected reaction. The resulting reaction product (complex II) is filtered through a gel filter column and diluted with reagent storage solution to obtain the second working solution; S4. The second working solution is subjected to freeze spot treatment to form frozen microspheres, which are then subjected to vacuum freezing treatment to obtain the second lyophilized microspheres.

[0050] Preparation of sample processing solution: The sample processing solution is obtained by mixing and dissolving buffer, sodium chloride, emulsifier, surfactant, preservative and ultrapure water.

[0051] In this invention, in step S1, the preferred mass ratio of the rubella virus antigen to the epoxy magnetic beads is (0.1~0.3):10, such as 0.1:10, 0.15:10, 0.2:10, 0.25:10, 0.3:10 or any ratio therebetween.

[0052] In this invention, in step S1, the particle size of the epoxy-based magnetic beads is preferably 2.4~3.0 μm, such as 2.5 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3.0 μm, or any value between them; the surface epoxy density is preferably 400~600 μmol / g, such as 400 μmol / g, 450 μmol / g, 500 μmol / g, 550 μmol / g, 600 μmol / g, or any value between them; the specific surface area is preferably 15~25 m². 2 / g, such as 15m 2 / g、18m 2 / g、20m 2 / g、22m 2 / g、25m 2 / g or any value between them, is more conducive to improving the ability to capture IgM and improve detection sensitivity.

[0053] In this invention, in step S1, the first buffer solution preferably contains disodium hydrogen phosphate and sodium dihydrogen phosphate. The pH value of the first buffer solution is preferably 7.5-8.5, such as 7.5, 7.8, 8.0, 8.2, 8.5, or any value between them. This not only facilitates the reaction between the epoxy magnetic beads and the amino and / or hydroxyl groups of the RV antigen to complete the coating, but also ensures the stability of the antigen properties, reduces false positives, and improves accuracy. This invention does not impose particular restrictions on the content of disodium hydrogen phosphate and sodium dihydrogen phosphate in the first buffer solution, as long as the pH value of the first buffer solution is 7.5-8.5. In one specific embodiment, with the volume of the first buffer solution being 1 L, the content of disodium hydrogen phosphate is preferably 0.03~0.05 mol, such as 0.03 mol, 0.04 mol, 0.05 mol or any value between them; the content of sodium dihydrogen phosphate is preferably 0.0015~0.025 mol, such as 0.0015 mol, 0.005 mol, 0.010 mol, 0.015 mol, 0.020 mol, 0.025 mol or any value between them.

[0054] In this invention, the conditions for the coupling coating reaction in step S1 preferably include: a temperature of 20~30℃, such as 20℃, 22℃, 25℃, 28℃, 30℃ or any value between them; and a time of 2~5h, such as 2h, 3h, 4h, 5h or any value between them.

[0055] In this invention, in step S1, the concentration of epoxy-based magnetic beads in the first working solution is preferably 0.3~0.7 mg / mL, such as 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL or any value between them.

[0056] In this invention, in step S3, the preferred ratio of acridine ester to anti-human IgM antibody is (1~3) μL:0.1 mg, such as 1 μL:0.1 mg, 1.5 μL:0.1 mg, 2 μL:0.1 mg, 2.5 μL:0.1 mg, 3 μL:0.1 mg or any ratio thereof.

[0057] In this invention, specific examples of the acridine ester in step S3 include, but are not limited to, at least one of NSP-DMAE-NHS, DMAE-NHS, Me-DMAE-NHS, and NSP-SA-NHS, with NSP-DMAE-NHS being particularly preferred.

[0058] In this invention, in step S3, the second buffer solution preferably contains sodium carbonate and sodium bicarbonate. This invention does not impose particular restrictions on the content of sodium carbonate and sodium bicarbonate in the second buffer solution, as long as it provides a suitable reaction environment for the reaction between acridine ester and anti-human IgM antibody. In a specific embodiment, with a volume of 1 L of the second buffer solution, the content of sodium carbonate is preferably 0.01~0.07 mol, such as 0.001 mol, 0.002 mol, 0.003 mol, 0.004 mol, 0.005 mol, 0.006 mol, 0.007 mol, or any value between them; the content of sodium bicarbonate is preferably 0.030~0.050 mol, such as 0.030 mol, 0.035 mol, 0.040 mol, 0.045 mol, 0.050 mol, or any value between them.

[0059] In this invention, the conditions for the light-avoidance reaction in step S3 preferably include: a temperature of 20~30℃, such as 20℃, 22℃, 25℃, 28℃, 30℃ or any value between them; and a time of 1~3h, such as 1h, 1.5h, 2h, 2.5h, 3h or any value between them.

[0060] In this invention, in step S3, the concentration of the anti-human IgM antibody in the second working solution is preferably 0.001~0.003 mg / mL, such as 0.001 mg / mL, 0.0015 mg / mL, 0.002 mg / mL, 0.0025 mg / mL, 0.003 mg / mL, or any value between them. This provides advantages such as high specificity, high detection sensitivity, and low background signal. This is because optimizing the concentration of the anti-human IgM antibody provides sufficient binding sites to capture low-abundance target IgM in the sample, ensuring the required detection sensitivity, while preventing unbound antibody residue due to excessively high antibody concentration, thereby reducing the experimental background signal and improving the detection signal-to-noise ratio.

[0061] In this invention, the reagent storage solution preferably contains amino acids, mannitol, trehalose, bovine serum albumin (BSA), antioxidants, surfactants, gelatin, polyethylene glycol, sodium chloride, antibacterial agents, and buffers. The pH value of the reagent storage solution is preferably 7.0 to 8.0, such as 7.0, 7.2, 7.5, 7.8, 8.0, or any value between them.

[0062] In this invention, there are no particular limitations on the specific content and type of each component in the reagent storage solution, as long as it is a solution that can be used as a diluent to prepare a first working solution or a second working solution and can ensure the stability of the reactants contained therein. The amino acids, antioxidants, surfactants, antibacterial agents, and buffers can be conventional choices in the art. In this invention, proline, glutathione-ergothioneine complex, Tween 20, antibacterial agent Proclin 300, and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) are particularly preferred.

[0063] In this invention, the preferred method of freezing and spotting the sample includes: spotting the working solution at a rate of 15-25 μL / drop (which can be 15 μL / drop, 18 μL / drop, 20 μL / drop, 22 μL / drop, 25 μL / drop or any value between them), and after each drop forms an independent droplet, freezing it in liquid nitrogen until solidified, thus forming frozen microspheres.

[0064] In this invention, the first and second lyophilized microspheres are each individually packaged and sealed with nitrogen gas. The sample processing solution is packaged in reaction flasks at 3.5 mL / part and stored away from light.

[0065] Furthermore, the terms "first," "second," "I," and "II" used in this invention are for ease of description only and should not be construed as specific limitations on the technical features.

[0066] The present invention will be described in detail below through specific embodiments. These embodiments are intended to explain the invention and should not be construed as limiting it. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0067] The preparation methods and raw material sources of the first buffer solution, second buffer solution, reagent storage solution, washing buffer, blocking buffer, coupling buffer, and activation buffer involved in the following examples and Comparative Example 1 are as follows: First buffer solution: Weigh out disodium hydrogen phosphate (Na₂HPO₄) 12H₂O (0.047 mol) 17.00 g, sodium dihydrogen phosphate (NaH₂PO₄) 0.380 g of 2H2O (0.0024 mol) was dissolved in ultrapure water and brought to a final volume of 1 L. The pH was adjusted to 8.0 ± 0.1 with 1 mol / L HCl or 1 mol / L NaOH. The solution was then filtered through a 0.22 μm filter membrane to remove impurities before use. Second buffer solution: Weigh 1.590g of sodium carbonate (anhydrous Na2CO3, 0.015mol) and 2.930g of sodium bicarbonate (NaHCO3, 0.035mol), dissolve them in ultrapure water and bring the volume to 1L. Filter the solution through a 0.22μm filter membrane to remove impurities and set aside for later use. Reagent storage solution: Weigh out 10.000g proline, 5.000g mannitol, 50.000g trehalose, 30.000g bovine serum albumin (BSA), 1.000g antioxidant glutathione-ergothioneine complex, 0.500g Tween 20, 1.000g gelatin, 3.000g PEG 8000, 9.000g sodium chloride, 1.000g antibacterial agent proclin 300, and 4.986g 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES). Dissolve HEPES in 800mL of ultrapure water (resistivity ≥18.2MΩ) before preparation. (cm), adjust the pH to 7.5 with 1mol / L HCl or 1mol / L NaOH, add other raw materials in sequence and ensure complete dissolution, finally add ultrapure water to make up to 1L, adjust the pH to 7.5±0.1 with 1mol / L HCl or 1mol / L NaOH, filter through a 0.22μm filter membrane to remove impurities and set aside for use; Washing buffer: Weigh out disodium hydrogen phosphate (Na2HPO4) 12.250g of sodium dihydrogen phosphate (NaH2PO4) 2.250g of 2H2O, 0.500g of Tween 20, 9.00g of sodium chloride, and 0.5mL of preservative Proclin 300 were dissolved in ultrapure water and brought to a final volume of 1L. The mixture was then filtered through a 0.22μm filter membrane to remove impurities before use. Blocking buffer: Weigh 100,000 g of bovine serum albumin, dissolve it in washing buffer and bring the volume to 1 L. Filter the solution through a 0.22 μm filter membrane to remove impurities and set aside. Coupling buffer: Weigh 10.600g of 2-(N-morpholino)ethanesulfonic acid (MES), add about 800mL of ultrapure water, adjust the pH to 6.0±0.1, add ultrapure water to make up to 1L, filter through a 0.22μm filter membrane to remove impurities and set aside. Activation buffer (EDC / NHS activation buffer): Weigh 10 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 20 mg of NHS (N-hydroxysuccinimide), add 1 mL of coupling buffer and mix well. Filter through a 0.22 μm filter membrane to remove impurities and set aside for later use.

[0068] Epoxy-based magnetic beads, purchased from Changsha Meiniu Co., Ltd., model MGE03, with a particle size of 2.8 μm, a surface epoxy density >200 μmol / g, and a specific surface area of ​​20 m². 2 / g; Rubella virus antigen (RV antigen) is rubella virus spike protein (E1-E2), purchased from Verun Cellun, catalog number BA129R01; Acridinium ester (NSP-DMAE-NHS), purchased from Meikai Biotechnology Co., Ltd., model number Intlus-B01, catalog number 201137; Anti-human IgM antibody is anti-human IgM (μ-chain specific) monoclonal antibody produced by Sigma-Aldrich, clone number MB-11, catalog number SAB4200780; Carboxyl magnetic beads, purchased from JSR Life Sciences, catalog number J-MS-S300C, particle size 3.0μm, surface carboxyl density 10μmol / g; Tosyl magnetic beads, purchased from JSR Life Sciences, catalog number J-MS-S300T, particle size 3.0μm, surface carboxyl density 80μmol / g.

[0069] Example 1 This embodiment illustrates the preparation and assembly of a rapid test kit for rubella virus-IgM antibodies, as detailed below: (1) Preparation of lyophilized magnetic beads: Take 10 mg of epoxy-based magnetic beads, add 1 mL of pH 8.0 buffer solution, sonicate for 5 min, and then add 0.1 mg of... RV antigen was mixed by rotation at room temperature (25℃) for 3 hours, then 100 μL of blocking buffer was added, and the mixture was mixed by rotation at room temperature (25℃) for 1 hour. The mixture was then washed three times with washing buffer using magnetic separation. After each wash, the supernatant was discarded, and the solid phase was collected and resuspended in 20 mL of reagent stock to obtain a working solution containing 0.5 mg / mL epoxy magnetic beads coated with RV antigen. Using an automated liquid nitrogen spotter, the working solution was spotted at a rate of 20 μL / drop. Each drop formed an independent droplet and was then quickly placed in liquid nitrogen to freeze for 1 minute until completely solidified, forming frozen microspheres. The plate was pre-cooled to -45℃, and the frozen microspheres were transferred to a lyophilizer. The pre-freezing temperature was set to -50℃ and maintained for 2 hours. Then, the vacuum pump was turned on, the vacuum was reduced to 10 Pa, and the temperature was raised to -20℃ for sublimation drying for 16 hours. Subsequently, the vacuum was reduced to 1 Pa, the temperature was raised to 25℃, and desorption drying was carried out for 6 hours. After lyophilization, the tray was removed, and the lyophilized magnetic bead microspheres with a particle size of 3.2~3.5 mm were obtained. like Figure 1 As shown, the microspheres are round and spherical, without cracks, and the moisture content calculated by weighing is ≤3wt%.

[0070] (2) Preparation of acridine ester lyophilized microspheres: Weigh 0.5 mg of anti-human IgM antibody and 0.5 mL of labeling buffer, and 10 μL of acridine ester (NSP-DMAE-) The acridine ester was mixed by rotating at 25°C in the dark for 2 hours; free acridine ester was removed by passing it through a gel filtration column, and the solution was diluted with reagent stock to a concentration of 0.001 mg / mL to obtain the acridine ester-labeled anti-human IgM working solution; using an automated liquid nitrogen spotter, the working solution was spotted at a rate of 20 μL / drop, and each drop was quickly placed in liquid nitrogen for 1 minute to completely solidify, forming frozen microspheres; the plate was pre-cooled to -45°C, and the frozen microspheres were transferred to a lyophilizer. The pre-freezing temperature was set to -50°C and maintained for 2 hours. Then, the vacuum pump was turned on, the vacuum was reduced to 10 Pa, the temperature was raised to -20°C, and the microspheres were sublimated and dried for 16 hours. Subsequently, the vacuum was reduced to 1 Pa, the temperature was raised to 25°C, and the microspheres were desorbed and dried for 6 hours. After lyophilization, the tray was removed, and the acridine ester lyophilized microspheres with a particle size of 3.2~3.5 mm were obtained. Figure 1 As shown, the microspheres are round and spherical, without cracks, and the moisture content calculated by weighing is ≤3wt%.

[0071] (3) Preparation of sample processing solution: Weigh 10.6g of 2-(N-morpholino)ethanesulfonic acid (MES), 9.0g of sodium chloride, 0.5g of Triton 100, 1.0g of Tween 80, 0.5g of preservative PC950, and 0.5g of preservative BND-10. When preparing the solution, first dissolve MES in 800mL of ultrapure water, adjust the pH to 6.0, add other raw materials to dissolve, add ultrapure water to make up the volume to 1L, adjust the pH to 6.0±0.1, filter through a 0.22μm filter membrane to remove impurities, and store in the dark.

[0072] (4) Reagent kit assembly: Packaging of reaction cups: Take one lyophilized magnetic bead microsphere prepared in step (1) and one lyophilized acridinium ester microsphere prepared in step (2) with tweezers and place them into the corresponding reaction cup 1 and reaction cup 2 respectively, ensuring that the microsphere is located in the center of the bottom of the cup. Fill the reaction cup with nitrogen gas and immediately heat seal the cup mouth with aluminum-plastic film. Reagent assembly: Dispense the sample processing solution into reagent tubes at 3.5 mL / part using a dispenser, heat-seal the cup mouth with aluminum-plastic film, each bottle corresponds to 20 sets of reagents (one set of reagents: 1 reaction cup 1 + 1 reaction cup 2), pack into aluminum-plastic packaging, label and complete the reagent kit assembly.

[0073] Example 2 This embodiment illustrates the preparation and assembly of a rapid test kit for rubella virus-IgM antibodies, as detailed below: The kit was prepared according to the method of Example 1, except that in step (1): the solid phase was collected and resuspended in 33.3 mL of reagent storage medium to obtain a working solution containing 0.3 mg / mL epoxy magnetic beads coated with RV antigen. All other conditions were the same as in Example 1. The rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0074] Example 3 This embodiment illustrates the preparation and assembly of a rapid test kit for rubella virus-IgM antibodies, as detailed below: The kit was prepared according to the method of Example 1, except that in step (2): reagent stock solution was added to dilute to a concentration of 0.0015 mg / mL for anti-human IgM antibody. All other conditions were the same as in Example 1. The rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0075] Example 4 This embodiment illustrates the preparation and assembly of a rapid test kit for rubella virus-IgM antibodies, as detailed below: The kit was prepared according to the method of Example 1, except that the amount of RV antigen added in step (1) was 0.3 mg and the amount of acridine ester (NSP-DMAE-NHS) used in step (2) was 5 μL. All other conditions were the same as in Example 1. The rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0076] Example 5 This embodiment illustrates the preparation and assembly of a rapid test kit for rubella virus-IgM antibodies, as detailed below: The kit was prepared according to the method of Example 1, except that the amount of RV antigen added in step (1) was 0.2 mg and the amount of acridine ester (NSP-DMAE-NHS) used in step (2) was 15 μL. All other conditions were the same as in Example 1. The rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0077] Comparative Example 1 This comparative example illustrates the preparation and assembly of a reference rubella virus-IgM antibody rapid detection kit, as detailed below: The kit was prepared according to the method in Example 1, except that step (1) was as follows: 10 mg of carboxyl magnetic beads were weighed, 1 mL of coupling buffer at pH 6.0 was added, and the mixture was sonicated for 5 min. Then, EDC / NHS activation buffer was added for activation reaction for 30 min. The mixture was then washed twice with coupling buffer by magnetic separation. 1 mL of coupling buffer and 0.1 mg of RV antigen were added, and the mixture was rotated and mixed at room temperature (25°C) for 3 h. 100 μL of blocking buffer was added, and the mixture was rotated and mixed at room temperature (25°C) for 1 h. The mixture was washed three times with washing buffer by magnetic separation. After each wash, the supernatant was discarded, and the solid phase was collected and resuspended in 20 mL of reagent stock to obtain a working solution containing 0.5 mg / mL carboxyl magnetic beads coated with RV antigen. All other conditions were the same as in Example 1. The reference rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0078] Comparative Example 2 This comparative example illustrates the preparation and assembly of a reference rubella virus-IgM antibody rapid detection kit, as detailed below: The kit was prepared according to the method in Example 1, except that step (1) was as follows: 10 mg of Tosyl-based magnetic beads were added to 1 mL of pH 8.0 first buffer solution, and the mixture was ultrasonically dispersed for 5 min. Then, the mixture was magnetically washed twice with the first buffer solution. Next, 1 mL of the first buffer solution and 0.1 mg of RV antigen were added, and the mixture was rotated and mixed at 37°C for 24 h. Then, 100 μL of blocking buffer was added, and the mixture was rotated and mixed at 37°C for 12 h. The mixture was magnetically washed three times with washing buffer. After each washing, the supernatant was discarded, and the solid phase was collected and resuspended in 20 mL of reagent stock solution to obtain a working solution containing 0.5 mg / mL Tosyl-based magnetic beads coated with RV antigen. All other conditions were the same as in Example 1. The reference rubella virus-IgM antibody rapid detection kit was thus prepared and assembled.

[0079] Test case 1. Establishment of RV-IgM standard curve (1-1) A series of calibrator solutions were prepared using RV-IgM antibody (purchased from Phytobio Biotechnology Co., Ltd., catalog number RV-REAB-E1-002) with calibrator diluent, at concentrations of 0.000 AU / mL, 1.021 AU / mL, 4.882 AU / mL, 22.483 AU / mL, 102.579 AU / mL, 253.367 AU / mL, and 503.643 AU / mL. The calibrator diluent consisted of 0.05 M PBS buffer (pH=7.4) + 200 mL / L newborn calf serum + 0.1 wt% Proclin-300.

[0080] The RV-IgM antibody at a concentration of 153.781 AU / mL (purchased from Phytobio Biotechnology Co., Ltd., catalog number RV-REAB-E1-002) was diluted with calibrator diluent to 77.026 AU / mL, 53.173 AU / mL and 29.391 AU / mL, and used as high-value quality control (C1), medium-value quality control (C2) and low-value quality control (C3), respectively.

[0081] (1-2) Testing was performed using a self-produced fully automated chemiluminescence immunoassay analyzer: One sample of rubella virus-IgM single-person test reagent obtained from the examples or comparative examples was taken, and 10 μL of calibrator solution was reacted with 90 μL of sample processing solution for 2 min to obtain a sample mixture. After puncturing the aluminum foil sealing the reaction cups 1 and 2 using the puncture technique, 125 μL of sample processing solution and 10 μL of the processed sample mixture were added to reaction cup 1, and 125 μL of sample processing solution was added to reaction cup 2. After incubation at 37°C for 10 min, 75 μL of the solution from reaction cup 2 was added to reaction cup 1. After incubation at 37°C for 10 min, magnetic separation and washing were performed to remove unbound substances. Reaction cup 1 was transferred to a dark room and excitation solution (0.1 mL of pre-excitation solution A + 0.1 mL of excitation solution B) was added for reaction. The luminescence value (RLU) was read and recorded. The detection results of the examples and comparative examples are shown in Tables 1-1, 1-2, 1-3, 1-4, 1-5 and 2-1 and 2-2, respectively. The fitting type is four-parameter Logistic fitting, and the standard curve results are shown in... Figures 2-8 The results showed that the detection gradients of Examples 1-5 were superior, with lower background signal values.

[0082] The pre-activation solution A is: 976.4 mL of ultrapure water + 5 mL of 65% nitric acid + 18.6 mL of 30% hydrogen peroxide, stored in a brown bottle protected from light; the activation solution B is: 989 mL of ultrapure water + 10.000 g of sodium hydroxide + 1 mL of Triton 100, stored at room temperature.

[0083] Table 1-1 Luminescence values ​​from the RV-IgM standard curve test in Example 1

[0084] Table 1-2 Luminescence values ​​from the RV-IgM standard curve test in Example 2

[0085] Table 1-3 Luminescence values ​​from the RV-IgM standard curve test in Example 3

[0086] Table 1-4 Luminescence values ​​from the RV-IgM standard curve test in Example 4

[0087] Table 1-5 Luminescence values ​​from the RV-IgM standard curve test in Example 5

[0088] Table 2-1 Luminescence values ​​from the RV-IgM standard curve test in Comparative Example 1

[0089] Table 2-2 Luminescence values ​​from the RV-IgM standard curve test in Comparative Example 2

[0090] 2. Anti-interference test: The following samples containing endogenous interfering substances were selected: hemolyzed sample (hemoglobin 20g / L), lipemia sample (triglyceride concentration 20mmol / L), jaundice sample (513μmol / L), rheumatoid factor (RF) concentration 500IU / mL, antinuclear antibody (ANA) positive sample (titer 1:1000), and albumin (50g / L). The rubella virus IgM negative serum containing the above interfering samples was tested using the rubella virus-IgM single-person test reagent obtained in the examples or comparative examples according to the method described in (1-2) above (three parallel samples). The luminescence value (RLU) results read and recorded are shown in Tables 3 and 4.

[0091] The test results in Tables 3 and 4 show that the kit in Example 1 has strong anti-interference ability, and the influence of interfering substances on the samples is less than 2%.

[0092] Table 3. Anti-interference test results of Example 1

[0093] Table 4. Results of anti-interference test for Comparative Example 1

[0094] 3. Clinical testing Clinical testing of clinical serum samples: At least 30 serum samples (IgM positive) from hospital-diagnosed rubella virus infection patients and at least 200 serum samples (IgM negative) from healthy individuals were collected. These serum samples were tested using the rubella virus-IgM single-sample test reagents obtained in the examples and comparative examples, following the methods described in (1-2). The luminescence value of the sample was read and input into the corresponding standard curve formula to calculate the concentration value. The positive or negative result of the serum sample was then determined based on the concentration value, and the positive / negative concordance rate was calculated. Specific results are shown in Tables 5-1, 5-2, 5-3, 5-4, 5-5, 6-1, and 6-2. The results indicate that the kits in Examples 1-5 have a low false positive probability, a higher positive / negative concordance rate, and significantly higher detection accuracy than the comparative examples.

[0095] The criteria for judging positive and negative results for samples are as follows: a sample concentration value < 5 AU / mL is considered negative; a concentration value ≤ 5 AU / mL and < 8 AU / mL is considered suspicious; and a concentration value > 8 AU / mL is considered positive. The symbols TP (true positive) and TN (false negative) represent the correct positive result, FN (false negative) and FP (false positive) respectively, respectively.

[0096] Table 5-1 Clinical test results of Example 1

[0097] Table 5-2 Clinical test results of Example 2

[0098] Table 5-3 Clinical test results of Example 3

[0099] Table 5-4 Clinical test results of Example 4

[0100] Table 5-5 Clinical test results of Example 5

[0101] Table 6-1 Clinical test results of Comparative Example 1

[0102] Table 6-2 Clinical test results of Comparative Example 2

[0103] 4. Sensitivity Testing: Using the same calibrator diluent as used in (1-1) as the blank sample, the test was performed according to the method described in (1-2). Each example and comparative example was tested 20 times, and the luminescence value (RLU) was recorded. The specific results of the examples and comparative examples are shown in Tables 7-1, 7-2, 7-3, 7-4, 7-5 and 8-1, 8-2, respectively. The mean (X) and standard deviation (SD) of the 20 test results were calculated. The calculated value of X+2SD was substituted into the standard curve established for each example and comparative example to calculate the concentration value. The obtained concentration value was used as the analytical sensitivity (blank limit) of the reagent. The analytical sensitivities (blank limits) of Examples 1-5 were 0.055 AU / mL, 0.061 AU / mL, 0.060 AU / mL, 0.056 AU / mL, and 0.055 AU / mL, respectively, which were much lower than 0.285 AU / mL of Comparative Example 1 and 0.134 AU / mL of Comparative Example 2. That is, the analytical sensitivities of Examples 1-5 all met the requirement that the minimum detection limit should not be higher than 0.1 AU / mL, and the sensitivity was much higher than that of the comparative examples.

[0104] Table 7-1 Sensitivity Results of Example 1

[0105] Table 7-2 Sensitivity results of Example 2

[0106] Table 7-3 Sensitivity results of Example 3

[0107] Table 7-4 Sensitivity results for Example 4

[0108] Table 7-5 Sensitivity results of Example 5

[0109] Table 8-1 Sensitivity results of Comparative Example 1

[0110] Table 8-2 Sensitivity results of Comparative Example 2

[0111] 5. Precision testing Intra-batch precision: The kit was prepared according to the method in Example 1. The same batch of kits was used to test quality control samples at low, medium, and high levels of rubella virus-IgM antibody. Each quality control level was tested in parallel 10 times in the same test. The tests were performed according to the kit's operating procedures, and the test results for each concentration sample were recorded. The coefficient of variation (CV) of each quality control level's 10 test results was calculated using the formula: CV = (standard deviation / mean) × 100%. The test results are shown in Table 9.

[0112] Inter-batch precision: The kit was prepared according to the method in Example 1. Three batches of the kit were used to test the three levels of quality control samples. Each quality control level was tested in parallel 10 times within the same batch. The tests were performed according to the kit operation steps, and the test results for each concentration sample were recorded. The coefficient of variation (CV) of the 30 test results for each quality control level was calculated. The formula for calculating the coefficient of variation is: CV = (standard deviation / mean) × 100%. The test results are shown in Table 9.

[0113] The high concentration of RV-IgM antibody (153.781 AU / mL) was diluted with calibrator diluent to 77.026 AU / mL, 53.173 AU / mL and 29.391 AU / mL, respectively, and used as high-value quality control (C1), medium-value quality control (C2) and low-value quality control (C3).

[0114] As shown in Table 9, the coefficients of variation (CV) within and between batches are both within 5%, indicating that the precision of the kit in Example 1 meets the detection requirements (intra-batch coefficient of variation CV ≤ 10%; inter-batch coefficient of variation CV ≤ 15%).

[0115] Table 9

[0116] 6. Stability Testing Accelerated stability test: The kit was prepared according to the method in Example 1. Three batches of kits were placed in a constant temperature and humidity incubator at 45±2℃. The kits were removed on days 0, 7, 14, 28, 60, and 90, respectively. Quality control samples at low, medium, and high levels of rubella virus-IgM antibody were tested. Each quality control level was tested in parallel 10 times within the same batch. The tests were performed according to the kit operation procedure, and the test results for each concentration sample were recorded. The degradation rate was calculated using the formula: [(mean of test results - mean of test results on day 0) / mean of test results on day 0] × 100%. The results are shown in Table 10.

[0117] Long-term stability test: The kit was prepared according to the method in Example 1. Three batches of kits were stored at 2-8℃, 25℃, and 30℃. The kits were retrieved at 0 months, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months, respectively. Quality control samples at low, medium, and high levels of rubella virus-IgM antibody were tested. Each quality control level was tested in parallel 10 times within the same batch. The tests were performed according to the kit's operating procedures, and the test results for each concentration sample were recorded. The degradation rate was calculated using the formula: [(mean of test results - mean of test results in month 0) / mean of test results in month 0] × 100%. The results are shown in Table 11.

[0118] As can be seen from the results in Tables 10 and 11, the shelf life of the kit in Example 1 is much longer than that of commonly available luminescent reagents (1 year under cold chain conditions).

[0119] Table 10 Accelerated stability test results

[0120] Table 11 Results of Long-Term Stability Tests

[0121] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A rapid detection kit for rubella virus-IgM antibody, characterized in that, The sample comprises a first lyophilized microsphere, a second lyophilized microsphere, and a sample processing solution, each stored independently. The first lyophilized microsphere contains a complex I formed by coupling and coating rubella virus antigen with epoxy magnetic beads. The second lyophilized microsphere contains a complex II formed by reacting acridinium ester with anti-human IgM antibody in the dark. The sample processing solution includes a buffer, sodium chloride, a surfactant, and a preservative.

2. The rapid detection kit for rubella virus-IgM antibody according to claim 1, characterized in that, The particle size of the first and second freeze-dried microspheres is independently 3.0~3.5 mm; Preferably, the water content of both the first and second freeze-dried microspheres is less than 3 wt%.

3. The rapid detection kit for rubella virus-IgM antibody according to claim 1, characterized in that, The mass ratio of rubella virus antigen to epoxy magnetic beads in the first freeze-dried microspheres is (0.1~0.3):10; Preferably, the ratio of acridine ester to anti-human IgM antibody in the second lyophilized microspheres is (1~3) μL:0.1 mg.

4. The rapid detection kit for rubella virus-IgM antibody according to claim 1, characterized in that, The pH value of the sample processing solution is 6.0~7.0; Preferably, the buffer is selected from at least one of 2-(N-morpholino)ethanesulfonic acid, phosphate buffer, and tris(hydroxymethyl)aminomethane-hydrochloric acid buffer. Preferably, the surfactant is selected from at least one of Tween 80, Tween 60, Triton 405, and Triton 100.

5. The rapid detection kit for rubella virus-IgM antibody according to claim 1 or 4, characterized in that, Based on a sample processing solution volume of 1L, the content of the buffer is 8.0~15.0g, the content of the sodium chloride is 8.0~10.0g, the content of the surfactant is 0.5~3.0g, and the content of the preservative is 0.5~1.5g.

6. The method for preparing the rubella virus-IgM antibody rapid detection kit according to any one of claims 1 to 5, characterized in that, The preparation method includes: Preparation of the first lyophilized microspheres: S1. Rubella virus antigen, epoxy magnetic beads and first buffer solution are mixed and coupled and coated. The product of the coupling and coating reaction is magnetically separated and washed and then resuspended in reagent storage solution to obtain the first working solution; S2. The first working solution is frozen and spotted to form frozen microspheres, and then vacuum frozen to obtain the first lyophilized microspheres. Preparation of the second lyophilized microspheres: S3. Acridinium ester, anti-human IgM antibody, and second buffer solution are mixed and reacted in the dark. The resulting reaction product is filtered through a gel filter column and diluted with reagent storage solution to obtain the second working solution; S4. The second working solution is subjected to freeze spot treatment to form frozen microspheres, which are then subjected to vacuum freezing treatment to obtain the second lyophilized microspheres. Preparation of sample processing solution: The sample processing solution is obtained by mixing and dissolving buffer, sodium chloride, surfactant, preservative and ultrapure water.

7. The rapid detection kit for rubella virus-IgM antibody according to claim 6, characterized in that, In step S1, the mass ratio of the rubella virus antigen to the epoxy magnetic beads is (0.1~0.3):10; Preferably, the particle size of the epoxy-based magnetic beads is 2.4-3.0 μm, the surface epoxy group density is 400-600 μmol / g, and the specific surface area is 15-25 m 2 / g. Preferably, the first buffer solution contains disodium hydrogen phosphate and sodium dihydrogen phosphate, and has a pH value of 7.5 to 8.5; Preferably, the conditions for the coupling coating reaction include: a temperature of 20~30℃ and a time of 2~5h; Preferably, the concentration of epoxy-based magnetic beads in the first working solution is 0.3~0.7 mg / mL.

8. The rapid detection kit for rubella virus-IgM antibody according to claim 6, characterized in that, In step S3, the ratio of acridine ester to anti-human IgM antibody is (1~3) μL: 0.1 mg; Preferably, the acridine ester is selected from at least one of NSP-DMAE-NHS, DMAE-NHS, Me-DMAE-NHS, and NSP-SA-NHS; Preferably, the second buffer solution contains sodium carbonate and sodium bicarbonate; Preferably, the conditions for the light-protected reaction include: a temperature of 20~30℃ and a time of 1~3h; Preferably, the concentration of anti-human IgM antibody in the second working solution is 0.001~0.003 mg / mL.

9. The rapid detection kit for rubella virus-IgM antibody according to claim 6, characterized in that, The reagent storage solution contains amino acids, mannitol, trehalose, bovine serum albumin, antioxidants, surfactants, gelatin, polyethylene glycol, sodium chloride, antibacterial agents, and buffers, with a pH value of 7.0~8.

0. Preferably, the method of freezing and spotting includes: spotting the working solution at a rate of 15~25 μL / drop, with each drop forming an independent droplet, and then freezing it in liquid nitrogen until solidified, thus forming frozen microspheres.

10. The rapid detection kit for rubella virus-IgM antibody according to claim 6, characterized in that, The first and second lyophilized microspheres were each individually packaged and sealed with nitrogen gas. The sample processing solution was packaged in reaction flasks at 3.5 mL / part and stored away from light.

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