Preparation method of HCG Ab detection reagent magnetic bead working solution
The HCGβCF antigen was treated with buffer dilution and thiol modification, and then combined with magnetic bead coupling technology to prepare the HCGAb detection reagent magnetic bead working solution. This solved the problems of biosafety risks and low sensitivity, and achieved efficient and safe detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- URIT MEDICAL ELECTRONICS CO LTD
- Filing Date
- 2024-01-02
- Publication Date
- 2026-07-03
AI Technical Summary
Existing HCG antigen-coated carboxyl magnetic beads pose biosafety risks during production and have poor low-sensitivity in clinical settings, affecting detection results.
The antigen HCGβCF was diluted to 2 mg/mL with buffer solution, reacted with the working solution using a thiol-modified Traut's reagent, and then desalted and purified before being diluted to 1 mg/mL with dimethyl sulfoxide aqueous solution. Subsequently, the solution was coated with SA magnetic beads, carboxyl magnetic beads, or Tosyl magnetic beads to prepare the HCGβ detection reagent working solution.
It reduces biosafety risks during the production process, improves the sensitivity and clinical concordance rate of detection, reduces enterprise production costs, and prolongs the stability of magnetic beads.
Smart Images
Figure CN117969864B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemiluminescence technology for in vitro diagnostic reagents, and particularly to a method for preparing a working solution of magnetic beads for HCGAb detection. Background Technology
[0002] Human chorionic gonadotropin (HCG) is a glycoprotein composed of α and β subunits, secreted by the trophoblast cells of the placenta after conception. Its main function is to stimulate hormone secretion, playing a crucial role in maintaining pregnancy and the maternal-fetal barrier. In individuals with a history of miscarriage or ovulation induction, HCG may act as an antigen to stimulate the body to produce anti-human chorionic gonadotropin antibodies (HCGAb). HCG b blocks HCG, and the resulting decrease in HCG concentration cannot maintain gamete implantation and pregnancy, leading to miscarriage. Studies have shown a significant correlation between anti-human chorionic gonadotropin antibodies and the pathogenesis of immunological infertility; therefore, detecting anti-HCG antibodies is an important auxiliary diagnostic tool for immunological infertility.
[0003] In the diagnosis of infertility, enzyme-linked immunosorbent assay (ELISA) is the mainstream clinical method for detecting anti-human chorionic gonadotropin (hCG) antibodies. However, its cumbersome operation and poor sensitivity cannot be ignored. Currently, chemiluminescence immunoassay (chemiluminescence) uses an indirect method to detect hCG antibodies by capturing them with hCG antigen, which then forms an immune complex with a secondary antibody conjugated to alkaline phosphatase (AMPPD). Although this method can solve the aforementioned problems of ELISA, its low sensitivity in clinical applications remains a challenge. However, there is very little research on anti-hCG antibody detection kits. Chinese patent CN107561293A provides a method for preparing an anti-hCG antibody detection kit, which simply uses hCG antigen to coat carboxyl magnetic beads, providing an option for bead coating. However, the biosafety risks that inevitably arise during the production of bioactive raw materials deserve attention. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing the working solution of HCGb detection reagent magnetic beads, which aims to solve the biosafety risks that may arise during the production of existing raw materials for HCG antigen-coated carboxyl magnetic beads.
[0005] To achieve the above objectives, the present invention provides a method for preparing the working solution of HCGAb detection reagent magnetic beads, comprising the following steps:
[0006] The antigen HCGβCF was diluted to 2 mg / mL with buffer solution to obtain the working solution;
[0007] The thiol-modified Traut's reagent was reacted with the working solution at 37°C under closed conditions for 30 min, and the reaction solution was then desalted, purified and concentrated to obtain the reaction solution.
[0008] The reaction solution was diluted to 1 mg / mL with dimethyl sulfoxide aqueous solution, reacted at room temperature for 4 h, and then concentrated and purified by changing the solution to obtain HCGβCF conjugate.
[0009] The HCGβCF conjugate is coated using any one of SA magnetic bead coupling technology, carboxyl magnetic bead coupling technology, and Tosyl magnetic bead coupling technology to obtain the HCGβ detection reagent magnetic bead working solution.
[0010] The step of coating the HCGβCF conjugate using carboxylated magnetic bead coupling technology includes:
[0011] Wash the magnetic beads three times with coupling buffer in the pH range of 5-7, and dilute to 10 mg / mL;
[0012] The HCGβCF conjugate was added at a coating ratio of 5–40 μg / mg and incubated for 15 min, with 0.05% surfactant added for catalysis.
[0013] Add EDC to catalyze the reaction, and rotate the reaction at room temperature for 4 hours. After the reaction is complete, remove the supernatant.
[0014] Add 1% blocking agent for blocking, rotate the reaction at room temperature for 2 hours, remove the supernatant after the reaction, add an equal volume of buffer containing primary amino acids to quench the reaction, and repeat the washing 3 times.
[0015] Remove the supernatant, wash three times with magnetic bead working solution, and dilute to a magnetic bead concentration of 0.2 mg / mL to obtain the HCGAb detection reagent magnetic bead working solution.
[0016] The buffer solution contains 0.9% sodium chloride in PB buffer, MES buffer or CB buffer, with a pH range of 7-8;
[0017] The reaction mass ratio of the thiol-modified Traut's reagent to the working solution is 1:0.5 to 1:8;
[0018] The concentration of the dimethyl sulfoxide aqueous solution is 5% to 30%.
[0019] The coupling buffer is a 100mM MES buffer with pH=6.0;
[0020] The coating ratio is 10 μg / mg, and the surfactant is S21;
[0021] EDC reaction mass ratio 1:15 to 1:3;
[0022] The blocking agent is Blockmaster DB1130 or casein;
[0023] The primary amino-containing buffer comprises 25 mM Tris buffer, pH 7, containing 0.2% S14, 0.9% sodium chloride and 1% glycerol;
[0024] The working solution of the magnetic beads is a 10mM MES buffer solution with a pH of 6.5, containing 0.9% sodium chloride, 0.5%–1% trehalose, 0.5%–1% bovine serum albumin, 0.2% surfactant S14, 0.05%–0.2% preservative and 0.05% L-cysteine.
[0025] The preservatives include any two or more of PC300, PC950, BND-10, and Krovin 300.
[0026] This invention discloses a method for preparing a magnetic bead working solution for HCGb detection reagent. The method involves diluting the antigen HCGβCF to 2 mg / mL using a buffer solution to obtain a working solution; reacting the working solution with a thiol-modified Traut's reagent at 37°C under sealed conditions for 30 min; desalting, purifying, and concentrating the reaction solution to obtain a reaction solution; diluting the reaction solution to 1 mg / mL using a dimethyl sulfoxide aqueous solution; reacting at room temperature for 4 h; and then purifying by changing the solution to obtain an HCGβCF conjugate; and coating the HCGβCF conjugate using any one of SA magnetic bead coupling technology, carboxyl magnetic bead coupling technology, and Tosyl magnetic bead coupling technology to obtain the HCGb detection reagent magnetic bead working solution. This invention provides a raw material solution for preparing a low-cost and safe capture antigen, reducing enterprise production costs, reducing biosafety risks during production, improving clinical sensitivity, and improving clinical concordance rates. It also solves the biosafety risks associated with the production of existing HCG antigen-coated carboxyl magnetic beads. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 This is a flowchart of a method for preparing the working solution of HCGAb detection reagent magnetic beads provided by the present invention. Detailed Implementation
[0029] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.
[0030] Please see Figure 1 This invention provides a method for preparing the working solution of magnetic beads for HCGAb detection, comprising the following steps:
[0031] S1 uses buffer to dilute antigen HCGβCF to 2 mg / mL to obtain working solution;
[0032] Specifically, the buffer solution contains 0.9% sodium chloride in PB buffer, MES buffer, or CB buffer, with a pH range of 7-8. Preferably, it is a 100mM MES buffer with a pH of 7.4.
[0033] S2 was reacted with the thiol-modified Traut's reagent and the working solution at 37°C under closed conditions for 30 min, and the reaction solution was then desalted, purified and concentrated to obtain the reaction solution.
[0034] Specifically, the reaction mass ratio of the thiol-modified Traut's reagent to the working solution is 1:0.5 to 1:8, preferably 1:2.
[0035] S3 diluted the reaction solution to 1 mg / mL with dimethyl sulfoxide aqueous solution, reacted at room temperature for 4 h, and then concentrated and purified by changing the solution to obtain HCGβCF conjugate;
[0036] Specifically, the concentration of the dimethyl sulfoxide aqueous solution is 5% to 30%, preferably 10%.
[0037] S4. The HCGβCF conjugate is coated using any one of SA magnetic bead coupling technology, carboxyl magnetic bead coupling technology, and Tosyl magnetic bead coupling technology to obtain the HCGβ detection reagent magnetic bead working solution.
[0038] The specific method is as follows:
[0039] S41 was washed three times with coupling buffer in the pH range of 5-7 and diluted to 10 mg / mL.
[0040] Specifically, the coupling buffer is a 100mM MES buffer with pH=6.0.
[0041] S42 The HCGβCF conjugate is added to step S41 at a coating ratio of 5-40 μg / mg and incubated for 15 min, and 0.05% surfactant is added for catalysis.
[0042] Specifically, the coating ratio is 10 μg / mg, and the surfactant is S21.
[0043] S43 Add EDC to step S42 for catalytic reaction, rotate the reaction at room temperature for 4 hours, and remove the supernatant after the reaction is completed;
[0044] Specifically, the EDC reaction mass ratio is 1:15 to 1:3. Preferably, the EDC reaction mass ratio is 1:10.
[0045] S44 Add 1% blocking agent to step S43 for blocking, rotate reaction at room temperature for 2 hours, remove supernatant after reaction, add equal volume of buffer containing primary amino acid to quench reaction, and repeat washing 3 times.
[0046] Specifically, the blocking agent is Blockmaster DB1130 or casein. The primary amino buffer comprises 25 mM Tris buffer, pH 7, containing 0.2% S14, 0.9% sodium chloride, and 1% glycerol.
[0047] S45 Remove the supernatant from step S44, wash three times with magnetic bead working solution, and dilute to a magnetic bead concentration of 0.2 mg / mL to obtain the HCGAb detection reagent magnetic bead working solution.
[0048] Specifically, the working solution for the magnetic beads is a 10mM MES buffer solution with a pH of 6.5, containing 0.9% sodium chloride, 0.5%–1% trehalose, 0.5%–1% bovine serum albumin, 0.2% surfactant S14, 0.05%–0.2% preservative, and 0.05% L-cysteine. In particular, the addition of 0.05% L-cysteine as a protective agent effectively extends the shelf-life stability of the magnetic beads. The preservatives include any two or more of PC300, PC950, BND-10, and Krovin300.
[0049] The specific implementation method is as follows:
[0050] Example 1: Conventional process for coating magnetic beads with HCG (control group)
[0051] Using intact HCG antigen as the capture antigen, a one-step carboxyl group coating method was employed, with the following steps:
[0052] Select 10 mg of MS 300 carboxyl magnetic beads, wash 3 times with 2 mL of coating buffer (0.1 M MES buffer), remove the supernatant, and resuspend in 1 mL of coating buffer;
[0053] Add 100 μg of antigen and mix thoroughly by rotating at room temperature for 15 min.
[0054] Add 1 mg of coupling reagent EDC and mix by rotation at room temperature for 4 h.
[0055] Add 1% bovine serum albumin as a blocking agent and mix by rotating at room temperature for 2 hours.
[0056] After the reaction, the supernatant was removed, and 1 mL of elution buffer (25 mM Tris buffer, pH 7.2, 0.15 M NaCl, 0.05% S14) was added to resuspend the magnetic beads and quench the reaction. This process was repeated three times. The magnetic beads were then diluted to a concentration of 0.2 mg / mL using magnetic bead diluent buf-0. The magnetic bead diluent buf-0 was prepared as follows: 10 mM PBS buffer, pH 7.2, containing 0.9% sodium chloride, 0.5% trehalose, 0.5% bovine serum albumin, 0.2% surfactant S14, and 0.05% preservative PC300.
[0057] Example 2: Conventional process magnetic beads coated with HCGβCF couplings (Experimental Group 1)
[0058] HCGβCF conjugates were used as the capture antigen, and a one-step carboxyl coating method was employed.
[0059] The steps for HCGβCF coupling are as follows:
[0060] Working solution A is prepared by diluting 200 μg of antigen HCGβCF to 2 mg / mL using a coupling buffer (100 mM MES buffer at pH 7.4, 0.9% sodium chloride).
[0061] Dilute 2 mg Traut's reagent with 100 μL of coupling buffer to prepare a 20 mg / mL crosslinking agent working solution. Add 20 μL of crosslinking agent working solution at an antigen-reagent mass ratio of 1:2. React with working solution A at 37°C under sealed conditions for 30 min. Desalt, purify and concentrate to obtain reaction solution B.
[0062] The reaction solution B was diluted to 1 mg / mL with 10% dimethyl sulfoxide (DMSO) aqueous solution, and the reaction was carried out under mild catalysis at room temperature for 4 h. The solution was then changed and concentrated to purify the solution, yielding HCGβCF conjugate.
[0063] The one-step carboxyl coating is as follows:
[0064] Select 10 mg of MS 300 carboxyl magnetic beads, wash 3 times with 2 mL of coating buffer (0.1 M MES buffer), remove the supernatant, and resuspend in 1 mL of coating buffer;
[0065] Add 100 μg of HCGβCF conjugate and mix by rotation at room temperature for 15 min.
[0066] Add 1 mg of coupling reagent EDC and mix by rotation at room temperature for 4 h.
[0067] Add 1% bovine serum albumin as a blocking agent and mix by rotating at room temperature for 2 hours.
[0068] After the reaction was complete, the supernatant was removed, and 1 mL of elution buffer (i.e., 25 mM Tris buffer, pH 7.2, 0.15 M NaCl, 0.05% S14) was added to resuspend the magnetic beads and quench the reaction. This was repeated 3 times, and then diluted to a magnetic bead concentration of 0.2 mg / mL using magnetic bead dilution buffer buf-0.
[0069] Example 3: Optimized process for coating HCGβCF couplings with magnetic beads (Experimental Group 2)
[0070] Using HCGβCF conjugates as the capture antigen, a one-step carboxyl-based coating method was employed, with the following steps:
[0071] Select 10 mg of MS 300 carboxyl magnetic beads, wash 3 times with 2 mL of coating buffer (0.1 M MES buffer), remove the supernatant, and resuspend in 1 mL of coating buffer;
[0072] Add 100 μg of HCGβCF conjugate and 0.05% surfactant S21, and mix by rotation at room temperature for 15 min.
[0073] Add 1 mg of coupling reagent EDC and mix by rotation at room temperature for 4 h.
[0074] Add 1% Blockmaster DB1130 as a blocking agent and rotate to mix and react at room temperature for 2 hours.
[0075] After the reaction was complete, the supernatant was removed, and 1 mL of elution buffer (25 mM Tris buffer, pH 7.2, 0.15 M NaCl, 0.05% S14 and 1% glycerol) was added to resuspend the magnetic beads to quench the reaction. This was repeated 3 times, and then the magnetic beads were diluted to a concentration of 0.2 mg / mL with magnetic bead dilution buffer buf-0.
[0076] The magnetic bead working solutions prepared in Examples 1-3 above were combined with the AP enzyme-labeled anti-human IgG enzyme-labeled working solution produced by our company. The signal-to-noise ratio and potency of different magnetic bead working solutions were evaluated by testing a set of gradient samples (including blank sample S0, negative samples S1-2, sample with approximate CO value S3, weak positive sample S4, and positive sample S5).
[0077] Example 4: Optimized diluent (Experimental Group 3)
[0078] The magnetic beads coated in Example 3 were diluted with the optimized magnetic bead diluent buf-1 to a magnetic bead concentration of 0.2 mg / mL.
[0079] Weigh 1.95g of morpholinoethanesulfonic acid (MES) and dissolve it in 800mL of pure water. Adjust the pH to 6.50. Add 9g of sodium chloride, 10g of trehalose, 10g of bovine serum albumin, 0.2% surfactant S14, 0.05% PC300, 0.05% Krovin 300 and 0.5g of L-cysteine.
[0080] The magnetic bead working solutions prepared in the above embodiments were combined with the AP enzyme-labeled anti-human IgG enzyme-labeled working solution produced by our company. A set of gradient samples (including blank sample S0, negative samples S1-2, samples with approximately CO value S3, weakly positive samples S4, and positive samples S5-6) were used to evaluate the signal-to-noise ratio and titer of different magnetic bead working solutions. The test data are as follows:
[0081] Table 1. Signal-to-noise ratio and potency results of magnetic bead working fluids under different processes.
[0082]
[0083] As shown in Table 1, changes in raw materials and processes can significantly optimize the signal-to-noise ratio at the low end of the reagent; improved sample titer near the gray zone can reduce the gray zone range and improve clinical concordance.
[0084] The above-mentioned magnetic bead working solution was placed at 37℃ for 6 days to evaluate its accelerated stability. The test data are as follows:
[0085] Table 2 Signal retention rate of working fluids for magnetic beads produced by different processes
[0086]
[0087] As shown in Table 2, optimizing the diluent has a significant effect on improving stability.
[0088] Beneficial effects
[0089] 1. Provide raw material solutions to prepare low-cost and safe capture antigens, reduce enterprise production costs, reduce biosafety risks in production, improve clinical sensitivity, and improve clinical concordance rates;
[0090] 2. Optimize the carboxyl coating process to improve antigen coating efficiency and reduce enterprise production costs.
[0091] 3. Provide a working fluid for magnetic beads that effectively extends the stability of magnetic beads in the HCGAb project.
[0092] The above-disclosed method is merely a preferred embodiment of the preparation method of the HCGAb detection reagent magnetic bead working solution of the present invention. Of course, it should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that all or part of the process of the above-described embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.
Claims
1. A method for preparing a magnetic bead working solution for HCGAb detection reagent, characterized in that, Includes the following steps: Step 1: Dilute the antigen HCGβCF to 2 mg / mL with buffer solution to obtain the working solution; Step 2: The Traut's reagent modified with thiol group is reacted with the working solution from Step 1 at 37°C under sealed conditions for 30 min, and the reaction solution is then desalted, purified, and concentrated to obtain the reaction solution. Step 3: Dilute the reaction solution to 1 mg / mL with dimethyl sulfoxide aqueous solution, react at room temperature for 4 h, change the solution and concentrate and purify to obtain HCGβCF conjugate; Step 4: Coat the HCGβCF conjugate using any one of SA magnetic bead coupling technology, carboxyl magnetic bead coupling technology, and Tosyl magnetic bead coupling technology to obtain the HCGβ detection reagent magnetic bead working solution.
2. The method for preparing the HCGAb detection reagent magnetic bead working solution as described in claim 1, characterized in that, The process of coating the HCGβCF conjugate using carboxylated magnetic bead coupling technology includes: Wash the magnetic beads three times with coupling buffer in the pH range of 5-7, and dilute to 10 mg / mL; The HCGβCF conjugate was added at a coating ratio of 5–40 μg / mg and incubated for 15 min, with 0.05% surfactant added for catalysis. Add EDC to catalyze the reaction, and rotate the reaction at room temperature for 4 hours. After the reaction is complete, remove the supernatant. Add 1% blocking agent for blocking, rotate the reaction at room temperature for 2 hours, remove the supernatant after the reaction, add an equal volume of buffer containing primary amino groups to quench the reaction, and repeat the washing 3 times. Remove the supernatant, wash three times with magnetic bead working solution, and dilute to a magnetic bead concentration of 0.2 mg / mL to obtain the HCGAb detection reagent magnetic bead working solution.
3. The method for preparing the HCGAb detection reagent magnetic bead working solution as described in claim 1, characterized in that, The buffer solution is a PB buffer, MES buffer, or CB buffer containing 0.9% sodium chloride, with a pH range of 7–8. The reaction mass ratio of the thiol-modified Traut's reagent to the working solution is 1:0.5 to 1:8; The concentration of the dimethyl sulfoxide aqueous solution is 5% to 30%.
4. The method for preparing the HCGAb detection reagent magnetic bead working solution as described in claim 2, characterized in that, The coupling buffer is a 100mM MES buffer with pH=6.0; The coating ratio is 10 μg / mg, and the surfactant is S21; The blocking agent is Blockmaster DB1130 or casein; The primary amino-containing buffer comprises 25 mM Tris buffer, pH=7, containing 0.2% S14, 0.9% sodium chloride and 1% glycerol; The working solution for the magnetic beads is a 10mM MES buffer solution with a pH of 6.5, containing 0.9% sodium chloride, 0.5%–1% trehalose, 0.5%–1% bovine serum albumin, 0.2% surfactant S14, 0.05%–0.2% preservative, and 0.05% L-cysteine.
5. The method for preparing the HCGAb detection reagent magnetic bead working solution as described in claim 4, characterized in that, The preservative is any two or more of PC300, PC950, BND-10 and Krovin 300.