Preparation method of magnetic bead coated antibody working solution and kit

Abstract

The invention relates to the technical field of kits, in particular to a preparation method of a magnetic bead coated antibody working solution and a kit, activated carboxyl magnetic beads are pretreated by using a pretreatment solution containing CMEA, and the magnetic bead coated antibody working solution is obtained by combining a coating solution and a confining solution of a specific formula and then cleaning and resuspending by using a PBS buffer solution, cMEA and Tween 20 in the pretreatment liquid have a synergistic effect, so that the problem of magnetic bead agglomeration can be effectively solved, and meanwhile, the interference of pH fluctuation on subsequent reaction is avoided; the dispersibility, reactivity and stability of the magnetic bead working solution are further improved through the optimal proportion of the Tris concentration in the pretreatment solution and the coating solution and the combination of fish skin gelatin and mannose in the confining liquid; and finally, the kit obtained by combining the magnetic bead coated antibody working solution with the alkaline phosphatase labeled antibody working solution has high sensitivity and stability, can reduce a test background signal value, and overcomes the defects in the prior art.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, specifically to a method for preparing a magnetic bead-coated antibody working solution and a reagent kit. Background Technology

[0002] Immunomagnetic beads (IMBs), also known as immunomagnetic microspheres, are uniform, superparamagnetic, and protective spherical particles, primarily composed of carrier microspheres and immunoligands. The carrier microspheres mainly contain magnetic materials (small metal particles, such as iron oxide and magnetite) and polymer layers (such as polyethyleneimine, polyvinyl alcohol, and polyacrylic acid). Common functional groups include toluenesulfonyl magnetic beads, carboxyl magnetic beads, amino magnetic beads, epoxy magnetic beads, thiol magnetic beads, silicon-based magnetic beads, and physisorption magnetic beads, which impart different physical properties to their surfaces, such as hydrophobic-hydrophilic, nonpolar-polar, and positively-negatively charged. Carboxyl magnetic beads are a type of magnetic microsphere coated with carboxyl groups (-COOH). Under the action of special chemical reagents (such as EDC and NHS), they can be rapidly, efficiently, sensitively, and specifically covalently coupled to biomolecules such as peptides, proteins, antibodies, and oligonucleotides. However, in current coating methods, the coating solution for carboxyl magnetic beads generally uses the MES (2-morpholinoethanesulfonic acid) zwitterionic buffer system. Before and after coating, the magnetic beads often exhibit agglomeration, which is not conducive to their subsequent application in kits. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a working solution of magnetic beads coated with antibodies and a kit, which aims to solve the technical problem that the aggregation phenomenon of existing carboxyl magnetic beads in immunoassay affects the subsequent coupling efficiency with antibodies and reduces the detection sensitivity of the kit.

[0004] To achieve the above objectives, the present invention provides a method for preparing a magnetic bead-coated antibody working solution, comprising the following steps:

[0005] Step 1: Pre-treat the magnetic beads that have been activated by EDC and NHS using a pretreatment solution;

[0006] Step 2: The pretreated magnetic beads and antibodies are mixed in the coating solution and coated.

[0007] Step 3: Seal with sealing liquid;

[0008] Step 4: Wash and resuspend with PBS buffer to obtain the working solution of magnetic bead-coated antibody.

[0009] Optionally, the pretreatment solution comprises 3-6 mM Tris, 10-20 mM CMEA, 0.1-0.2% Tween 20, 1-3 MMES, and has a pH of 6.0.

[0010] Optionally, in step 1, 1000 μl of pretreatment solution is added to every 0.2-0.4 mg of magnetic beads.

[0011] Optionally, the coating solution in step 2 may include 5-50 mM Tris, 0.1-0.2% Tween 20, 0.2-1 M MES, and pH 6.0.

[0012] Optionally, the mass ratio of the magnetic beads to the antibody is 100:(0.3-2).

[0013] Optionally, the blocking solution is a 50mM MES buffer solution with a pH of 7.4 containing 0.1-5% fish skin gelatin and 0.05-0.2% mannose.

[0014] Optionally, the concentration of magnetic beads in the working solution for coating antibodies is 0.3-0.5 mg / mL.

[0015] The present invention also provides a kit prepared using the aforementioned magnetic bead-coated antibody working solution. Specifically, the kit is prepared by coating and blocking carboxyl magnetic beads with the aforementioned magnetic bead-coated antibody working solution, diluting them to a certain working concentration, and then combining them with alkaline phosphatase-labeled antibody working solution diluted to a certain working concentration.

[0016] This invention provides a method and kit for preparing a magnetic bead-coated antibody working solution. The method involves pretreating activated carboxyl magnetic beads with a pretreatment solution containing CMEA, combining this with a specific formulation of coating and blocking solutions, and then washing and resuspending the beads with PBS buffer to obtain the magnetic bead-coated antibody working solution. The CMEA in the pretreatment solution synergistically works with Tween 20 to effectively solve the problem of magnetic bead aggregation and avoids interference from pH fluctuations in subsequent reactions. The optimized ratio of Tris concentration in the pretreatment and coating solutions, and the combination of fish skin gelatin and mannose in the blocking solution, further improve the dispersibility, reactivity, and stability of the magnetic bead working solution. Finally, the kit obtained by combining the magnetic bead-coated antibody working solution with an alkaline phosphatase-labeled antibody working solution exhibits high sensitivity and stability, and also reduces background signal values, overcoming the shortcomings of existing technologies. Attached Figure Description

[0017] 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.

[0018] Figure 1 This is a schematic flowchart of the preparation method of a magnetic bead-coated antibody working solution according to the present invention. Detailed Implementation

[0019] 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.

[0020] Please see Figure 1 This invention provides a method and kit for preparing a magnetic bead-coated antibody working solution, comprising the following steps:

[0021] Step 1: Pre-treat the magnetic beads that have been activated by EDC and NHS using a pretreatment solution;

[0022] Step 2: The pretreated magnetic beads and antibodies are mixed in the coating solution and coated.

[0023] Step 3: Seal with sealing liquid;

[0024] Step 4: Wash and resuspend with PBS buffer to obtain the working solution of magnetic bead-coated antibody.

[0025] The pretreatment solution comprises 3-6 mM Tris, 10-20 mM CMEA, 0.1-0.2% Tween 20, 1-3 M MES, and has a pH of 6.0.

[0026] Preferably, the pretreatment solution includes 5-6 mM Tris, 14-16 mM CMEA, 0.15-0.2% Tween 20, and 2-3 MMES.

[0027] Furthermore, the only surfactant used in the pretreatment solution of this invention is CMEA, because CMEA is a nonionic surfactant. Compared with the alkaline aqueous solution of amino acid-type amphoteric surfactants, the aqueous solution of CMEA will not change pH, and it can work together with Tween to disperse the clustered magnetic beads due to the action of CMEA.

[0028] In step 1, 1000 μl of pretreatment solution was added to every 0.2-0.4 mg of magnetic beads.

[0029] The coating solution in step 2 includes 5-50 mM Tris, 0.1-0.2% Tween 20, and 0.2-1 M MES, with a pH of 6.0.

[0030] Preferably, the concentration ratio of Tris in the pretreatment solution and the coating solution is 1:(2-4).

[0031] The CMEA concentration in the pretreatment solution is 10-15 mM / mL.

[0032] The mass ratio of the magnetic beads to the antibody is 100:(0.3-2).

[0033] The blocking solution is a 50mM MES buffer solution with a pH of 7.4 containing 0.1-5% fish skin gelatin and 0.05-0.2% mannose.

[0034] The concentration of magnetic beads in the working solution of the antibody coated with magnetic beads is 0.3-0.5 mg / mL.

[0035] The present invention also provides a kit prepared using the aforementioned magnetic bead-coated antibody working solution, specifically obtained by combining the magnetic bead-coated antibody working solution with alkaline phosphatase-labeled antibody working solution using conventional methods.

[0036] The present invention will be further described below with reference to several specific embodiments:

[0037] Example 1

[0038] The activated 2.5 mg carboxyl magnetic beads were resuspended in 500 μL of pretreatment solution (pH 6.0) containing 5 mM Tris, 15 mM CMEA, and 2 M MES. After thorough mixing, the centrifuge tube was placed on a magnetic separator until the supernatant was completely clear. The supernatant was carefully removed with a pipette. The operation was repeated three times. The mixture was then transferred to a blood mixer and mixed for 20 min.

[0039] Example 2-10

[0040] Compared with Example 1, the difference lies in the different components in the pretreatment solution, as shown in Table 1 below. The dispersibility is good in Examples 1-10, and Example 1 is the best.

[0041] Example 11

[0042] Immediately after pretreatment with the pretreatment solution from Example 1, resuspend the magnetic beads to the target volume in a coating buffer containing 15 mM Tris, 1 M MES, and 0.1% Tween 20. Add antibody at a ratio of 20 μg / mg of antibody to magnetic beads and mix thoroughly. Incubate the coating solution at 37°C using a blood homogenizer for 1 hour. After the reaction, place the centrifuge tube on a magnetic separator until the supernatant is completely clear. Carefully discard the supernatant with a pipette. Add 500 μL of MES blocking buffer containing 5% fish skin gelatin at pH 6.0 and mix thoroughly. Incubate the blocking solution at 37°C using a blood homogenizer for 4 hours. After blocking, place the centrifuge tube on a magnetic separator until the supernatant is completely clear. Carefully discard the supernatant with a pipette. Incubate the blocking solution using 0.1% BSA in 0.1 M MES blocking buffer. Wash the magnetic beads with PBS buffer at pH 7.4 (5 mg / ml). Place the centrifuge tube on a magnetic separator until the supernatant is completely clear. Carefully remove the supernatant with a pipette. Repeat the washing process twice. Resuspend the magnetic beads in the same buffer and store at 2-8°C.

[0043] Examples 12-24

[0044] Compared with Example 11, the difference lies in the composition of the coating solution, as shown in Table 2 below. Example 19 exhibits the best overall performance in terms of reactivity and dispersibility. Examples 14 and 15 show slightly poor reactivity due to excessive Tris concentration. Examples 22-23 use higher pH values, resulting in lower reactivity values, and use lower pH values, resulting in higher reactivity values. Therefore, the optimal pH value is 6.0.

[0045] Examples 25-33

[0046] Compared to Example 19, the difference lies in the pretreatment solution being the same as that used in Examples 2-10. Based on the dispersibility and reactivity data, the overall performance of Examples 25-33 remains superior.

[0047] Examples 34-50

[0048] Compared with Example 11, the difference lies in the composition of the sealing liquid and the feeding ratio. In the selection of the feeding ratio, the ratio results obtained by different feeding ratios in Examples 37 and 40-42 are similar, but Example 40 has a lower background and is therefore better.

[0049] Examples 51-59

[0050] Compared to Example 40, the difference lies in that the solution pretreated with the pretreatment solution is from Examples 2-10. Based on the dispersibility and reactivity data, the overall performance of Example 40 remains superior.

[0051] Comparative Example 1

[0052] Compared to Example 40, the difference lies in that the pretreatment solution is only a 0.1M MES solution at pH 6.5, the coating solution is a 1M Tris buffer system at pH 7.4, and the blocking solution is a PBS solution at pH 7.4 containing 0.5% BSA. The performance is shown in Table 3.

[0053] Comparative Example 2

[0054] The difference from Example 40 is that the pretreatment solution is a 0.1M sodium borate solution with pH 6.0. The performance is shown in Table 3.

[0055] Table 1 Composition and Performance of Pretreatment Solution

[0056]

[0057] Table 2 Composition and Properties of Coating and Sealing Fluids

[0058]

[0059] Table 3 Reactivity Test Table for Different Pretreatment Processes

[0060]

[0061] Table 2 shows that the magnetic bead working solution exhibits good dispersibility in a pretreatment solution containing 5-6 mM Tris, 14-16 mM CMEA, 0.15-0.2% Tween 20, and 2-3 M MES at pH 6.0. The working solution also shows good reactivity in a coating solution containing 5-50 mM Tris, 0.1-0.2% Tween 20, 0.2-1 M MES, and pH 6.0. Furthermore, the working solution shows good reactivity in a blocking solution containing 50 mM Tris at pH 7.4, with 0.1-5% fish skin gelatin and 0.05-0.2% mannose. Comparative Example 1, using a conventional coating method, showed poor dispersibility and reactivity. Comparative Example 2, using a conventional pretreatment solution and only the coating and blocking solutions of this invention (without the pretreatment solution), showed some improvement in dispersibility and reactivity compared to Comparative Example 1, but still lower than the dispersibility and reactivity in Example 1 of this invention. The working fluid prepared in this invention has a reactivity ratio greater than 24 and a background value maintained at a slightly low level, with good agglomeration.

[0062] In summary, the preparation method of the present invention enables carboxyl magnetic beads to be uniformly dispersed in the working solution and exhibits good reactivity.

[0063] Therefore, compared with the prior art, the present invention has the following beneficial effects:

[0064] 1. The pretreatment solution of the present invention can uniformly disperse carboxyl magnetic beads, which is beneficial to the subsequent reaction.

[0065] 2. Through the combined effects of the pretreatment liquid, coating liquid, and sealing liquid of this invention, the magnetic bead working solution prepared by this invention has good reactivity.

[0066] 3. This invention can improve reagent stability and reduce test background signal values.

[0067] The above description discloses only one or more preferred embodiments of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that implementing all or part of the above embodiments and making equivalent changes 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-coated antibody working solution, characterized in that, Includes the following steps: Step 1: Pre-treat the magnetic beads that have been activated by EDC and NHS using a pretreatment solution; Step 2: The pretreated magnetic beads and antibodies are mixed in the coating solution and coated. Step 3: Seal with sealing solution; Step 4: Wash and resuspend with PBS buffer to obtain the working solution of magnetic bead-coated antibody.

2. The method for preparing the magnetic bead-coated antibody working solution as described in claim 1, characterized in that, The pretreatment solution comprises 3-6 mM Tris, 10-20 mM CMEA, 0.1-0.2% Tween 20, 1-3 M MES, and has a pH of 6.

0.

3. The method for preparing the magnetic bead-coated antibody working solution as described in claim 2, characterized in that, In step 1, 1000 μl of pretreatment solution was added to every 0.2-0.4 mg of magnetic beads.

4. The method for preparing the magnetic bead-coated antibody working solution as described in claim 3, characterized in that, The coating solution in step 2 includes 5-50 mM Tris, 0.1-0.2% Tween 20, and 0.2-1 M MES, with a pH of 6.

0.

5. The method for preparing the magnetic bead-coated antibody working solution as described in claim 4, characterized in that, The mass ratio of the magnetic beads to the antibody is 100:(0.3-2).

6. The method for preparing the magnetic bead-coated antibody working solution as described in claim 5, characterized in that, The blocking solution is a 50mM MES buffer solution with a pH of 7.4 containing 0.1-5% fish skin gelatin and 0.05-0.2% mannose.

7. The method for preparing the magnetic bead-coated antibody working solution according to claim 6, characterized in that, The concentration of magnetic beads in the working solution for coating antibodies is 0.3-0.5 mg / mL.

8. A reagent kit, prepared using the magnetic bead-coated antibody working solution according to any one of claims 1 to 7, characterized in that, The kit is composed of carboxyl magnetic beads coated and blocked with the magnetic bead-coated antibody working solution, diluted to a certain working concentration, and then combined with alkaline phosphatase-labeled antibody working solution diluted to a certain working concentration.

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