LCA magnetic bead and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an LCA magnetic bead and a preparation method and application thereof. According to the method, the alpha-methyl-D-mannoside is used for preparing the LCA magnetic beads, lectin is fixed while the magnetic beads are synthesized, the efficiency of coupling the LCA magnetic beads with the LCA protein is greatly improved and reaches 98% or above while operation steps are simplified, the binding amount of target protein is also obviously improved, and the storage stability of the magnetic beads is good. Meanwhile, the particle size D50 of the magnetic beads is mainly distributed within 40-50 microns, the particle size is uniform, the magnetic response is strong, and the consumed time is short.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to LCA magnetic beads, their preparation methods, and applications. Background Technology

[0002] In the biological field, the isolation and purification of glycoproteins is of great significance for subsequent scientific research (such as tumor marker screening and stem cell surface labeling studies). Traditional glycoprotein isolation and purification methods, such as boric acid affinity chromatography and hydrogen bonding adsorption, suffer from poor selectivity and high non-specific binding rates. This is because residual charges or hydrophobic groups on the carrier surface introduce background interference, affecting the separation and purification effect. Agarose lectin columns, on the other hand, face challenges such as slow flow rates, high non-specific adsorption, and poor reproducibility, making it difficult to meet the requirements for efficient separation. The use of magnetic beads for glycoprotein isolation and purification has become a research hotspot. This method eliminates the need for complex operations such as centrifugation, filtration, and chromatographic separation; only an external magnetic field is required to achieve the separation of the target protein. This eliminates numerous sample pretreatment and separation processes, and compared with conventional isolation and purification methods, it has advantages such as speed, efficiency, and high purity.

[0003] Currently, commonly used commercial lectin magnetic beads (such as Con A magnetic beads) have insufficient affinity for specific sugar types (such as fucose), and when lectin magnetic beads are prepared by covalent coupling, lectin activity is easily lost. For example, the protein inactivation rate of magnetic beads prepared by glutaraldehyde crosslinking exceeds 30%.

[0004] Lens Culinaris agglutinin (LCA) is a metalloprotein isolated from lentil seeds. It consists of two 17 kDa large subunits and two 8 kDa small subunits, with a molecular weight of approximately 49 kDa. It can specifically bind to molecules containing α-D-mannose, α-D-glucose, or spatially related residues. Based on this property of LCA, LCA magnetic beads, formed by coupling it with agarose magnetic spheres, have become a novel type of affinity magnetic bead for enriching or purifying glycoproteins. Currently, there are two main methods for preparing LCA magnetic beads. One is the lectin covalently coupled magnetic bead method, which selects superparamagnetic microspheres (such as carboxylated or aminated magnetic beads). The carboxyl groups of the carboxyl magnetic beads are activated by EDC / NHS to form amide bonds with the amino groups in lentil lectin. The amino magnetic beads are then coupled to the lectin via glutaraldehyde cross-linking. The remaining active sites are then blocked using ethanolamine or BSA. The other method is the streptavidin-biotin indirect coupling method, which first prepares streptavidin magnetic beads and then binds them with biotin-labeled lentil lectin (such as Biotinylated LentilLectin from Vector Laboratories) with high affinity. This method avoids the inactivation of lectin active sites due to direct coupling. Related techniques can be found in "Lectin Affinity Chromatography" (Volume 2, 1994, Molecular Biotechnology) and "Binding interantions of glycoproteins with lectins" (Volume 21, number 1, MOLECULAR). (CELLULAR BIOCHEMISTRY). In existing LCA magnetic bead preparation processes, the coupling efficiency between lentil lectin LCA and agarose magnetic beads remains low. Therefore, providing an LCA magnetic bead with high protein coupling efficiency and a high target protein adsorption and binding capacity, along with its preparation method, is of significant practical importance. Summary of the Invention

[0005] In view of this, the present invention provides LCA magnetic beads, their preparation methods, and applications.

[0006] Application of α-methyl-D-mannoside (α-D-MMP) in the preparation of LCA magnetic beads or in coupling buffers for the preparation of LCA magnetic beads.

[0007] Compared with other components in the prior art, this invention is the first to discover and use α-methyl-D-mannoside in the preparation of LCA magnetic beads, which significantly improves the coupling efficiency of LCA protein and the binding amount of target protein is also significantly increased.

[0008] The present invention also provides a coupling buffer for preparing LCA magnetic beads, comprising PBS buffer and 0.2 M to 0.4 M α-methyl-D-mannoside.

[0009] In some embodiments, the concentration of α-methyl-D-mannoside in the coupling buffer is specifically 0.2M, 0.3M, or 0.4M. The pH of the coupling buffer can specifically be 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0. In a specific embodiment of the present invention, the pH is 7.4.

[0010] In some embodiments, the coupling buffer comprises PBS buffer and α-methyl-D-mannoside, calcium chloride, and manganese chloride; wherein the concentration of α-methyl-D-mannoside is 0.2 M to 0.4 M.

[0011] This invention has found that adding calcium chloride and manganese chloride to the coupling buffer significantly improves both the LCA protein coupling efficiency and the amount of target protein bound.

[0012] In some embodiments, the coupling buffer comprises 10–20 mM PBS buffer, 0.2 M–0.4 M α-methyl-D-mannoside, 1–3 mM calcium chloride, and 1–3 mM manganese chloride.

[0013] This invention reveals that the concentration of α-methyl-D-mannoside has a significant impact on LCA protein coupling efficiency and target protein binding. Compared to other concentrations, 0.2 M to 0.4 M α-methyl-D-mannoside is more effective; among them, 0.2 M α-methyl-D-mannoside shows the best effect.

[0014] In some specific embodiments, the coupling buffer consists of the following components:

[0015] 10 mM PBS buffer, 0.2 M α-methyl-D-mannoside, 1 mM calcium chloride and 1 mM manganese chloride.

[0016] In some specific embodiments, the coupling buffer consists of the following components:

[0017] 10 mM PBS buffer, 0.4 M α-methyl-D-mannoside, 1 mM calcium chloride and 1 mM manganese chloride.

[0018] The present invention also provides LCA magnetic beads, which are prepared from coupling buffer and agarose magnetic beads as described above.

[0019] The present invention also provides a method for preparing the LCA magnetic beads, comprising: washing activated agarose magnetic beads with the coupling buffer described in the present invention, mixing them with LCA protein, incubating, and obtaining LCA magnetic beads.

[0020] In some implementations, the activator used for activation includes EDC / NHS.

[0021] In some embodiments, the activated agarose magnetic beads are NHS-activated agarose magnetic beads, which can be prepared according to conventional methods in the art, or can be commercially available NHS-activated agarose magnetic beads. In a specific embodiment of the present invention, the magnetic beads are commercially available NHS-activated agarose magnetic beads produced by Beaver Biotechnology, specifically NHS-activated agarose magnetic beads with an NHS group content of 50~150 μmol / mL.

[0022] In some implementations, the mixing method is vortex oscillation. The duration of the vortex oscillation is 10~30 s, specifically 10 s, 15 s, 20 s, 25 s, or 30 s, and in a specific embodiment of the present invention, 15 s is preferred.

[0023] In some implementations, the incubation is performed at room temperature. The incubation time at room temperature is preferably 20 to 24 hours, specifically 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0024] The present invention also provides the application of the LCA magnetic bead in any of the following:

[0025] (1) Enrichment and / or purification of glycoproteins;

[0026] (2) Preparation of glycoprotein detection reagents;

[0027] (3) Cell sorting.

[0028] The present invention also provides reagents for the enrichment, purification and / or detection of glycoproteins, comprising magnetic bead suspensions;

[0029] The magnetic bead suspension includes the LCA magnetic beads and magnetic bead preservation solution described in this invention.

[0030] In some implementations, the volume of the LCA magnetic beads is 10% of the total volume of the magnetic bead suspension.

[0031] In some embodiments, the magnetic bead preservation solution comprises: 20% ethanol, 0.15 M sodium chloride, 1 mM calcium chloride, and 1 mM manganese chloride.

[0032] In some embodiments, the reagent further includes washing buffer 1, washing buffer 2, and / or washing buffer;

[0033] The cleaning buffer 1 is a 1 mM hydrochloric acid solution;

[0034] The washing buffer 2 is a 10 mM PBS buffer with a pH of 7.4;

[0035] The washing buffer 3 is a 20 mM Tris-HCl solution containing 0.15 M sodium chloride, 0.2 M α-methyl-D-mannoside, 1 mM calcium chloride and 1 mM manganese chloride, with a pH of 7.4.

[0036] Washing buffer 1 is used before washing the magnetic beads with the coupling buffer. Washing buffers 2 and 3 are used after coupling is complete.

[0037] This invention utilizes α-methyl-D-mannoside to prepare LCA magnetic beads, immobilizing lectins simultaneously during the magnetic bead synthesis. This simplifies the operation steps and significantly improves the efficiency of LCA magnetic bead coupling with LCA proteins, reaching over 98%. The binding amount of the target protein is also significantly increased, and the magnetic beads exhibit good storage stability. Furthermore, the magnetic beads have a uniform particle size (D50) mainly distributed between 40-50 μm, strong magnetic response, and short processing time. Detailed Implementation

[0038] This invention provides LCA magnetic beads, their preparation methods, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0039] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0040] The present invention will be further illustrated below with reference to the embodiments:

[0041] Example 1: Preparation of the coupling buffer of the present invention

[0042] Add 0.2 M α-methyl-D-mannoside, 1 mM calcium chloride, and 1 mM manganese chloride to 10 mM PBS buffer at pH 7.4 and dissolve thoroughly.

[0043] LCA protein solution: Weigh 22 mg of LCA protein and dissolve it in 1.1 mL of coupling buffer.

[0044] Example 2: Preparation of the coupling buffer of the present invention

[0045] Add 0.4 M α-methyl-D-mannoside, 1 mM calcium chloride, and 1 mM manganese chloride to 10 mM PBS buffer at pH 7.4 and dissolve thoroughly.

[0046] LCA protein solution: Weigh 22 mg of LCA protein and dissolve it in 1.1 mL of coupling buffer.

[0047] Example 3: Preparation of LCA magnetic beads of the present invention

[0048] Prepare the following reagents:

[0049] Coupling buffer: Prepared according to the formulations in Examples 1-2.

[0050] Washing buffer: 1:1 mM hydrochloric acid solution.

[0051] Washing buffer 2: 10 mM PBS buffer, pH=7.4.

[0052] Washing buffer 3: Prepare a 20 mM Tris-HCl solution containing 0.15 M sodium chloride, adjust the pH to 7.4, and then add the following components and dissolve them completely until the final concentration of each component is 0.2 M or 0.4 M α-methyl-D-mannoside, 1 mM calcium chloride, and 1 mM manganese chloride.

[0053] Magnetic bead preservation solution: Add calcium chloride, sodium chloride and manganese chloride to a 20% ethanol solution and dissolve them completely until the final concentration is 0.15 M sodium chloride, 1 mM calcium chloride and 1 mM manganese chloride.

[0054] The preparation method includes the following steps:

[0055] 1) Pipette 5 mL of NHS activated agarose magnetic bead suspension (corresponding to 1 mL of activated magnetic beads) into a 15 mL centrifuge tube and remove acetone by magnetic adsorption. The activation density of the NHS activated agarose magnetic beads shall not be less than 50 μml / mL, and the cross-linking time of the raw materials shall not be less than 1.5 h.

[0056] 2) Use 5 mL of cooled washing buffer 1 to quickly wash the magnetic beads. After vortexing for 15 s, remove the liquid with magnetic suction and repeat the washing process twice.

[0057] 3) Wash the magnetic beads with 5 mL of coupling buffer, vortex for 15 s, remove the liquid with magnetic suction, and repeat the washing process twice.

[0058] 4) Use a 1000 μL pipette to transfer 1 mL of LCA protein solution into the above 15 mL centrifuge tube, vortex for 15 s (the remaining 100 μL of protein solution is reserved for later use), place the centrifuge tube containing the magnetic beads in a vertical mixer, and incubate at room temperature for 20-24 h.

[0059] 5) After the coupling reaction is complete, remove the centrifuge tube from the magnetic suction device, transfer 50 μL of flow-through solution and 100 μL of LCA solution to be used, measure the OD value and calculate the coupling efficiency.

[0060] 6) Add 100 μL of ethanolamine to the above centrifuge tubes and seal at room temperature for 2 h.

[0061] 7) Wash once with 5 mL of washing buffer 2, vortex for 15 s, remove the liquid with a magnetic suction, and repeat the washing process twice.

[0062] 8) Wash once with 5 mL of washing buffer 3, vortex for 15 s, remove the liquid with a magnetic suction, and repeat the washing process twice.

[0063] 9) Wash once with 5 mL of magnetic bead preservation solution, vortex for 15 s, and then magnetically remove the liquid.

[0064] 10) Add 9 mL of magnetic bead preservation solution, vortex to mix, and store at 4 °C. The final preservation concentration of magnetic beads is 10% v / v, that is, 1 mL of magnetic beads are contained in 10 mL of suspension.

[0065] Test Example 1

[0066] The coupling buffers for Comparative Examples 1 to 5 were prepared, with the following specific compositions:

[0067] Comparative Example 1: 10 mM PBS + 0.2 M α-D-MM;

[0068] Comparative Example 2: 10 mM PBS + 0.5 M α-D-MM;

[0069] Comparative Example 3: 10 mM PBS + 0.5 M α-D-MM + 1 mM Ca 2+ +1mM Mn 2+ ;

[0070] Comparative Example 4: 10 mM PBS + 0.8 M α-D-MM + 1 mM Ca 2+ +1mM Mn 2+

[0071] Comparative Example 5: 0.1 M NaHCO3;

[0072] Example 1: 10 mM PBS + 0.2 M α-D-MM + 1 mM Ca 2+ +1mM Mn 2+ ;

[0073] Example 2: 10 mM PBS + 0.4M α-D-MM + 1 mM Ca 2++1mM Mn 2+ ;

[0074] LCA magnetic beads were prepared using the coupling buffers of Comparative Examples 1-5 and Examples 1-2 respectively, according to the method in Example 2, and PTG protein was adsorbed. The components and conditions involved in the experiment were the same. The coupling efficiency and the amount of PTG protein bound were calculated, and the results are shown in Table 1.

[0075] Table 1

[0076]

[0077] The results showed that the coupling buffer provided by this invention has significant advantages in improving the coupling efficiency of LCA proteins and the binding amount of target proteins, and its effect is significantly better than the coupling solutions of comparative examples 1-5. Among them, the PBS buffer formulation containing 0.2 M α-methyl-D-mannoside, 1 mM calcium chloride, and 1 mM manganese chloride performed particularly well, with a coupling efficiency generally reaching over 98%, and the binding amount of PTG protein remained stable at a high level. In addition, this buffer system demonstrated good reproducibility and stability in different batches of experiments, providing reliable technical support for industrial production.

[0078] The above indicates that α-methyl-D-mannoside is a key component in the coupling solution, not only acting as a stabilizer to protect protein activity but also promoting the binding of the protein to the magnetic beads through specific intermolecular forces. Further addition of divalent metal ions (calcium and manganese) significantly enhanced this interaction, resulting in superior performance characteristics of the final LCA magnetic beads in practical applications. This demonstrates that the synergistic effect of the components in the buffer solution plays a crucial role in improving the performance of the magnetic beads.

[0079] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of α-methyl-D-mannoside in the preparation of LCA magnetic beads or in coupling buffers for the preparation of LCA magnetic beads.

2. A coupling buffer for preparing LCA magnetic beads, characterized in that, It includes PBS buffer and 0.2 M to 0.4 M α-methyl-D-mannoside, wherein the pH of the coupling buffer is 7-8.

3. The coupling buffer according to claim 2, characterized in that, The coupling buffer also includes calcium chloride and manganese chloride.

4. The coupling buffer according to claim 2 or 3, characterized in that, The coupling buffer comprises 10-20 mM PBS buffer, 0.2 M-0.4 M α-methyl-D-mannoside, 1-3 mM calcium chloride, and 1-3 mM manganese chloride.

5. An LCA magnetic bead, characterized in that, It is prepared from the coupling buffer and agarose magnetic beads as described in any one of claims 2 to 4.

6. The method for preparing LCA magnetic beads as described in claim 5, characterized in that, include: After washing NHS-activated agarose magnetic beads with coupling buffer, they were mixed with LCA protein and incubated to obtain LCA magnetic beads. The coupling buffer is the coupling buffer according to any one of claims 2 to 4.

7. The preparation method according to claim 6, characterized in that, The mixing method is vortex oscillation; the duration of the vortex oscillation is 10~30 s; And / or, the incubation is performed at room temperature for 20-24 hours.

8. The application of the LCA magnetic bead as described in claim 5 in any of the following: (1) Enrichment and / or purification of glycoproteins; (2) Preparation of glycoprotein detection reagents; (3) Cell sorting.

9. A reagent for the enrichment, purification, and / or detection of glycoproteins, characterized in that, Including magnetic bead suspensions; The magnetic bead suspension includes the LCA magnetic beads and magnetic bead preservation solution as described in claim 5.

10. The reagent according to claim 9, characterized in that, The volume of the LCA magnetic beads is 10% of the total volume of the magnetic bead suspension; And / or, the magnetic bead preservation solution comprises: 20% ethanol, 0.15 M sodium chloride, 1 mM calcium chloride and 1 mM manganese chloride; And / or, the reagents further include washing buffer 1, washing buffer 2 and / or washing buffer 3; The cleaning buffer 1 is a 1 mM hydrochloric acid solution; The washing buffer 2 is a 10 mM PBS buffer with a pH of 7.4; The washing buffer 3 is a 20 mM Tris-HCl solution containing 0.15 M sodium chloride, 0.2 M α-methyl-D-mannoside, 1 mM calcium chloride and 1 mM manganese chloride, with a pH of 7.4.

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