Methotrexate detection kit and preparation method thereof

Abstract

The invention relates to the technical field of methotrexate blood concentration in-vitro detection, in particular to a methotrexate detection kit and a preparation method thereof. The invention provides a preparation method of a methotrexate detection kit, the preparation method comprises the four steps of S1 surface modified magnetic bead preparation, S2R1 reagent preparation, S3R2 reagent preparation and S4 combination, and a molecular imprinting technology and a bimetallic chelation strategy are combined and applied to magnetic bead modification. High-selectivity capture of methotrexate is realized through a synergistic effect of spatial configuration matching and multivalent chemical bonding. The methotrexate detection kit prepared by the preparation method has good detection selectivity, sensitivity and accuracy.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology of methotrexate blood concentration, specifically to a methotrexate detection kit and its preparation method. Background Technology

[0002] Methotrexate (MTX) is a folic acid-based antitumor drug widely used clinically to treat malignant tumors such as acute lymphoblastic leukemia, lymphoma, choriocarcinoma, and osteosarcoma. High-dose methotrexate combined with leucovorin salvage is currently one of the important treatment regimens for acute lymphoblastic leukemia, malignant lymphoma, and osteosarcoma. High-dose methotrexate chemotherapy requires monitoring of MTX blood concentrations to guide the rational use of salvage drugs and avoid serious toxic side effects caused by MTX. Therefore, monitoring methotrexate blood concentrations is of great significance for guiding the rational clinical use of drugs in cancer patients.

[0003] Currently, the main methods for detecting methotrexate are liquid chromatography-tandem mass spectrometry (LC-MS / MS) and enzyme-linked immunosorbent assay (ELISA).

[0004] Chromatography-mass spectrometry (GC-MS) involves complex sample pretreatment and procedures, and the expensive instruments and testing costs make it unsuitable for large-sample testing and grassroots applications. Enzyme-linked immunosorbent assay (ELISA) has lower sensitivity and specificity.

[0005] To address the aforementioned deficiencies in the prior art, the main objective of this invention is to provide a chemiluminescent immunoassay kit for detecting methotrexate. This kit uses simple reagents, eliminates the need for expensive instruments, reduces costs, and simultaneously provides a rapid, convenient, and stable method for detecting methotrexate in samples. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to develop a methotrexate detection kit and its preparation method. It combines molecular imprinting technology with a bimetallic chelation strategy applied to magnetic bead modification, achieving highly selective capture of methotrexate through the synergistic effect of spatial configuration matching and multivalent chemical bonding. The methotrexate detection kit prepared using this method exhibits excellent detection selectivity, sensitivity, and accuracy.

[0007] This invention discloses a method for preparing a methotrexate detection kit, comprising the following steps:

[0008] Preparation of S1 surface-modified magnetic beads: Carboxylated magnetic beads were washed three times with MES buffer; a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added, and the mixture was activated by shaking at 37°C for 30 min to obtain activated magnetic beads; methotrexate standard was mixed with methacrylic acid in a certain proportion, and ethylene glycol dimethacrylate and azobisisobutyronitrile were added as crosslinking agent and azobisisobutyronitrile as initiator, and nitrogen was used to deoxygenate for 15 min to obtain an imprint solution; the activated magnetic beads were mixed with the imprint solution, and thermal polymerization was initiated at 70°C for 4 h to form a polymer coating layer; the imprinted magnetic beads were obtained by elution with a methanol / acetic acid mixture; the imprinted magnetic beads were dispersed in a copper sulfate and zinc nitrate mixture and chelated by shaking at 37°C for 2 h; the beads were washed with deionized water until no metal ion residue was found, and bovine serum albumin was added to block the beads at 37°C for 1 h to obtain surface-modified magnetic beads, which were stored at 4°C in PBS buffer containing 0.05% ProClin 300;

[0009] Preparation of S2 R1 reagent: Add the surface-modified magnetic beads prepared in step S1 to the diluent, then add methotrexate monoclonal antibody, and incubate at 4°C for 12 h to prepare R1 reagent;

[0010] Preparation of S3 R2 reagent: Methotrexate synthetic antigen and alkaline phosphatase were placed in a centrifuge tube at a concentration ratio of 1:4, mixed well, dialyzed with TBS, glutaraldehyde was added in proportion, and the mixture was reacted for 3 hours. After dialyzed with TBS, glycerol was added and mixed. Then, R2 reagent was prepared with mouse IgG, methylisothiazolinone, Tween-20, sodium azide, and MOPS buffer.

[0011] S4 combination: The R1 reagent obtained in step S2 and the R2 reagent obtained in step S3 are combined to obtain a methotrexate detection kit.

[0012] Preferably, in the S1 surface-modified magnetic bead preparation step, the particle size of the hydroxyl magnetic beads is 1-3 μm.

[0013] Preferably, in the S1 surface-modified magnetic bead preparation step, the concentration of the MES buffer is 0.05M and the pH value is 6; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.1mM; and the concentration of N-hydroxysuccinimide is 0.04M.

[0014] Preferably, in the S1 surface-modified magnetic bead preparation step, the concentration of the methotrexate standard is 0.01M; the concentration of methacrylic acid is 0.04M; the concentration of ethylene glycol dimethacrylate is 0.2M; and the concentration of azobisisobutyronitrile is 0.01M.

[0015] Preferably, in the S1 sample diluent preparation step, the concentration of the composite chelating agent in the sample diluent is 0.01M, the concentration of Tween 80-deoxycholate sodium is 0.001M, and the concentration of Proclin 300 is 0.0005M.

[0016] Preferably, in the S1 surface-modified magnetic bead preparation step, the volume ratio of the methotrexate standard to methacrylic acid is 1:1.

[0017] Preferably, in the preparation step of the S1 surface-modified magnetic beads, the volume ratio of methanol to acetic acid in the methanol / acetic acid mixed solution is 9:1.

[0018] Preferably, in the S1 surface-modified magnetic bead preparation step, the concentration of copper sulfate is 0.005M; the concentration of zinc nitrate is 0.002M; and the mass fraction of bovine serum albumin is 1%.

[0019] Preferably, in the S2 R1 reagent preparation step, the diluent is composed of 0.1% Tween-20, 2% trehalose and 0.02% sodium azide, with a pH of 7.4.

[0020] Preferably, in the S4 assembly step, the volume ratio of reagent R1 to reagent R2 is 1:1.

[0021] A methotrexate detection kit, prepared by any of the above-described methods for preparing a methotrexate detection kit.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) In the preparation step of S1 surface modified magnetic beads, a methotrexate molecular imprint cavity is formed on the surface of the magnetic beads to achieve target molecule spatial configuration matching; through the synergistic effect of Cu2+ / Zn2+ bimetallic ions, the electrostatic interaction with methotrexate carboxylic acid groups is strengthened; the spatial recognition of molecular imprints and the chemical bonding of metal chelation are combined to break through the limitation of single action force and significantly improve detection selectivity and sensitivity.

[0024] (2) When MOPS buffer is used in combination with mouse IgG and methylisothiazolinone, the activity of alkaline phosphatase can be guaranteed, while non-specific binding in methotrexate detection and reducing the binding of methotrexate to proteins in blood samples, thereby further improving the accuracy of this kit.

[0025] (3) It innovatively proposes to use chemiluminescence to detect methotrexate. It uses simple reagents, does not require expensive instruments or professional personnel, reduces costs, and can automatically analyze and detect samples through closed reagent kits and instrument detection systems, reducing systematic errors and eliminating errors introduced by manual operation. It has high detection efficiency and broad application prospects. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] Example 1: A method for preparing a methotrexate detection kit, comprising the following steps:

[0029] Preparation of S1 surface-modified magnetic beads: Carboxylated magnetic beads with a particle size of 1 μm were washed three times with MES buffer; a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added, and the mixture was activated by shaking at 37℃ for 30 min to obtain activated magnetic beads. The concentration of MES buffer was 0.05 M and the pH value was 6; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 0.1 mM; and the concentration of N-hydroxysuccinimide was 0.04 M. Methotrexate standard and methacrylic acid were mixed at a volume ratio of 1:1, and crosslinking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile were added. The mixture was deoxygenated under nitrogen for 15 min to prepare an imprinting solution, wherein the concentration of methotrexate standard was 0.01 M, methacrylic acid was 0.04 M, ethylene glycol dimethacrylate was 0.2 M, and azobisisobutyronitrile was 0.01 M. Activated magnetic beads were mixed with the imprinting solution and thermally polymerized at 70 °C for 4 h to form a polymer coating layer. The polymer was then eluted with a methanol / acetic acid mixture (volume ratio of methanol to acetic acid 9:1) to obtain molecularly imprinted magnetic beads. The molecularly imprinted magnetic beads were dispersed in a copper sulfate and zinc nitrate mixture and chelated at 37 °C with shaking for 2 h. The beads were washed with deionized water until no metal ion residue remained, and bovine serum albumin was added for blocking at 37 °C for 1 h to obtain surface-modified magnetic beads. These beads were stored at 4 °C in PBS buffer containing 0.05% ProClin 300. The concentration of copper sulfate was 0.005 M; the concentration of zinc nitrate was 0.002 M; and the mass fraction of bovine serum albumin was 1%.

[0030] Preparation of S2 R1 reagent: The surface-modified magnetic beads prepared in step S1 were added to a diluent consisting of 0.1% Tween-20, 2% trehalose and 0.02% NaN3, with a pH of 7.4; then methotrexate monoclonal antibody was added, and the mixture was incubated at 4°C for 12 h to prepare R1 reagent with a magnetic bead concentration of 0.05 mg / mL by mass.

[0031] Preparation of S3 R2 reagent: Methotrexate synthetic antigen and alkaline phosphatase were placed in a centrifuge tube at a concentration ratio of 1:4, mixed well, dialyzed with TBS, and then glutaraldehyde was added in proportion. The mixture was reacted for 3 hours and dialyzed with TBS again. Glycerol of equal volume to pentylene glycol was added and mixed. Then, R2 reagent with a protein concentration (excluding mouse IgG) of 0.1-1 mg / mL was prepared by using MOPS buffer with a concentration of 0.05-5 μg / ml mouse IgG, a mass fraction of 0.1%-5% methylisothiazolinone, a mass fraction of 0.05-0.2% Tween-20, a mass fraction of 0.01-0.1% sodium azide, and a pH of 6-8.

[0032] S4 Combination: Combine the R1 reagent obtained in step S2 and the R2 reagent obtained in step S3 at a volume ratio of 1:1 to obtain a methotrexate detection kit.

[0033] Example 2: The particle size of the carboxylated magnetic beads required for the preparation of S1 surface modified magnetic beads was changed to 1.5, and the remaining steps and conditions were the same as in Example 1.

[0034] Example 3: The particle size of the carboxylated magnetic beads required for the preparation of S1 surface modified magnetic beads was changed to 2, and the remaining steps and conditions were the same as in Example 1.

[0035] Example 4: The particle size of the carboxylated magnetic beads required for the preparation of S1 surface modified magnetic beads was changed to 2.5, and the remaining steps and conditions were the same as in Example 1.

[0036] Example 5: The particle size of the carboxylated magnetic beads required for the preparation of S1 surface modified magnetic beads was changed to 3, and the remaining steps and conditions were the same as in Example 1.

[0037] Example 6: A method for preparing a methotrexate detection kit, comprising the following steps:

[0038] Preparation of S1 surface-modified magnetic beads: Carboxylated magnetic beads and hydroxyl magnetic beads with a particle size of 1 μm were washed three times with MES buffer. A mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added, and the mixture was activated at 37°C with shaking for 30 min to obtain activated magnetic beads. The concentration of MES buffer was 0.05 M and the pH was 6; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 0.1 mM; and the concentration of N-hydroxysuccinimide was 0.04 M. The activated magnetic beads were dispersed in a mixture of copper sulfate and zinc nitrate solution and chelated at 37°C with shaking for 2 h. The beads were washed with deionized water until no metal ion residue was found, and bovine serum albumin was added to block the beads at 37°C for 1 h to obtain surface-modified magnetic beads. The beads were stored at 4°C in PBS buffer containing 0.05% ProClin 300. The concentration of copper sulfate was 0.005 M; the concentration of zinc nitrate was 0.002 M; and the mass fraction of bovine serum albumin was 1%.

[0039] The remaining steps and conditions are the same as in Example 1.

[0040] Example 7: A method for preparing a methotrexate detection kit, comprising the following steps:

[0041] Preparation of S1 surface-modified magnetic beads: Carboxylated magnetic beads with a particle size of 1 μm were washed three times with MES buffer; a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added, and the mixture was activated by shaking at 37℃ for 30 min to obtain activated magnetic beads. The concentration of MES buffer was 0.05 M and the pH value was 6; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 0.1 mM; and the concentration of N-hydroxysuccinimide was 0.04 M. Methotrexate standard and methacrylic acid were mixed at a volume ratio of 1:1, and crosslinking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile were added. The mixture was deoxygenated under nitrogen for 15 min to prepare a blot solution, wherein the concentration of methotrexate standard was 0.01 M, methacrylic acid was 0.04 M, ethylene glycol dimethacrylate was 0.2 M, and azobisisobutyronitrile was 0.01 M. Activated magnetic beads were mixed with the blot solution and thermally polymerized at 70 °C for 4 h to form a polymer coating layer. The mixture was eluted with a methanol / acetic acid mixture (volume ratio of methanol to acetic acid 9:1), and 1% bovine serum albumin was added. The mixture was then blocked at 37 °C for 1 h to obtain surface-modified magnetic beads, which were stored at 4 °C in PBS buffer containing 0.05% ProClin 300.

[0042] The remaining steps and conditions are the same as in Example 1.

[0043] The selectivity, sensitivity, stability, and detection limit of the methotrexate detection kits prepared in Examples 1-7 were tested, and the results are shown in the table below:

[0044]

[0045] The data in the table above show that the cross-reactivity rates of Examples 1 to 5 are low and similar, indicating that the magnetic bead size (1–3 μm) has little impact on selectivity. The cross-reactivity rates of Examples 6 and 7 are significantly higher than other examples, indicating that omitting the metal chelation or molecular imprinting steps significantly reduces the selectivity of the kit and increases the risk of false positives. Example 1 has the lowest limit of detection and half-maximum inhibition, indicating the highest sensitivity, capable of detecting lower concentrations of methotrexate. Examples 6 and 7 have lower sensitivity, indicating that metal chelation and molecular imprinting steps are crucial for improving sensitivity. Magnetic bead size has some influence on sensitivity, but the differences between Examples 1 to 5 are not significant, indicating that within the 1–3 μm range, particle size is not decisive for sensitivity. Example 1 exhibits the best stability at 4°C and the lowest percentage performance degradation, making it suitable for long-term storage. Examples 6 and 7 have poor stability, especially at high temperatures, with significant performance degradation, indicating that metal chelation and molecular imprinting steps help improve stability. The effect of magnetic bead size on stability is not significant, and Examples 1 to 5 show similar stability.

[0046] In summary, Example 1 demonstrates the best performance in terms of selectivity, sensitivity, stability, and detection limit, making it the preferred method for preparing a methotrexate detection kit. The metal chelation and molecular imprinting steps are crucial for improving kit performance; omitting these steps significantly reduces the kit's selectivity, sensitivity, and stability. Within the 1–3 μm range, the magnetic bead size has a relatively small impact on kit performance; an appropriate particle size can be selected based on actual needs.

[0047] The kit prepared in Example 1 of this invention was compared with liquid chromatography-tandem mass spectrometry. Blood samples with concentrations ranging from 0.04 μmol / L to 2.0 μmol / L were tested simultaneously, and the deviation between the concentration detected by the methotrexate chemiluminescent immunoassay kit and the liquid chromatography-tandem mass spectrometry method was calculated.

[0048]

[0049]

[0050] The data in the table above shows that the deviation between the data detected by this kit and the results detected by liquid chromatography-tandem mass spectrometry is within ±9%, indicating that this kit can maintain high accuracy and stability at different concentrations.

[0051] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a methotrexate detection kit, characterized in that, Includes the following steps: Preparation of S1 surface-modified magnetic beads: Carboxylated magnetic beads were washed three times with MES buffer; a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added, and the mixture was activated by shaking at 37°C for 30 min to obtain activated magnetic beads; methotrexate standard was mixed with methacrylic acid in a certain proportion, and ethylene glycol dimethacrylate and azobisisobutyronitrile were added as crosslinking agent and nitrogen deoxygenation for 15 min to obtain an imprint solution; the activated magnetic beads were mixed with the imprint solution, and thermal polymerization was initiated at 70°C for 4 h to form a polymer coating layer; the imprinted magnetic beads were obtained by elution with a methanol / acetic acid mixture; the imprinted magnetic beads were dispersed in a copper sulfate and zinc nitrate mixture and chelated by shaking at 37°C for 2 h; the beads were washed with deionized water until no metal ion residue was found, and bovine serum albumin was added to block the beads at 37°C for 1 h to obtain surface-modified magnetic beads, which were then stored at 4°C in PBS buffer containing 0.05% ProClin 300; Preparation of S2 R1 reagent: Add the surface-modified magnetic beads prepared in step S1 to the diluent, then add methotrexate monoclonal antibody, and incubate at 4°C for 12 h to prepare R1 reagent; Preparation of S3 R2 reagent: Methotrexate synthetic antigen and alkaline phosphatase were placed in a centrifuge tube at a concentration ratio of 1:4, mixed well, dialyzed with TBS, glutaraldehyde was added in proportion, and the mixture was reacted for 3 hours. After dialyzed with TBS, glycerol was added and mixed. Then, R2 reagent was prepared with mouse IgG, methylisothiazolinone, Tween-20, sodium azide, and MOPS buffer. S4 combination: The R1 reagent obtained in step S2 and the R2 reagent obtained in step S3 are combined to obtain a methotrexate detection kit.

2. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the S1 surface-modified magnetic bead preparation step, the particle size of the hydroxyl magnetic beads is 1-3 μm.

3. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the S1 surface-modified magnetic bead preparation step, the concentration of the MES buffer is 0.05M and the pH value is 6; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.1mM; and the concentration of N-hydroxysuccinimide is 0.04M.

4. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the S1 surface-modified magnetic bead preparation step, the concentration of the methotrexate standard is 0.01M; the concentration of methacrylic acid is 0.04M; the concentration of ethylene glycol dimethacrylate is 0.2M; and the concentration of azobisisobutyronitrile is 0.01M.

5. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the preparation step of S1 surface-modified magnetic beads, the volume ratio of the methotrexate standard to methacrylic acid is 1:

1.

6. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the preparation step of S1 surface-modified magnetic beads, the volume ratio of methanol to acetic acid in the methanol / acetic acid mixed solution is 9:

1.

7. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the S1 surface-modified magnetic bead preparation step, the concentration of copper sulfate is 0.005M; the concentration of zinc nitrate is 0.002M; and the mass fraction of bovine serum albumin is 1%.

8. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In the S2 R1 reagent preparation step, the diluent is composed of 0.1% Tween-20, 2% trehalose and 0.02% sodium azide, with a pH of 7.

4.

9. The method for preparing a methotrexate detection kit according to claim 1, characterized in that, In step S4, the volume ratio of reagent R1 to reagent R2 is 1:

1.

10. A methotrexate detection kit prepared by the method described in any one of claims 1 to 9.

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