Methotrexate latex immunoturbidimetry kit and preparation method thereof

A technology of methotrexate and latex immunization, which is applied in the direction of measuring devices, material analysis by observing the influence on chemical indicators, instruments, etc., can solve problems that cannot meet clinical needs, and achieve good clinical application prospects and large applications value, easy preparation

Inactive Publication Date: 2018-10-02
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are methotrexate assay kits that can be applied to biochemical analyzers on the market, they are far from meeting clinical needs
At present, there is still a lack of methotrexate detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automated test reagents

Method used

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  • Methotrexate latex immunoturbidimetry kit and preparation method thereof
  • Methotrexate latex immunoturbidimetry kit and preparation method thereof
  • Methotrexate latex immunoturbidimetry kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Synthesis of KLH-Methotrexate Immunogen

[0056] KLH-methotrexate immunogen is the antigen used in the preparation process of the antibody raw material in reagent R2. It is formed by linking hemocyanin (KLH) with the terminal carboxyl group of methotrexate. The specific synthesis steps of the immunogen are as follows :

[0057] (1) Weigh 2.13g of 2-(N-morpholine)ethanesulfonic acid, 8.5g of sodium chloride, and 0.95g of magnesium chloride, dissolve them in 1L of deionized water, adjust the pH to 6.1, and prepare buffer solution A;

[0058] (2) Weigh 3mg of KLH and dissolve it in 3mL of the above-mentioned buffer solution A at room temperature to make a KLH solution;

[0059] (3) Weigh 3 mg of methotrexate, dissolve it in 300 ul of the above-mentioned buffer solution A, and make a methotrexate solution;

[0060] (4) Add 5 mg NHS and 1 mg EDC·HCl to the methotrexate solution, react for 30 minutes, add the above KLH solution dropwise into the methotrexate solution, and t...

Embodiment 2

[0063] Preparation of Anti-Methotrexate Specific Antibody

[0064] Anti-methotrexate specific antibody is the specific antibody used in reagent R2. The KLH-methotrexate immunogen prepared above was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0065] (1) Dilute the KLH-methotrexate immunogen synthesized above to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with Freund's complete adjuvant, and inject the experimental animal rabbit.

[0066] (2) After 3 weeks, inject once more the same antigen solution and Freund's incomplete adjuvant with 1.0ml of the same antigen solution and Freund's incomplete adjuvant, and then inject once every four weeks, a total of 4 injections.

[0067] (3) Blood was collected from the above experimental animal rabbit, separated and purified to obtain anti-methotrexate specific antibody with a...

Embodiment 3

[0069] Preparation of methotrexate conjugates

[0070] (1) Weigh 2.13g of 2-(N-morpholine)ethanesulfonic acid and 8.5g of sodium chloride, dissolve them in 1L of deionized water, adjust the pH to 6.1, and make buffer solution A

[0071] (2) Dissolve 1.0 g of methotrexate (MTX) in buffer solution A, add 50 mg of N-hydroxysuccinimide (NHS) and 30 mg of coupling agent 1-(3-dimethylpropyl)-3-ethane Carbodiimide hydrochloride (EDC·HCl), reacted for 30 minutes to form MTX-NHS activated middle fat;

[0072] (2) add 50mg chitosan in above-mentioned solution, react at room temperature for 2 hours, methotrexate and chitosan condensation reaction form the conjugate of methotrexate-chitosan;

[0073] (3) add 0.5ml ethanolamine to inactivate the middle liposome;

[0074] (4) The reaction solution is dialyzed in the dialysis bag, in the phosphate buffered saline solution of 20mmol / L, 2-8 ℃ of dialyzing 12 hours, freeze-drying, obtain the pure product of methotrexate-chitosan conjugate, na...

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Abstract

The invention provides a methotrexate latex immunoturbidimetry kit and a preparation method thereof. The kit is used for determining the content of plasma drug methotrexate in human serum, and is composed of a reagent R1 and a reagent R2 in a ratio of 1 to 1. R1 is prepared from the following components by weight percentage: 0.01% to 4% of an emulsifier, 0.01% to 4% of a methotrexate conjugate, 0.1% to 2% of sodium chloride, 0.05% to 2% of a sensitizer, 0.02% of a preservative, and a 30 to 100 mmol / L buffer. R2 is prepared from the following components by weight percentage: 0.1% to 0.5% of latex particles combined with an anti-methotrexate antibody, 0.1% to 1.0% of bovine serum albumin, 0.02% to 2% of Tween 20, 0.02 % to 2% of a stabilizer 2, 0.02% to 2% of a protein protectant 2, 0.02% ofpreservative, and a 20 to 50 mmol / L buffer. The kit provided by the invention has high sensitivity and stability, and has a good clinical application prospect, and the invention provides the preparation method.

Description

technical field [0001] The invention relates to a biological reagent, in particular to a detection kit, in particular to a high-sensitivity and specific latex immunoturbidimetric method for measuring the content of methotrexate in human serum and a preparation method thereof. Background technique [0002] Methotrexate (Methotrexate, MTX) is a folate reductase inhibitor, which is an anti-folate antineoplastic drug. It mainly inhibits the synthesis of tumor cell DNA by inhibiting dihydrofolate reductase, thereby inhibiting the growth of tumor cells. and reproduce. Its anti-tumor mechanism is: methotrexate is similar in structure to folic acid, both of which belong to pteridine derivatives, so MTX can bind to the active site of dihydrofolate reductase to block the formation of tetrahydrofolate. It acts selectively on the S phase (DNA synthesis phase) and belongs to cell cycle specific drugs. Clinically, it has a good therapeutic effect on acute leukemia (especially acute lymp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/82
CPCG01N21/82G01N33/54313
Inventor 张龙平张跃建孙卫兵
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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