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Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof

A homogeneous enzyme immunoassay and methotrexate technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem that the number of products cannot meet clinical needs, and achieve high-throughput rapid determination, high potency, and accuracy The effect of increasing

Active Publication Date: 2015-04-29
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although foreign manufacturers have launched methotrexate assay kits that can be applied to biochemical analyzers, the number of products is 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 homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof
  • Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof
  • Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Synthesis and structure confirmation of methotrexate derivatives

[0066] The chemical structure of the methotrexate derivative used in the following examples is shown in formula (II):

[0067]

[0068] The synthetic route of this methotrexate derivative is as follows:

[0069]

[0070] Concrete synthetic steps are as follows:

[0071] Synthesis of compound 2

[0072]

[0073] Weigh 5.0 g of compound 1, dissolve it in 50 mL of MeOH, and add 4.0 g (34.1 mmol) of SOCl dropwise at 0°C 2 , then the reaction mixture was stirred overnight at 70 °C, the solvent was evaporated by reduced pressure, and the residue was passed through 200 mL of DCM and 200 mL of NaHCO 3 Saturated aqueous solution was separated, the organic phase was washed with brine, and then passed through Na 2 SO 4 It was dried and concentrated to obtain 5.1 g of white solid compound 2 with a yield of 96%.

[0074] Synthesis of Compound 3

[0075]

[0076] Weigh 5.1g of compound 2,...

Embodiment 2

[0080] Example 2: Synthesis of BSA-methotrexate immunogen

[0081] The BSA-methotrexate immunogen is formed by linking bovine serum albumin (BSA) to the terminal carboxyl group of methotrexate shown in formula (Ⅲ), and the specific synthesis steps of the immunogen are as follows:

[0082] a. Weigh 2.72g of potassium dihydrogen phosphate, 4.26g of disodium hydrogen phosphate, 8.5g of sodium chloride, and 0.95g of magnesium chloride, dissolve them in 1L of deionized water, adjust the pH to 7.4, and make buffer solution A;

[0083] b. Weigh 3 mg of BSA and dissolve it in 3 mL of the above buffer solution A at room temperature to make a BSA solution;

[0084] c. Weigh 3mg of methotrexate derivatives and dissolve in 300ul of the above buffer solution A to make methotrexate derivatives solution;

[0085] d. When the above-mentioned methotrexate derivative solution just became clear, it was added dropwise to the above-mentioned BSA solution, and then the mixed solution was stirred a...

Embodiment 3

[0087] Example 3: Preparation of Anti-Methotrexate Specific Antibody

[0088] The BSA-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:

[0089] The above synthesized BSA-methotrexate immunogen was diluted to 1.0 mg / ml with PBS to obtain an antigen solution, and then 1.0 ml of the antigen solution was mixed with Freund's complete adjuvant, and then injected into experimental animal rabbits.

[0090] After 2 to 3 weeks, the rabbits were injected once with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant, and then injected once every four weeks, for a total of 4 injections.

[0091] Blood was collected from the above-mentioned experimental animal rabbit, separated and purified to obtain anti-methotrexate specific antibody with a titer of 1:30000-1:50000.

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Abstract

The invention relates to a methotrexate detection reagent as well as a preparation method and a detection method thereof, and specifically relates to a methotrexate homogenous enzyme immunoassay reagent as well as a preparation method and a detection method thereof. The methotrexate homogenous enzyme immunoassay reagent comprises an anti-methotrexate specific antibody, and an indication reagent for detecting an anti-methotrexate specific antibody-methotrexate compound, wherein the anti-methotrexate specific antibody is obtained from immune animals with methotrexate immunogen. The methotrexate homogenous enzyme immunoassay reagent disclosed by the invention has the following beneficial effects: the methotrexate immunogen is high in specificity and immunogenicity, and the prepared anti-methotrexate specific antibody is high in specificity and valence, and free from any cross reaction with 62 common medicines; the homogenous enzyme immunoassay reagent containing the anti-methotrexate specific antibody is capable of conveniently, rapidly and accurately determining the content of methotrexate in a sample and measuring a plurality of samples on a fully-automatic biochemical analyser to realize high-flux rapid measurement for methotrexate, is high in accuracy and high in specificity, and is capable of greatly improving the accuracy and the detection efficiency.

Description

technical field [0001] The invention relates to a methotrexate detection reagent and its preparation and detection method, in particular to a methotrexate homogeneous enzyme immunological detection reagent and its preparation and detection method. Background technique [0002] Methotrexate (Methotrexate) structural formula is shown in formula (Ⅲ): [0003] [0004] Methotrexate is a folate reductase inhibitor, which is an anti-folate antineoplastic drug. It mainly hinders the synthesis of tumor cell DNA by inhibiting dihydrofolate reductase, thereby inhibiting the growth and reproduction of tumor cells. It acts selectively in S phase and belongs to cell cycle specific drugs. Clinically, it is effective in the treatment of acute leukemia (especially acute lymphoblastic leukemia), choriocarcinoma and malignant mole. It has a certain effect on head and neck tumors, breast cancer, lung cancer and pelvic cavity tumors, and can also be used in combination with other drugs to ...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/544
CPCG01N33/531G01N33/544
Inventor 虞留明卢忠心胡瑜娄金丽成志鹏
Owner 苏州博源医疗科技有限公司
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