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Beta-receptor blocker homogeneous enzyme immunoassay reagent and preparation and detection methods thereof

A technology of receptor blocker and homogeneous enzyme immunity, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inapplicability to the rapid detection of large quantities of clinical samples, etc., and achieve easy implementation and popularization and use, and high potency , High immunogenic effect

Active Publication Date: 2015-04-22
AISIJIEKE GLOBAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The analysis and determination of β-receptor blockers are mostly carried out for the prototype drugs and metabolites in biological samples. At present, the commonly used determination methods at home and abroad are: gas-liquid chromatography, high performance liquid chromatography, reversed-phase liquid Chromatography, gas chromatography, mass spectrometry, gas chromatography, liquid phase tandem mass spectrometry, fluorescence spectrophotometry, thin layer chromatography, capillary electrophoresis, carbon paste electrode method, radioimmunoassay, etc., these methods have their own advantages and disadvantages. characteristics and advantages and disadvantages, but they are not suitable for the rapid detection of a large number of clinical samples, and the commonly used detection methods can only be determined for a single β-receptor blocker
At present, there is no β-receptor blocker detection reagent on the market that can simultaneously measure a variety of common β-receptor blockers, and has good stability, high sensitivity, and strong specificity, especially a good quality automated one. Detection reagents, therefore, the research and development and production quality meet clinical requirements, strong practicability, and high cost performance. The β-receptor blocker assay reagents that can be applied to automatic biochemical analyzers have become a hot spot in the in vitro diagnostic reagent industry at home and abroad.

Method used

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  • Beta-receptor blocker homogeneous enzyme immunoassay reagent and preparation and detection methods thereof
  • Beta-receptor blocker homogeneous enzyme immunoassay reagent and preparation and detection methods thereof
  • Beta-receptor blocker homogeneous enzyme immunoassay reagent and preparation and detection methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Synthesis and structure confirmation of pindolol derivatives

[0078] The chemical structure of the pindolol derivatives used in the following examples is shown in formula (V):

[0079]

[0080] The synthetic route of this pindolol derivative is as follows:

[0081]

[0082] Concrete synthetic steps are as follows:

[0083] Synthesis of compound 2

[0084]

[0085] 1) Weigh 20.0g (150mmol) of compound 1 and 15.3g (225mmol) of imidazole, dissolve them together in 200mL of dimethylformamide (DMF), weigh 27.0g (180mmol) of tert-butyldimethylsilyl chloride (TBSCl) was dissolved in 200 mL of DMF, and the TBSCl solution was added dropwise to the mixed solution of compound 1 and imidazole at 0° C., and the resulting mixed solution was stirred overnight at room temperature. Thin layer chromatography (TLC) detection showed that the above mixed solution had reacted completely. The reaction synthesis solution was quenched with 200 mL of water to terminate t...

Embodiment 2

[0104] Example 2: Synthesis of BSA-Pindolol Immunogen

[0105] The BSA-pindolol immunogen consists of bovine serum albumin (BSA) and the -(CH 2 ) n The -COO- group is connected. In this embodiment, the synthesis method of the immunogen is described in detail by taking n=3 as an example. The specific steps are as follows:

[0106] (1) 20mg of BSA was dissolved in 5ml of 0.2M, pH8.5 phosphate buffer solution (Phosphate buffer solution, PBS), and the above solution was placed in beaker A;

[0107] (2) Add the following chemicals into beaker B and stir to dissolve: 20mg pindolol derivatives, 0.35ml dimethylformamide (dimethylformamide, DMF), 0.35ml ethanol, 0.7ml 10mM potassium phosphate buffer solution at pH 5.0 . 40mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxysulfosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), stirred and dissolved at room temperature, reacted 30 minutes;

[0108] (3) Add the solution in beaker B to beaker A dropwise to obtain a mix...

Embodiment 3

[0111] Example 3: Preparation of anti-β-receptor blocker-specific antibody

[0112] The BSA-pindolol 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:

[0113] Dilute the above synthesized BSA-pindolol immunogen 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.

[0114] 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.

[0115] Blood was collected from the above-mentioned experimental animal rabbits, separated and purified to obtain anti-β-receptor blocker specific antibodies with a titer of 1:30000-1:50000.

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Abstract

The invention relates to a beta-receptor blocker detection reagent and preparation and detection methods thereof, and specifically relates to a beta-receptor blocker homogeneous enzyme immunoassay reagent and preparation and detection methods thereof. The beta-receptor blocker homogeneous enzyme immunoassay reagent comprises an anti-beta-receptor blocker specific antibody, and an indication reagent for detecting an anti-beta-receptor blocker specific antibody-beta-receptor blocker compound; the anti-beta-receptor blocker specific antibody is obtained from a pindolol immunogen immune animal. The beta-receptor blocker detection reagent has the following beneficial effects: the pindolol immunogen is strong in specificity and high in immunogenicity, the prepared antibody is the anti-beta-receptor blocker specific antibody which can be specially bonded with various common beta-receptor blockers such as pindolol, bisoprolol, atenolol, metoprolol and propranolol, is strong in specificity and high in titer, and has no cross reaction with common 62 drugs; the homogeneous enzyme immunoassay reagent containing the anti-beta-receptor blocker specific antibody is capable of determining the content of various beta-receptor blockers in a sample conveniently, quickly and accurately; besides, a plurality of samples can be measured simultaneously on a full-automatic biochemical analyzer, and high-flux fast measurement of the beta-receptor blockers can be realized; the homogeneous enzyme immunoassay reagent is high in accuracy and strong in specificity, and are greatly improved in accuracy and detection efficiency in contrast with other methods.

Description

technical field [0001] The invention relates to a beta-receptor blocker detection reagent and its preparation and detection method, in particular to a beta-receptor blocker homogeneous enzyme immunoassay reagent and its preparation and detection method. Background technique [0002] β-receptor blockers (Beta Blockers) are called β-adrenergic receptor blockers, mainly by blocking the combination of β-receptors (β1-receptors or β2-receptors) and catecholamines, Thus, it can regulate the cardiovascular system, metabolic system, and bronchial smooth muscle. The emergence of β-receptor blockers is a major advance in modern pharmacology. In 1962, the first β-receptor blocker propranolol (propranolol) came out and was used in clinical practice. So far, thousands of β-receptor blockers have been synthesized in the world, and dozens of them have been approved for the treatment of human diseases and have been produced and marketed. At present, the most commonly used β-receptor bloc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/566
CPCG01N33/566
Inventor 虞留明
Owner AISIJIEKE GLOBAL
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