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A kind of mycophenolic acid homogeneous enzyme immunoassay reagent and its preparation and detection method

A homogeneous enzyme immunoassay and detection reagent technology, applied in chemical instruments and methods, immunoglobulins, measurement devices, etc. specific effect

Active Publication Date: 2016-08-17
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 such as Siemens and Roche Diagnostics have launched mycophenolic acid assay kits that can be applied to biochemical analyzers, they are far from meeting the growing demand for clinical testing
At present, there is still a lack of mycophenolic acid detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic detection reagents

Method used

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  • A kind of mycophenolic acid homogeneous enzyme immunoassay reagent and its preparation and detection method
  • A kind of mycophenolic acid homogeneous enzyme immunoassay reagent and its preparation and detection method
  • A kind of mycophenolic acid homogeneous enzyme immunoassay reagent and its preparation and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Synthesis and structure confirmation of mycophenolic acid derivatives

[0065] The chemical structure of the mycophenolic acid derivative used in the following examples is shown in formula (II):

[0066]

[0067] The synthetic route of this mycophenolic acid derivative is as follows:

[0068]

[0069] Concrete synthetic steps are as follows:

[0070] Synthesis of compound 2

[0071]

[0072] 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 4.9 g of white solid compound 2 with a yield of 91%.

[0073] Synthesis of compound 3

[0074]

[0075] Weigh 4.9g...

Embodiment 2

[0079] Example 2: Synthesis of BSA-mycophenolic acid immunogen

[0080] The BSA-mycophenolic acid immunogen is formed by linking bovine serum albumin (BSA) to the terminal carboxyl group of mycophenolic acid represented by formula (Ⅲ). The specific synthesis steps of the immunogen are as follows:

[0081] 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;

[0082] 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;

[0083] c. Weigh 3mg of mycophenolic acid derivatives and dissolve them in 300ul of the above buffer solution A to make a mycophenolic acid derivative solution;

[0084] d. When the above-mentioned mycophenolic acid derivative solution just becomes clear, add it dropwise to the above-mentioned BSA solution, and then...

Embodiment 3

[0086] Example 3: Preparation of anti-mycophenolic acid specific antibody

[0087] The BSA-mycophenolic acid immunogen prepared above was used to vaccinate experimental animal rabbits by conventional methods, and antiserum was collected after booster immunization. The specific steps were as follows:

[0088] Dilute the synthesized BSA-mycophenolic acid 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.

[0089] 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.

[0090] Blood was collected from the above-mentioned experimental animal rabbits, separated and purified to obtain anti-mycophenolic acid specific antibodies with a titer of 1:30000-1:50000.

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Abstract

The invention relates to a mycophenolic acid detection reagent as well as preparation and detection methods thereof and particularly relates to a mycophenolic acid homogeneous phase enzyme immunological detection reagent as well as preparation and detection methods thereof. The mycophenolic acid homogeneous phase enzyme immunological detection reagent comprises an anti-mycophenolic acid specific antibody, and an indication reagent for detecting an anti-mycophenolic acid specific antibody-mycophenolic acid compound; and the anti-mycophenolic acid specific antibody is prepared from mycophenolic acid immunogen immunological animals. The mycophenolic acid detection reagent has the beneficial effects that mycophenolic acid immunogen has strong specificity and high immunogenicity; the prepared anti-mycophenolic acid specific antibody has strong specificity and high valence and has no crossed reaction with common 62 types of medicines; the homogeneous phase enzyme immunological detection reagent containing the anti-mycophenolic acid specific antibody can be used for conveniently, rapidly and accurately determining the content of mycophenolic acid in a sample, and determining a plurality of samples on a full-automatic biochemical analyzer at the same time, so that the high-flux rapid determination for the mycophenolic acid is realized; and the accuracy is high, the specificity is strong, and the accuracy and the detection efficiency can be relatively greatly improved.

Description

technical field [0001] The invention relates to a mycophenolic acid detection reagent and its preparation and detection method, in particular to a mycophenolic acid homogeneous enzyme immunological detection reagent and its preparation and detection method. Background technique [0002] The structural formula of mycophenolic acid is shown in formula (Ⅲ): [0003] [0004] As an immunosuppressant, mycophenolic acid is widely used clinically to inhibit the rejection of kidney, liver, heart and other organ transplants. Mycophenolic acid can non-competitively bind to hypoxanthine mononucleotide dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides during the proliferation of T and B lymphocytes. Its clinical effective blood drug concentration is narrow (clinical effective blood drug concentration is 1.0-3.5μg / mL), lower than its effective blood drug concentration can not play an immunosuppressive effect, and can not reach the correspond...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/02G01N33/544C07K16/44
CPCC07K16/44G01N33/544
Inventor 虞留明伍勇邓拥军封飒飒成志鹏
Owner 苏州博源医疗科技有限公司
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