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Cyclosporine A immunological detection reagent as well as preparation and detection methods thereof

A cyclosporine and immunodetection technology, which is applied in biological testing, material inspection products, etc., can solve the problems of low measurement accuracy and complicated operation of cyclosporine A, and achieve high sensitivity, strong specificity, and accurate quantification. Effect

Inactive Publication Date: 2018-03-09
太原瑞盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of complex operation and low measurement accuracy in the detection process of cyclosporine A in the prior art. Cyclosporin A homogeneous enzyme immunoassay reagent and preparation method thereof

Method used

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  • Cyclosporine A immunological detection reagent as well as preparation and detection methods thereof
  • Cyclosporine A immunological detection reagent as well as preparation and detection methods thereof
  • Cyclosporine A immunological detection reagent as well as preparation and detection methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of glucose dehydrogenase-antigen conjugate

[0042] 1) Coupling of glucose dehydrogenase (GDH) and cyclosporine A (CsA)

[0043] a. Accurately weigh 240 mg of cyclosporine A and 50 mg of 4-benzoylbenzoic acid (BBa) into a quartz cuvette, add 10 mL of tert-butanol, and dissolve by ultrasound;

[0044] b. Ultraviolet light irradiation for 3 hours, the reaction is complete, and it is allowed to stand at room temperature to form a freeze-dried powder;

[0045] c. Accurately weigh 4.5 mg GDH dissolved in 0.5 mL 10mM pH 7.4 PBS solution, take 15 mg of the above powder (CsA-BBa) dissolved in 0.7 mL dimethylformamide (DMF) solution, and stir at 25℃ Add CsA-BBa dropwise to the PBS solution with GDH;

[0046] d. Adjust the pH of the solution to keep it at 6.0-8.0, then add 120 μL of 0.5% carbodiimide (EDC) dropwise, and stir overnight at 4°C.

[0047] 2) Purify the coupled enzyme-labeled antigen

[0048] The coupled enzyme-labeled antigen is purified by a G-25 gel chr...

Embodiment 2

[0049] Example 2: Preparation of cyclosporin A homogeneous enzyme immunoassay reagent

[0050] Cyclosporin A homogeneous enzyme immunoassay reagents, including: enzyme-labeled cyclosporine A, indicator reagents for detecting cyclosporin A antibody-enzyme-labeled cyclosporin A complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents and chemiluminescent reagents. Preferably, the indicator reagent is an enzyme reagent, including: an enzyme label conjugate and an enzyme substrate. Wherein, the enzyme-labeled conjugate includes glucose dehydrogenase-antigen conjugate, which can be obtained by chemical synthesis.

[0051] Before using the cyclosporin A homogeneous enzyme immunoassay reagent, in order to avoid the reaction between the enzyme-labeled conjugate in the indicator reagent and the enzyme substrate, the enzyme-labeled conjugate and enzyme substrate are placed separately. Cyclosporin A homogeneous enzyme immunoassay reagent i...

Embodiment 3

[0060] Example 3: Cyclosporine A homogeneous enzyme immunoassay reagent reaction calibration curve.

[0061] 1) Preparation of CsA calibrator: Dissolve commercially available human cyclosporin A recombinant protein in a solution similar to human serum matrix (NaCl 0.9%, BSA 0.2%, NaN 3 0.1%, Tris-HCl pH 7.4), made into different concentrations of calibrators. Taking the cyclosporin A calibrator of Shenzhen New Industry Biomedical Engineering Co., Ltd. as the original standard, the cyclosporin A kit was used to test the calibrators of different concentrations for 10 times, and the average value was obtained to obtain cyclosporin The concentration of A calibrator: 25, 50, 100, 200, 400, 800 ng / mL.

[0062] 2) Biochemical analyzer detection: Take Hitachi 7170 operation as an example: the measurement wavelength is 340nm, and the calibrator solutions (20 μL) of different concentrations are respectively taken, and cyclosporine A R is added 1 Reagents (160 μL), mix well, and add cyclospo...

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Abstract

The invention discloses a cyclosporine A immunological detection reagent as well as preparation and detection methods thereof. The cyclosporine A immunological detection reagent comprises cyclosporineA and an indication reagent for detecting a cyclosporine A antibody-enzyme labeled cyclosporine A compound, wherein enzyme labeled cyclosporine A is prepared by coupling cyclosporine A and glucose dehydrogenase. The cyclosporine A immunological detection reagent disclosed by the invention can be used for accurately and rapidly determining the content of cyclosporine A in samples of human blood and the like. Compared with existing detection reagents in the market, the detection reagent disclosed by the invention has the advantages of convenience and rapidness, high sensitivity, strong specificity, accurate quantification and the like, and clinical popularization and application are facilitated.

Description

Technical field [0001] The invention relates to the field of biological detection, in particular to a cyclosporine A immunoassay reagent and a preparation and detection method thereof. Background technique [0002] Cyclosporin A (Cyclosporin, CsA) is a small cyclic peptide compound containing 11 amino acids. CsA is a powerful immunosuppressant for rejection of allogeneic organ or tissue transplantation commonly used in clinical practice. It has a highly selective inhibitory effect on cell and humoral immunity and is a lymphocyte activation inhibitor. It mainly inhibits T helper lymphocytes, and it also has a strong inhibitory effect on T wounded cells. CsA is increasingly widely used, but it has complex pharmacokinetics and pharmacodynamic characteristics, many side effects and high incidence, individual differences, toxic side effects and rejection reactions are not easy to distinguish, difficult to monitor, and its blood concentration is too high High can cause toxic and side...

Claims

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Application Information

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IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 许殊荣张轩马运乐杨钰祥杜爱铭徐兵
Owner 太原瑞盛生物科技有限公司
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