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Immunodetection method of 25-hydroxyvitamin D and application of immunodetection method

An immunoassay method and hydroxyvitamin technology, which are applied in the field of immunoassays, can solve the problems of low detection sensitivity, high difficulty of hapten-conjugated markers, and great influence on product development.

Active Publication Date: 2021-09-14
深圳健丰源科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Haptens are usually small molecular substances. Due to the small molecular weight of haptens, the coating and labeling processes of macromolecules such as antigens and antibodies in immune diagnostic reagents are quite different, so they have a greater impact on the development of related products, such as layer The product hapten of the analysis method cannot be directly immobilized on the nitrocellulose membrane, and the difficulty of coupling the hapten to the label is relatively high, etc.
[0003] In the prior art, chemical methods are often used to couple haptens to macromolecular proteins (such as BSA), and then use them as raw materials for IVD reagents, such as immobilizing VD-BSA on NC membranes, but the detection sensitivity is low and the linear range is narrow , sample matrix effects and other issues

Method used

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  • Immunodetection method of 25-hydroxyvitamin D and application of immunodetection method
  • Immunodetection method of 25-hydroxyvitamin D and application of immunodetection method
  • Immunodetection method of 25-hydroxyvitamin D and application of immunodetection method

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Experimental program
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Effect test

Embodiment 1

[0048] Preparation of 25-hydroxyvitamin D and macromolecular protein conjugates with two binding sites;

[0049] 1. Preparation of C3-position conjugates of 25-hydroxyvitamin D3 (purchased from Toronto Research Chemicals) and macromolecular protein (bovine serum albumin, purchased from Thermo);

[0050] Dissolve 1mg of 25-hydroxyvitamin D3 in an organic solvent (DMSO), add 10mg of succinic anhydride, under the protection of a suitable inert gas, react in the dark at room temperature for 12 hours, add the mixture to 20ml of ethyl acetate, and then Wash with ultrapure water, hydrochloric acid of appropriate concentration, ultrapure water successively, filter and vacuum dry to obtain 1mg 25-hydroxyvitamin D3 C3-hemisuccinate, dissolve in 1ml dichloromethane, add 0.2mg NHS and 0.4mg EDC , stirred at room temperature in the dark for 12 hours, filtered and vacuum-dried to obtain the activated ester of 25-hydroxyvitamin D3 C3-hemisuccinate, dissolved in 0.5ml organic solvent (DMSO) f...

Embodiment 2

[0054] ELISA indirect method to verify the activity of 25-hydroxyvitamin D and macromolecular protein conjugates with two binding sites;

[0055] 1. Dilute the above conjugate with coating solution (50mM / L carbonate buffer pH9.6) to 0.2-1μg / mL and add to 96-well microtiter plate (purchased from Xiamen Jincanhua), 100μL / well, 4 React overnight at ℃, wash the plate 3 times with washing solution (PBST); add 200 μL of blocking solution (1% bovine serum albumin in 10 mM / L pH7.4 phosphate buffer) to each well, incubate at 37 °C for 0.5 hours, wash with washing solution Wash the plate 3 times and dry it;

[0056] 2. Add an appropriate concentration of anti-25-hydroxyvitamin D antibody (the antibody comes from a number of IVD raw material companies) (diluted with blocking solution) 100 μL / well, incubate at 37°C for 0.5 hours, and wash the plate 3 times with washing solution;

[0057] 3. Add 100 μL / well of enzyme-labeled secondary antibody (purchased from Thermo, diluted with blocking...

Embodiment 3

[0062] Fluorescence lateral flow immunoassay method to screen antibody pairing raw materials for 25-hydroxyvitamin D detection kit;

[0063] Prepare the following components using conventional methods:

[0064] 1. Label the anti-25-hydroxyvitamin D 3 (C3) antibody and the anti-25-hydroxyvitamin D 22 (C22) antibody with fluorescein, and prepare the corresponding conjugate pads; select the following method as an example: Adjust the antibody concentration to 10 mg / ml with 0.02mol / LPBS (pH7.4); take FITC (purchased from Sigma Company) equivalent to 1 / 50 of the protein amount, and dissolve it in 0.2mol / L carbon dioxide equivalent to 1 / 10 of the volume of the protein solution. FITC was quickly added to the antibody under stirring conditions, and the reaction was stirred for 1 hour. After the labeling was completed, the fluorescein-labeled antibody was purified by dialysis, subpackaged and freeze-dried, and stored at -20°C for future use.

[0065] 2. Coating anti-25-hydroxyvitamin D ...

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Abstract

The invention discloses an immunodetection method of 25-hydroxyvitamin D. The immunodetection method comprises the following steps of pairing a No.3 antibody of anti-25-hydroxyvitamin D and a No.22 antibody of anti-25-hydroxyvitamin D, and respectively capturing and detecting a conjugate of 25-hydroxyvitamin D-macromolecular protein to form a double-antibody sandwich compound; the application comprises a lateral chromatography platform, an enzyme-linked immunosorbent assay (ELISA) platform, a chemiluminescence platform, a microfluidic platform and an immunoturbidimetric platform. The invention discloses an immunodetection method of 25-hydroxyvitamin D and an application thereof and relates to the field of immunodetection methods. The immunodetection method is advantaged in that project development difficulty is reduced, development cost is saved, development efficiency is improved, and the immunodetection method can be combined with 25-hydroxyvitamin D in a sample, so reagent performance is improved, the amount of effective competitive antibodies is much larger than that of conventional methods, so both sensitivity and reactivity can be considered, and the linear range can also be increased.

Description

technical field [0001] The invention relates to the field of immunoassay methods, in particular to an immunoassay method for 25-hydroxyvitamin D and its application. Background technique [0002] Vitamin D is a fat-soluble vitamin whose main form in the human body is vitamin D2 (ergocalciferol) ( figure 1 ) and vitamin D3 ( figure 2 ) (cholecalciferol). Food sources and self-synthesized vitamin D2 and vitamin D3 are converted into 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 respectively under the action of 25-hydroxylase, collectively referred to as 25-hydroxyvitamin D. As an active metabolite of vitamin D, 25-hydroxyvitamin D is often detected to reflect the metabolic level of vitamin D in the human body. It has important clinical significance in monitoring the signs of children and elderly metabolic diseases, menopausal women's diseases, and severe patients. At present, the determination methods of 25-hydroxyvitamin D mainly include colloidal gold chromatography, flu...

Claims

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Application Information

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IPC IPC(8): G01N33/82G01N33/577G01N33/558G01N33/543
CPCG01N33/82G01N33/577G01N33/558G01N33/54393
Inventor 叶绿盛
Owner 深圳健丰源科技有限公司
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