Chemiluminiscence immuno-assay kit for 25-hydroxyl vitamin D and preparation method thereof

A chemiluminescence immunity, hydroxyvitamin technology, applied in chemiluminescence/bioluminescence, biological testing, analysis by chemical reaction of materials, etc. Affect the accuracy of test results and other issues

Inactive Publication Date: 2016-12-07
SHENZHEN YHLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] (1) Use 12×8 type, 6×8 type, 8×12 type or full-plate type 96-well special microwell plate as antigen coating equipment and reaction container, which can only be divided into 12 batches and 6 batches when used , 8 batches or the whole board can be used at one time, and independent and single-person testing cannot be carried out;
[0011] (2) There are many kinds of reagents used in quantitative determination, and each detection reagent must be contained in a reagent bottle, and each time a reagent is used, the suction nozzle needs to be replaced to fill the microwells of the microwell plate respectively , not only there are many types of reagent bottles, but also the operation of filling reagents is extremely cumbersome;
[0012] (3) There is a lack of corresponding labeling of the testing information, and the production batch number and expiration date information of the testing reagent can only be known or known by checking the label on the outer packaging box of the kit, and the known information is not controlled during the testing process, which has great potential large randomness;
[0013] (4) The detection reagent is in an open space during the detection process, which may easily cause cross-contamination between various reagents and affect the accuracy of the detection result;
[0014] (5) Manual operation is mostly used in the detection process, the addition of reagen

Method used

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  • Chemiluminiscence immuno-assay kit for 25-hydroxyl vitamin D and preparation method thereof
  • Chemiluminiscence immuno-assay kit for 25-hydroxyl vitamin D and preparation method thereof
  • Chemiluminiscence immuno-assay kit for 25-hydroxyl vitamin D and preparation method thereof

Examples

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preparation example Construction

[0056] Such as figure 1 The preparation method of the above-mentioned 25-hydroxyvitamin D chemiluminescent immunoassay kit shown comprises the following steps:

[0057] Take the suspension of carboxylated magnetic particles, remove the supernatant by magnetic separation, resuspend with MES buffer, then add EDC aqueous solution to activate the carboxyl groups on the surface of carboxylated magnetic particles, then add 25-hydroxyvitamin D monoclonal antibody, room temperature Suspend for 2h-10h, remove the supernatant by magnetic separation, and then resuspend with Tris buffer to obtain carboxylated magnetic particles coated with 25-hydroxyvitamin D monoclonal antibody.

[0058] The MES (2-(N-morpholine)ethanesulfonic acid) buffer had a concentration of 0.02M and a pH of 5.5.

[0059] The Tris buffer has a concentration of 0.1 M and contains 2% BSA, pH 8.0.

[0060] The concentration of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) aqueous solution is 10mg / mL~20mg / mL, a...

Embodiment 1

[0076] Example 1: Preparation of 25-hydroxyvitamin D chemiluminescent immunoassay kit

[0077] (1) Preparation of carboxylated magnetic particles coated with 25-hydroxyvitamin D monoclonal antibody:

[0078] Take carboxylated magnetic particles (MagnaBind TM , Cat. No. 21353) suspension, magnetically separated to remove the supernatant, resuspended with 0.02 M, pH 5.5 MES buffer, added 1 mL of newly prepared 10 mg / mL EDC aqueous solution to activate the carboxyl groups on the surface of the magnetic beads, and added 4 mg of 25-hydroxyvitamin D Monoclonal antibody (biorbyt, Cat. No. orb48780), suspended at room temperature for 6 h, magnetically separated, removed supernatant, resuspended to 1 mg / mL with 0.1 M Tris buffer solution containing 2% BSA, pH 8.0, to obtain 25-hydroxy Carboxylated magnetic particles coated with vitamin D monoclonal antibody, 5mL per bottle and stored at 4°C for future use.

[0079] (2) Preparation of vitamin D derivative-labeled acridinium esters:

...

Embodiment 2

[0083] Example 2: 25-hydroxyvitamin D chemiluminescence immunoassay method

[0084] The automatic chemiluminescence immunoassay analyzer (YHLO, catalog number iFlash3000) was used as the detection tool, and the methodological model was the double-antibody sandwich method, that is, the instrument added 50 μL of sample and 50 μL of 25-hydroxyvitamin D monoclonal antibody-coated carboxylated The magnetic particles and 50 μL of 25-hydroxyvitamin D treatment solution were reacted for 20 minutes, and then 50 μL of 25-hydroxyvitamin D-coated acridinium ester was added. After 20 minutes of reaction, magnetic separation was carried out, and the instrument sent the reaction mixture into the dark room. Add the luminescent substrate A solution (H 2 o 2 ) and liquid B (NaOH) for a luminescence reaction, and finally record the luminescence value.

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Abstract

The invention discloses a chemiluminiscence immuno-assay kit for 25-hydroxyl vitamin D and a preparation method thereof. The chemiluminiscence immuno-assay kit herein includes: carboxylated magnetic micro-particles that are coated by a 25-hydroxyl vitamin D monoclonal antibody and a chemiluminiscence marker that is marked by a vitamin D derivative. The chemiluminiscence immuno-assay kit, with a full-auto chemiluminiscence immuno-analysis instrument as a detection tool, completes the detection on the 25-hydroxyl vitamin D. A test proves that the kit reaches 0.2 ng/ml in detection sensitivity, which is improved by at least 10 times than that of a conventional detection method of the 25-hydroxyl vitamin D. The chemiluminiscence immuno-assay kit has high detection precision.

Description

technical field [0001] The invention relates to the field of in vitro detection, in particular to a 25-hydroxyvitamin D chemiluminescent immunoassay kit and a preparation method thereof. Background technique [0002] Vitamin D (vitamin D) is a sterol derivative with anti-rickets effect, also known as anti-rickets vitamin. Can be divided into vitamin D2 and vitamin D3. Vitamin D2 is mostly contained in plant foods. It is synthesized by plant ergosterol through sunlight. Vitamin D3 can be synthesized by human skin and fat tissue from 7-dehydrocholesterol through sunlight. Vitamin D2 is a fat-soluble vitamin. Vitamin D from food is absorbed through the small intestine together with fat, forms chylomicrons with the assistance of bile, enters the blood from lymphatic vessels, and is transported to the liver together with self-synthesized vitamin D3. In the liver, 25-hydroxyvitamin D is formed through the action of the monooxygenase system (25-hydroxylase) in the hepatocyte micr...

Claims

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Application Information

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IPC IPC(8): G01N33/82G01N33/543G01N33/532G01N21/76
CPCG01N21/76G01N33/532G01N33/543G01N33/82G01N33/54326
Inventor 刘冬舟代洪飞夏福臻钱纯亘王刚
Owner SHENZHEN YHLO BIOTECH
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