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Magnetic-particle chemiluminescent detection kit for 25-hydroxyvitamin D

A chemiluminescence detection and hydroxyvitamin technology, applied in the field of immunoassay, can solve the problems of instrument operation, high personnel quality requirements, inability to accurately quantify low-concentration samples, and high cost of instrument purchase and use. Accuracy, good capture effect

Active Publication Date: 2018-08-03
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Liquid-mass detection has high sensitivity and accuracy, but its disadvantages are: 1. The sample needs to be pre-treated to dissociate 25-hydroxyvitamin D from the binding protein; 2. Only one sample can be detected at a time, and the detection throughput is low; 3. .Liquid-mass analyzer is a precision analytical instrument, the cost of purchasing and using the instrument is high, and the requirements for the operation of the instrument and the quality of personnel are high; 4. The state and maintenance of the instrument have a great impact on the test results, and there are relatively large differences in parallel testing between laboratories. Big
However, the use of immunochromatography and plate immunoassay has lower cost and mature instruments, but the disadvantages are: 1. The plate spotting operation is complicated, and the requirements for the operator to add samples are high; 2. It takes a long time; 3. The repeatability and accuracy are poor; 4. Limited by the reaction surface area, the analytical sensitivity is low, and it is impossible to accurately quantify low-concentration samples

Method used

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  • Magnetic-particle chemiluminescent detection kit for 25-hydroxyvitamin D
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  • Magnetic-particle chemiluminescent detection kit for 25-hydroxyvitamin D

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 125

[0042] Example 1 Composition of 25-Hydroxyvitamin D Magnetic Particle Chemiluminescence Detection Kit

[0043] In this embodiment, the 25-hydroxyvitamin D magnetic particle chemiluminescent detection kit includes R1 reagent, R2 reagent, R3 reagent, magnetic separation reagent, calibrator solution series and quality control solution series; wherein,

[0044] In this embodiment, the R1 reagent is sodium hydroxide with a molar concentration of 0.2-0.5M; preferably, the R1 reagent is sodium hydroxide with a molar concentration of 0.5M.

[0045] In this embodiment, the R2 reagent includes alkaline phosphatase-labeled anti-25 hydroxyvitamin D monoclonal antibody, bovine serum albumin, animal serum, preservatives and buffer; alkaline phosphatase-labeled anti-25 hydroxyvitamin D monoclonal antibody The antibody concentration is 0.5-2.0 μg / mL; preferably, the concentration of alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody is 1.0 μg / mL; the mass percentage con...

Embodiment 225

[0068] Example 2 Determination method of 25-hydroxyvitamin D magnetic particle chemiluminescence detection kit and standard curve drawing

[0069] Take the kit out of the storage condition and equilibrate to room temperature before using it for sample detection; mix thoroughly before the actual use of magnetic separation to ensure that the magnetic particles are evenly suspended, and do not use a magnetic stirrer for stirring; prepare the test tube and prepare it according to actual needs. mark.

[0070] Step 1. Take 2.0mL of venous blood into a glass test tube without adding anticoagulant, let it stand at room temperature, then centrifuge at 3000rpm for 5min, and take the supernatant;

[0071] Step 2. Take 10-50 μL of calibrator solution series, quality control solution series and collected serum samples into corresponding test tubes; the pipette head needs to be replaced before each sampling to avoid cross-contamination;

[0072] Step 3. Add 10-50 μL of R1 reagent to each t...

Embodiment 325

[0082] Example 3 Methodological Verification of 25-Hydroxyvitamin D Magnetic Particle Chemiluminescence Detection Kit

[0083] The test kit in Example 1 is tested according to the conventional manufacturing and testing procedures in the art, and the results are as follows:

[0084] 1. Determination of kit precision

[0085] 1.1 Intra-batch precision analysis

[0086] A batch of kits in Example 1 were used to measure high and low concentration quality control solution series respectively, and 10 wells were measured in parallel to obtain intra-assay coefficients of variation of 3.58% and 4.93%, respectively. The results are shown in Table 2.

[0087] Target value (ng / mL)

Measurement times

Intra-analytical CV(%)

15.45

10

4.93

39.17

10

3.58

[0088] Table 2.

[0089] 1.2 Batch-to-batch precision analysis

[0090] Get three batches of the test kit in embodiment 1, each batch of kits all measures the quality control product liquid se...

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Abstract

The invention discloses a magnetic-particle chemiluminescent detection kit for 25-hydroxyvitamin D, belonging to the field of immunoassay. The kit comprises an R1 reagent, an R2 reagent, an R3 reagentand a magnetic separation reagent, wherein the R2 reagent comprises an alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody; and the magnetic separation reagent comprises a fluorescein isothiocyanate-labeled 25-hydroxyvitamin D protein derivative and an anti-fluorescein isothiocyanate antibody-labeled magnetic particle, and protein in the fluorescein isothiocyanate-labeled 25-hydroxyvitamin D protein derivative one is selected from the group consisting of human serum albumin, bovine serum albumin, egg albumin and gamma globulin. The kit dissociates 25-hydroxyvitamin D from binding protein by using a highly basic reagent, and then realizes magnetic-particle chemiluminescent detection via a neutralization reagent; and the kit has high sensitivity, specificity and a widedetection range, and can be used for evaluating the metabolism status of vitamin D in patients and prompting the metabolism status of calcium and bone.

Description

technical field [0001] The invention relates to the field of immune analysis, in particular to a chemiluminescence detection kit of 25-hydroxyvitamin D magnetic particles. Background technique [0002] 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. Since 25-hydroxyvitamin D is a hydrophobic molecule, it often exists in combination with binding proteins in the body, and since the antigen-antibody reaction and alkaline phosphatase-substrate luminescent reaction need to be carried out in an ionic environment, how to obtain free 25-hydroxyvitamin D , and keeping it in a free state in an ion environment is one of the difficulties in immunoassay detection. In addition, the molecular weight of 25-hydroxyvitamin D is small, only about 42...

Claims

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Application Information

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IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 王鹏
Owner BEIJING LEADMAN BIOCHEM
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