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Detection reagent kit for overall 25-hydroxy-vitamin-D

A technology for hydroxyvitamin and detection kit, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of inability to realize large-scale detection, high price of imported kits, slow response speed, etc., and achieve fast detection speed. , the effect of preventing toxic effects and the moderate price of reagents

Active Publication Date: 2016-02-24
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using ELISA to detect 25-hydroxyvitamin D has a long reaction time, usually 2-3 hours, and requires special equipment, so large-scale detection cannot be realized
Using chemiluminescence to detect 25-hydroxyvitamin D, although the detection sensitivity is high, the reaction speed is relatively slow, and the detection generally takes 30 minutes. It also requires an expensive closed immunoluminescence instrument to detect, and the imported kit is expensive.

Method used

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  • Detection reagent kit for overall 25-hydroxy-vitamin-D
  • Detection reagent kit for overall 25-hydroxy-vitamin-D
  • Detection reagent kit for overall 25-hydroxy-vitamin-D

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Reagent preparation

[0031] 1. Preparation of reagent 3:

[0032] (1) Dilute polystyrene latex particles with a particle size of 130nm to a concentration of 4mg / mL in buffer with 10mM, pH6.0MES buffer; then add 1-ethyl-3-( 3-Dimethylaminopropyl) carbodiimide hydrochloride (EDAC) solution, make the final concentration of EDAC in the above solution system reach 100 μg / mL, add 1 mg / ml of N-hydroxysuccinimide ( NHS) solution, make the final concentration of NHS in the above solution system reach 60μg / mL, react at 25°C for 1h, centrifuge at 15000rpm for 40min after the reaction, remove the supernatant, and then add 15mM, pH7.4 phosphate buffer after ultrasonic dispersion Then add cysteamine solution with a concentration of 1mg / ml to make the final concentration of cysteamine in the reaction system reach 250ug / ml, react at 25°C for 3h, after the reaction, centrifuge at 15000rpm for 40min, remove the supernatant , and then add 15 mM phosphate buffer solution w...

Embodiment 2

[0040] Embodiment 2: The detection method of total 25-hydroxyvitamin D

[0041] The determination method of total 25-hydroxyvitamin D is a two-point endpoint method, and the detection wavelengths are respectively the main wavelength of 546nm and the secondary wavelength of 700nm. The volumes of reagent 1, reagent 2 and reagent 3 are all 70 μl. The sample volume is 10 μl; the reaction direction is downward, and the measurement temperature is 37°C. After mixing the sample with reagent 1, incubate at 37°C for 5 minutes, then add reagent 2, incubate at 37°C for 5 minutes, then add reagent 3, start reading after 10 seconds of reaction, and read another point after 5 minutes of reaction. Use Hitachi 7180 automatic biochemical analyzer to measure the calibration curve (such as figure 1 shown), where the X-axis is the total 25-hydroxyvitamin D calibrator concentration in ng / ml, and the Y-axis is the reaction absorbance.

Embodiment 3

[0042] Embodiment 3: detection of reagent performance index

[0043] 1. Correlation experiment

[0044] Purchase total 25-hydroxyvitamin D detection kit and test kit of the present invention on the Hitachi 7180 full-automatic biochemical analyzer according to the upper machine parameter of setting respectively from the market, detect 40 parts of fresh human serum (serum is taken from hospital) , carry out correlation analysis on the measured values, the results are as follows figure 2 As shown, the regression equation is: Y=1.023X-2.762, the correlation coefficient R 2 =0.975, the result shows that this reagent has a good correlation with the reagents currently on the market.

[0045] 2. Opening stability experiment

[0046] Purchase pure total 25-hydroxyvitamin D (high value, low value) (dry powder) from the market, reconstitute and divide into tubes according to the requirements of the instructions, and store at -20°C for long-term preservation. The reagent of the prese...

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Abstract

A detection reagent kit for overall 25-hydroxy-vitamin-D is characterized by comprising a reagent 1, a reagent 2 and a reagent 3; the reagent 1 is dissociation liquid containing surfactant, the reagent 2 is formed by putting antibodies resisting human 25-hydroxy-vitamin-D2 and 25-hydroxy-vitamin-D3 in a proper buffer solution, and the reagent 3 is formed by putting polystyrene latex particles with the surfaces marked with human 25-hydroxy-vitamin-D2 and 25-hydroxy-vitamin-D3 in a proper buffer solution. The reagent kit has the advantages of being high in detection sensitivity and accuracy, good in precision, wide in detection linearity, good in stability, short in detection time and low in production cost.

Description

technical field [0001] The invention relates to a medical detection kit, in particular to a total 25-hydroxyvitamin D detection kit. Background technique [0002] 25-Hydroxyvitamin D (25-Hydroxyvitamin D) is a steroid hormone that plays an important role in promoting the absorption of calcium in the intestine and maintaining its dynamic balance, and plays a key role in the growth and health of human bones. Studies in recent years have shown that, in addition to skeletal effects, vitamin D also has non-skeletal effects related to autoimmune diseases, diabetes, cardiovascular diseases, and cell growth and differentiation. Vitamin D is available in two forms, vitamin D2 and D3. Vitamin D in the human body comes from diet, while vitamin D3 can be synthesized by skin tissue exposed to ultraviolet rays. In the liver, vitamin D undergoes the action of 25-hydroxylase to form 25-hydroxyvitamin D. After being transported to the kidney, it undergoes the action of 1-hydroxylase to fo...

Claims

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Application Information

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IPC IPC(8): G01N21/82G01N33/82G01N33/544
Inventor 方亮邹炳德邹继华
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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