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Quantitative-detection test paper strip for 25-hydroxyvitamin D and application of quantitative-detection test paper strip

A technology for hydroxyvitamin and quantitative detection, applied in measuring devices, instruments, scientific instruments, etc., can solve problems such as incomplete affinity, and achieve the effects of high accuracy, time saving, and accurate quantification

Active Publication Date: 2015-04-08
同昕生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, more than 80% of 25-OH VD is combined with VDBP in serum. The usual immunoassay method is to use sample pretreatment solution (such as protein denaturants such as organic solvents) to dissociate and release 25-OH VD from VDBP. ; Or use an acidic reaction system (pH4.0-6.0) to measure; or use a high concentration of VDBP affinity conjugates to compete for the release of 25-OH VD, these methods exist in the sample dilution process or 25-OH VD release is not complete or affinity And the affinity of the conjugate and other factors are limited

Method used

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  • Quantitative-detection test paper strip for 25-hydroxyvitamin D and application of quantitative-detection test paper strip
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  • Quantitative-detection test paper strip for 25-hydroxyvitamin D and application of quantitative-detection test paper strip

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Experimental program
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Effect test

Embodiment 1、25

[0056] Preparation of embodiment 1, 25-hydroxyvitamin D quantitative detection test strip

[0057] (1) Expression and verification of VDBP recombinant protein

[0058]The rProtein recombinant protein, that is, the 159-458 amino acids of the human VDBP partial sequence, was expressed in the E.coli system through the expression of the GST fusion protein (the expression vector was PET28a, and the E.coli cell used was BL21 (DE3), which was purchased from Beijing Quanshijin Biotechnology Co., Ltd. Technology Co., Ltd). Bacteria were cultured in LB medium containing 100 mg / ml ampicillin (23YT-Amp), and scaled up at a ratio of 1:10 after overnight culture at 37°C. When the bacterial solution concentration (A600) reaches 0.8-0.9, add 0.5mM IPTG to induce expression. After culturing at 25°C for 12-15 hours, the cells were collected by centrifugation and resuspended with TBS buffer. Sonicate the bacterial liquid, 40w, 10s, 10s, 50 times; centrifuge at 12000rpm, 4°C for 15min, and col...

Embodiment 2

[0098] Embodiment 2, the selection of sample pad coating

[0099] The performance of the test strip detection system of the present invention is improved by optimizing the coating of the sample pad. According to the measurement results, 2 clinical samples (25-OH vitamin D content high value H, low value L) were used to prepare and add recovered samples according to Table 1, calculate the recovery rate, and select the optimal coating material.

[0100] Table 1 Addition recovery sample preparation method

[0101]

[0102] The screening method is as follows:

[0103] (1) Use LP-25-OH VD and LP-1,25-(OH) respectively 2 VD or 25-OH VD-BSA coated sample pad (2.0ug / cm 2 ), coating method is with embodiment 1;

[0104] (2) Chromatography test strip preparation method is the same as embodiment 1;

[0105] (3) Add 100 μL sample and react at room temperature for 15 minutes;

[0106] (4) Calculate the 25-OH VD according to the strip reader, and then calculate the recovery rate R ...

Embodiment 3

[0117] Coating concentration selection of embodiment 3, LP-25-OH VD

[0118] Since the coating concentration of LP-25-OH VD affects the capture efficiency of free VDBP in human serum, its coating concentration needs to be further optimized. The optimization method is the same as in Example 2.

[0119] The screening method is as follows:

[0120] (1) Select different concentrations of LP-25-OH VD to coat the sample pad, and the coating steps are the same as in Example 1;

[0121] (2) Chromatography test strip preparation method is the same as embodiment 1;

[0122] (3) Add 100 μL sample and react at room temperature for 15 minutes;

[0123] (4) Calculate the 25-OH VD according to the strip reader, and then calculate the recovery rate R by the formula (1).

[0124] The experimental results are shown in Table 3. The concentration of LP-25-OH VD is represented by the concentration of 25-OH VD-BSA, and the optimal concentration of LP-25-OH VD is 2.0ug / cm 2 .

[0125] Table 3 Th...

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Abstract

The invention relates to a quantitative-detection test paper strip for 25-hydroxyvitamin D and application of the quantitative-detection test paper strip and belongs to the field of medical reagents. The test paper strip comprises a sample pad, a combining pad, an NC film, a water absorbing pad and a PVC lining plate, wherein the sample pad is coated with latex microparticles activated by 25-OH VD and / or latex microparticles activated by 1, 25-(OH)2VD; the combining pad is coated with colloidal-gold-labeled anti-25-OH VD monoclonal antibodies and anti-VDBP monoclonal antibodies; the NC film comprises a T line and a C line in sequence; the T line is coated with 25-OH VD-BSA and partially-expressed human VDBP; and the C line is coated with goat-anti-mouse antibodies. With the adoption of the test paper strip, the content of vitamin D in a sample can be quickly and accurately determined without pretreating the sample.

Description

technical field [0001] The invention relates to a 25-hydroxyvitamin D quantitative detection test strip and an application thereof, belonging to the field of pharmaceutical preparations. Background technique [0002] In recent years, with the continuous deepening of research, people have more understanding of vitamin D (VD). VD is a very unique member of all biologically active substances with multiple functions. It is a vitamin, but in essence Hormones, and possibly cytokines. Because VD is an essential nutrient derived from food, and the human body needs a very small amount, it is called a vitamin. It is mainly transformed from 7-dehydrocholesterol in the skin under the irradiation of ultraviolet rays, and then travels with the blood to the whole body to play a role, so it belongs to steroid hormones. People gradually realize that it has a wider range of physiological functions. In addition to regulating calcium and phosphorus metabolism, it also has anti-proliferation, ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558
CPCG01N33/577
Inventor 肖智焦守恕李全
Owner 同昕生物技术(北京)有限公司
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