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Biotinylated liposome used for detecting pancreas cancer marker REG1A, and preparation method and applications thereof

A biotinylation and liposome technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, and the determination/inspection of microorganisms, can solve the problems of low specificity and high warning threshold, and achieve high sensitivity The effect of detection, easy operation and good stability

Inactive Publication Date: 2018-02-09
SHANGHAI IGENETEC DIAGNOSTICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, non-neoplastic diseases such as chronic pancreatitis, cholelithiasis, and diabetes are often accompanied by low concentrations, high warning thresholds, and low specificity.

Method used

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  • Biotinylated liposome used for detecting pancreas cancer marker REG1A, and preparation method and applications thereof
  • Biotinylated liposome used for detecting pancreas cancer marker REG1A, and preparation method and applications thereof
  • Biotinylated liposome used for detecting pancreas cancer marker REG1A, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] This embodiment is a method for preparing biotinylated liposomes for detecting pancreatic cancer marker REG1A, which includes the following steps:

[0053] a) Weigh DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin on an analytical balance (the molar ratio between the four is 95:95:9:1, the total mass is 36.5mg), and place them in a 10ml centrifuge tube, Add 3.43ml of chloroform and 571μl of methanol solvent (the volume ratio of the two is 6:1, the total volume is 4mL), and sonicate for 10min to accelerate the dissolution.

[0054] b) The above-mentioned liquid after ultrasonication was added to a 50ml eggplant-shaped flask, put into a built-up rotary evaporator, and evaporated the organic solvent (35°C) under reduced pressure to form a thin layer of lipid film on the inner wall of the flask.

[0055] c) Add the pre-prepared 2ml PBS solution containing 1μg DNA dropwise into the flask, and at the same time shake the flask in a water bath at 45°C to dissolve and detach...

Embodiment 2

[0061] This example is a non-diagnostic and non-therapeutic method for the detection of pancreatic cancer marker REG1A based on the biotinylated liposome above, and a schematic flow chart of the ultra-high sensitivity detection of REG1A protein based on liposome-encapsulated DNA is as follows figure 1 shown.

[0062] Specific steps are as follows:

[0063] 1) Coat Corning coast 9018 ELISA plate with 1 μg / ml monoclonal rat antibody of REG1A, overnight at 4°C.

[0064] 2) The plate was washed twice with PBST (0.05% Tween 20 PH7.4), and the detection plate was blocked with 250 μl of 23% BSA for 1 hour. Repeat plate washing 4 times.

[0065] 3) The recombinant protein was diluted 10 times and added to the assay plate, and incubated at 37°C for 1 hour. Repeat plate washing 4 times.

[0066] 4) Add REG1A polyclonal mouse antibody 1 μg / ml, and incubate at 37°C for 1 hour. Repeat plate washing 4 times.

[0067] 5) Add 100 μl of biotinylated goat anti-rabbit antibody at a ratio o...

Embodiment 3

[0076] This embodiment is the optimization of the concentration of biotinylated liposomes. The steps are the same as in Example 2. The concentrations of biotinylated liposomes used in this embodiment are 1.2mg / ml, 0.24mg / ml, 0.12mg / ml, 0.06mg / ml (corresponding to figure 2 Part A, Part B, Part C, Part D) of the figure 2 The amplification results show that when the concentration of biotinylated liposomes is 1.2mg / ml, both the REG1A protein group and the blank control group have "S" type amplification curves; when the concentration of biotinylated liposomes is When the concentration of biotinylated liposomes was 0.24 or 0.12mg / ml, only the REG1A protein group appeared "S" type expansion curve; increasing curve.

[0077] From the above experimental results, it can be seen that in the method for detecting the pancreatic cancer marker REG1A of the present invention, the preferred concentration of biotinylated liposomes is 0.12-0.24 mg lipid / ml.

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Abstract

The invention relates to a biotinylated liposome used for detecting pancreas cancer marker REG1A. The biotinylated liposome used for detecting pancreas cancer marker REG1A comprises coated reporter DNA fragments, and PEG-200 surface modified biotin; according to the preparation method, DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin film dispersion method is adopted to prepare the biotinylated liposome used for coating reporter DNA fragments, and the biotinylated liposome is taken as an immunolabelling biosensor. The invention also relates to a method used for detecting pancreas cancer marker REG1A based on the biotinylated liposome. The method comprises following steps: an ELSA plate is coated with REG1A murine monoclonal antibodies, a REG1A protein sample to be detected is added, reactions with REG1A rabbit source polyclonal antibodies, biotinylated goat anti rabbit IgG, and avidin are carried out respectively, at last the biotinylated liposome of an optimized concentration is added, fluorescence quantitative LAMP amplification is carried out, and the concentration of the REG1A protein is calculated based on amplification results. According to the preparation method, immunization LAMP reaction is combined with immunoliposome nano-particles, so that high sensitivity detection of pancreas cancer marker REG1A is realized, stability is high, cost is low, and the specificityis high.

Description

technical field [0001] The invention relates to the technical field of nano-biological detection, in particular to a biotinylated liposome for detecting pancreatic cancer marker REG1A, a preparation method and application thereof. Background technique [0002] Liposomes are spherical closed vesicles formed by phospholipids dispersed in water and encapsulating a part of the aqueous phase. It usually contains one or more layers of phospholipid membranes, and the size can range from 20nm to tens of microns. Liposomes are mostly used in drug delivery, in vitro transfection and so on. With the development of liposome technology, liposomes can encapsulate a large number of markers and combine various types of biorecognition molecules, and have a relatively high surface area, and the volume of the internal aqueous phase is also relatively large. Applied to immunoassay method, namely liposome immunoassay. In operation, surfactants can be used to promote liposome membrane rupture ...

Claims

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Application Information

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IPC IPC(8): C12N15/88C12Q1/6886C12Q1/6844
CPCC12N15/88C12Q1/6844C12Q1/6886C12Q2600/158C12Q2531/119C12Q2563/107
Inventor 刘鹏方雪恩曹宏梅邓安梅孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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