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ELISA kit for detecting exosome Wnt5a protein

A technology of exosomes and kits, applied in the field of biomedicine, can solve the problem of inability to meet the needs of clinical rapid, high-sensitivity and large-scale screening, the difficulty of meeting the needs of serum exosome Wnt5a detection, and the inability to achieve large-scale detection of clinical specimens etc. to achieve the effect of high accuracy, simple and fast operation, simple and fast accuracy

Pending Publication Date: 2022-03-18
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of Wnt5a at home and abroad is mainly limited to tissue and cell detection, and the detection methods often use quantitative PCR gene level detection and Western blot, immunohistochemical protein level detection, which cannot meet the needs of clinical rapid, high sensitivity and large-scale screening.
[0005] Serum is the most commonly used clinical screening sample. Wnt5a has been proven to be widely involved in the regulation of tumor development and is highly enriched in exosomes. The detection of Wnt5a in serum exosomes has become an ideal screening sample at present. However, the western blot method in the above methods The detection of Wnt5a protein is a qualitative and semi-quantitative detection, which is cumbersome and cannot meet the requirements of large-scale detection of clinical specimens, while PCR is used to detect the target gene at the transcription level, which cannot reflect the protein level, and it is difficult to meet the detection requirements of serum exosomal Wnt5a

Method used

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  • ELISA kit for detecting exosome Wnt5a protein
  • ELISA kit for detecting exosome Wnt5a protein
  • ELISA kit for detecting exosome Wnt5a protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation and condition optimization of an ELISA kit for detecting exosomal Wnt5a protein

[0043] The components of the ELISA kit for detecting exosomal Wnt5a protein are:

[0044] (1) Wnt5a coating antibody is a mouse anti-human monoclonal Wnt5a capture antibody from R&D Systems, the product number is MAB6452.

[0045] (2) The Wnt5a detection antibody is the mouse anti-human monoclonal Wnt5a antibody from R&D Systems, the product number is MAB6451.

[0046](3) The goat anti-mouse polyclonal antibody labeled with biotin is Goat anti-mouse IgG (H+L) biotin conjμgate from Proteintech Company, the product number is SA00004-1.

[0047] (4) HRP enzyme-labeled streptavidin is Streptavidin HRP conjugate of Proteintech Company, the article number is SA00001-0.

[0048] (6) The Wnt5a protein standard product is Recombinant Human / Mouse Wnt-5aProtein from R&D Systems, the catalog number is 645-WN.

[0049] (7) DuoSet ELISA auxiliary reagent is DuoSet ELISA Ancilla...

Embodiment 2

[0061] Embodiment 2: Wnt5a standard detection and fitting standard curve drawing

[0062] In this embodiment, the Wnt5a standard product is detected, and the equation of the standard curve of the Wnt5a protein is given according to the detection data, and the specific detection steps are as follows:

[0063] (1) Take out the required strips from the aluminum foil bag after equilibrating at room temperature, add 100 μl 5 μg / mL Wnt5a coating antibody to each well, seal the strips with parafilm, overnight at 4°C, and dry the bag the next day. Fill up with washing liquid after being soaked, let it stand for 3 minutes, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 3 times.

[0064] (2) Add 100 μL of blocking solution to each well and place it in an incubator at 37°C for 2 hours. After drying the blocking solution, fill it up with washing solution, let it stand for 3 minutes, shake off the washing solution, pat dry on absorbent paper, and w...

Embodiment 3

[0072] Embodiment 3: Wnt5a recovery rate analysis

[0073] In this implementation, the recovery rate of the ELISA methodology is calculated according to the DuoSet ELISA auxiliary reagent, and the specific method is as follows:

[0074] Three groups of experiments were set up, namely the unspiked group, the spiked group and the control group, wherein the unspiked group was 1 mL serum, the spiked group was 980 μL serum + 20 μL (500 ng / mL) Wnt5a standard; the control group was 980 μL diluted solution+20 μL (500ng / mL) Wnt5a standard substance, each group was set up with 3 duplicate wells, measured and calculated according to the method described in Example 2, and the average concentration.

[0075] According to the measurement, the average concentration obtained in the non-standardized group is 0.325ng / mL, the average concentration in the spiked group is 3.572ng / mL, and the average concentration in the control group is 3.134ng / mL. - Calculated by the formula of the average conce...

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Abstract

The invention provides an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting exosome Wnt5a protein, and belongs to the technical field of biological medicines. The ELISA kit for detecting the exosome Wnt5a protein, disclosed by the invention, can be used for quantitatively detecting the content of the Wnt5a protein in the serum exosome; the lowest detection limit of the kit is 156 pg / ml, a good linear relation exists at 156-10000 pg / ml, and the kit has high sensitivity and accuracy and is simple, convenient and rapid.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an ELISA kit for detecting exosome Wnt5a protein. Background technique [0002] Exosomes are a type of nanoscale vesicles with a diameter of about 30-150nm and rich in nucleic acids, proteins, lipids, etc. It is an important medium for intercellular signal transmission and is widely involved in the regulation of tumorigenesis and development. Tumor cell exosomes are highly enriched in non-coding RNAs and proteins abnormally expressed by tumor cells, which can remotely reshape the tumor microenvironment, transmit malignant phenotypes and drug resistance, and promote the malignant progression of tumors. Therefore, the detection of tumor-associated molecules in serum exosomes has become an important way for non-invasive detection of tumors. Tumor diagnosis, curative effect monitoring and prognosis judgment can be carried out by detecting related molecules in serum e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/574G01N33/543
CPCG01N33/6893G01N33/577G01N33/57488G01N33/54306G01N2333/47
Inventor 王梅俞万钧朱伟赵媛媛仇荣赵欣欣
Owner JIANGSU UNIV