A method for detecting exocysts in liquid samples

Abstract

The invention provides a method for detecting exocysts in a liquid sample to be tested, the liquid sample to be tested contains exocysts; the method comprises: passing the liquid sample to be tested on the surface of a microarray chip for contacting, the microarray chip The surface of the array chip is loaded with one or more specific antibodies to exocyst surface membrane proteins, and the surface plasmon resonance signal change value of the microarray chip before and after contacting the liquid sample to be tested is obtained; the surface of the microarray chip The change value of the plasmon resonance signal indicates the amount of exocysts in the liquid sample to be tested. The method provided by the present invention is based on the surface plasmon resonance imaging technology, does not need a complicated purification process for the liquid sample to be tested, and can accurately and reliably detect the internal and external cysts in the liquid sample quantitatively in real time.

Description

technical field

[0001] The invention relates to a method for quantitatively detecting exocysts in liquid samples. Background technique

[0002] Surface plasmon resonance (SPR) sensing technology is a surface-sensitive detection method. Compared with other biological detection methods, the SPR sensing analysis system has the advantages of label-free and real-time detection of interaction kinetics. . The surface plasmon resonance imaging (SPRi) analysis system based on SPR technology has increased the detection capacity on the basis of maintaining the advantages of SPR detection technology. SPRi can detect hundreds or even thousands of interactions at the same time. High-throughput detection technology.

[0003] The exosome is a flat spherical microvesicle surrounded by a phospholipid bilayer with a diameter in the range of 40-100 nm. The formation process of exocysts is relatively complicated. At present, scientific researchers generally believe that exocysts originate fro...

Examples

preparation example Construction

[0020] The preparation method of the microarray chip loaded with exocyst surface membrane protein specific antibody according to the present invention can be according to literature (Lausted, C.; Hu, Z.; Hood, L.; Campbell, C.T.Comb Chem High ThroughputScreen2009, 12, (8), 741-51.), preferably, the preparation process may include the following steps:

[0021] Printing: Print one or more specific antibodies to exocyst surface membrane proteins at a concentration of 0.1-10 μg / μL on a chip coated with a metal layer on the surface to form an antibody microarray. The chip can be a gold-plated chip or a silver-plated chip commonly used in SPRi technology. The size of the chip can be the size conventionally used in the art. Generally, the printing density of antibodies on the chip per square centimeter is 1-10000 dots, preferably 1-100 dots. In terms of protein amount, each The amount of antibody in the antibody spot is 0.02-2 μg, preferably 0.1-1 μg. After printing, place the chip...

preparation example 1

[0056] This preparation example is used to illustrate the preparation method of the microarray chip provided by the present invention. In this preparation example, the same microarray chips 1-6 are prepared for subsequent test examples. The specific preparation steps are:

[0057] (1) Printing: Mouse anti-CD63 IgG, mouse anti-CD9 IgG, mouse anti-CD41b IgG, mouse anti-E-cadherin IgG, mouse anti-EpCAM IgG, mouse anti-CD82 IgG and the control group IgG at a concentration of 5 μg / μL at 5000 cells / cm 2 The printing density was fixed to the surface of the plasmon resonance imaging gold-plated chip modified by polyethylene glycol to form an antibody microarray, and one spot was printed for each antibody (the amount of protein in each spot was 0.5 μg), and the microarray chip was placed Incubate at 40°C for 2 hours in a humid box with a humidity of 50%;

[0058] (2) Cleaning: Wash the chip with 100ml of 1M high-fold phosphate buffer, 100ml of 0.1M low-fold phosphate buffer and 200ml ...

technical field

[0001] The invention relates to a method for quantitatively detecting exocysts in liquid samples. Background technique

[0002] Surface plasmon resonance (SPR) sensing technology is a surface-sensitive detection method. Compared with other biological detection methods, the SPR sensing analysis system has the advantages of label-free and real-time detection of interaction kinetics. . The surface plasmon resonance imaging (SPRi) analysis system based on SPR technology has increased the detection capacity on the basis of maintaining the advantages of SPR detection technology. SPRi can detect hundreds or even thousands of interactions at the same time. High-throughput detection technology.

[0003] The exosome is a flat spherical microvesicle surrounded by a phospholipid bilayer with a diameter in the range of 40-100 nm. The formation process of exocysts is relatively complicated. At present, scientific researchers generally believe that exocysts originate fro...

Examples

preparation example Construction

[0020] The preparation method of the microarray chip loaded with exocyst surface membrane protein specific antibody according to the present invention can be according to literature (Lausted, C.; Hu, Z.; Hood, L.; Campbell, C.T.Comb Chem High ThroughputScreen2009, 12, (8), 741-51.), preferably, the preparation process may include the following steps:

[0021] Printing: Print one or more specific antibodies to exocyst surface membrane proteins at a concentration of 0.1-10 μg / μL on a chip coated with a metal layer on the surface to form an antibody microarray. The chip can be a gold-plated chip or a silver-plated chip commonly used in SPRi technology. The size of the chip can be the size conventionally used in the art. Generally, the printing density of antibodies on the chip per square centimeter is 1-10000 dots, preferably 1-100 dots. In terms of protein amount, each The amount of antibody in the antibody spot is 0.02-2 μg, preferably 0.1-1 μg. After printing, place the chip...

preparation example 1

[0056] This preparation example is used to illustrate the preparation method of the microarray chip provided by the present invention. In this preparation example, the same microarray chips 1-6 are prepared for subsequent test examples. The specific preparation steps are:

[0057] (1) Printing: Mouse anti-CD63 IgG, mouse anti-CD9 IgG, mouse anti-CD41b IgG, mouse anti-E-cadherin IgG, mouse anti-EpCAM IgG, mouse anti-CD82 IgG and the control group IgG at a concentration of 5 μg / μL at 5000 cells / cm 2 The printing density was fixed to the surface of the plasmon resonance imaging gold-plated chip modified by polyethylene glycol to form an antibody microarray, and one spot was printed for each antibody (the amount of protein in each spot was 0.5 μg), and the microarray chip was placed Incubate at 40°C for 2 hours in a humid box with a humidity of 50%;

[0058] (2) Cleaning: Wash the chip with 100ml of 1M high-fold phosphate buffer, 100ml of 0.1M low-fold phosphate buffer and 200ml ...

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