High-throughput simultaneous detection method for mucinophilic akkermansia efficacy proteins based on flow cytometry fluorescence-encoded microspheres

Abstract

This invention relates to a high-throughput simultaneous detection method for Akkermansia myxophilic proteins based on flow cytometry-encoded microspheres. The method uses different fluorescently encoded microspheres coated with specific antibodies against P9, Amuc_1100, and Amuc_1409 proteins, respectively, to achieve simultaneous detection of the three proteins in a single sample. This method offers high throughput and efficiency, making it suitable for large-scale product quality screening of Akkermansia myxophilic proteins.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a high-throughput method for the joint detection of Akkermansia myxophilic functional proteins based on flow cytometry-encoded microspheres. Background Technology

[0002] Flow cytometry-encoded microsphere technology enables simultaneous detection of multiple indicators in a single sampling, offering advantages such as high throughput, high speed, and low sample volume. Currently, there are no reports on the application of this technology to the joint detection of functional proteins in AKK bacteria. Summary of the Invention

[0003] ① Problem to be solved: Existing methods for detecting functional proteins in AKK bacteria have low throughput and cannot achieve simultaneous detection of multiple proteins.

[0004] ② Technical solution: Three different fluorescently encoded microspheres are used, each coated with a capture antibody for one of the three proteins, to achieve simultaneous detection of the three proteins in a single sampling.

[0005] ③ Beneficial effects: High throughput and high efficiency, suitable for large-scale product quality screening. Detailed Implementation

[0006] Example 1: Preparation of flow cytometry fluorescently encoded microspheres Three types of coded microspheres with different fluorescence intensities (coded #25, #45, and #65) were used for the detection of P9, Amuc_1100, and Amuc_1409 proteins, respectively. Microsphere activation: 1.25 × 10⁻⁶ microspheres were used. 6 Add 80 μL of activation buffer (0.1M NaH2PO4, pH 6.2) to each microsphere, then add 10 μL of 50 mg / mL Sulfo-NHS and 10 μL of 50 mg / mL EDC sequentially. Incubate at room temperature in the dark with shaking for 20 minutes, then wash twice with PBS. Antibody conjugation: Add 2 μg of capture antibody to the activated microspheres, incubate at room temperature in the dark with shaking for 2 hours, wash twice with PBS, add 1 mL of blocking buffer (PBS containing 1% BSA) and block for 30 minutes. After washing, resuspend in 150 μL of PBS and store at 4°C.

[0007] Example 2: Sample Detection (1) Sample pretreatment: Take 100 mg of the AKK bacteria sample to be tested, add 1 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% SDS, 1× protease inhibitor), incubate on ice for 30 minutes, sonicate (200W, 2s on and 2s off, 3 minutes in total), centrifuge at 12,000g for 20 minutes and take the supernatant.

[0008] (2) Combined detection procedure: Mix equal volumes of the three types of coded microspheres (approximately 500 microspheres / μL for each type), add 50 μL of the mixed microspheres to a 96-well filter plate, add 50 μL of the sample to be tested, and incubate at room temperature in the dark with shaking for 1 hour. Wash three times with washing buffer. Add 50 μL of the biotinylated detection antibody mixture and incubate at room temperature in the dark with shaking for 45 minutes. After washing three times, add 50 μL of PE-labeled streptavidin (2 μg / mL) and incubate at room temperature in the dark for 30 minutes. After washing three times, resuspend in 100 μL of PBS.

[0009] (3) Detection and quantification: Detection is performed using a flow cytometer or liquid phase chip analyzer. The instrument automatically identifies the microsphere code (to determine the protein type) and the PE fluorescence signal intensity (to determine the protein content), and calculates the content (ng / mL) of the three proteins based on the standard curve of each protein.

[0010] Example 3: Result Calculation and Judgment Efficacy Protein Comprehensive Index: EPI = 0.4×CP9 + 0.35×CAmuc1100 + 0.25×CAmuc1409.

[0011] Quality Grade EPI Value Range Determination Standard Grade S (Superior): EPI ≥ 80. High expression of all three functional proteins, exhibiting excellent functional activity. Grade A (Qualified Product): 60 ≤ EPI < 80. Main functional protein expression is normal, and functional activity is good. Grade B (Basic Qualified Product): 40 ≤ EPI < 60. Expression of some functional proteins is low, and functional activity is generally moderate. Grade C (Substandard Product): EPI < 40 indicates severely insufficient expression of functional proteins and poor functional activity. Acceptable product: EPI ≥ 60, and the detection value of any single protein is not less than 3 ng / mL (i.e., 3 times the lower limit of the standard curve). If any protein is <3 ng / mL, it should be downgraded or judged as unacceptable.

[0012] Non-conforming products: Products meeting any of the following conditions are considered non-conforming: (1) EPI < 40; (2) 40 ≤ EPI < 60, but the detection value of any protein is less than 3 ng / mL; (3) The detection value of any one of the three proteins is lower than the detection limit (0.1 ng / mL).

[0013] If the detection values ​​of two or more proteins are below the detection limit, it is considered a serious non-compliance.

Claims

1. A high-throughput method for detecting functional proteins of Akkermansia muciniphila, characterized in that, The P9 protein, Amuc_1100 protein, and Amuc_1409 protein were simultaneously detected using flow cytometry-encoded microsphere technology.

2. The joint inspection method according to claim 1, characterized in that, The method uses different fluorescently encoded microspheres coated with three different protein-specific antibodies.

3. The joint inspection method according to claim 1, characterized in that, The method involves detection using flow cytometry, distinguishing protein types based on microsphere coding, and quantifying protein content based on fluorescence intensity.

4. The joint inspection method according to claim 1, characterized in that, The method also includes calculating EPI = 0.4×CP9 + 0.35×CAmuc1100 + 0.25×CAmuc1409 based on the detection results of the three proteins. An EPI ≥ 60 is considered a qualified product, and an EPI < 40 is considered a non-qualified product.

5. The joint inspection method according to claim 1, characterized in that, The different fluorescently encoded microspheres refer to three types of encoded microspheres with different fluorescence intensities, which are respectively coated with specific capture antibodies of P9 protein, Amuc_1100 protein and Amuc_1409 protein by EDC / Sulfo-NHS conjugation method.

6. The joint inspection method according to claim 4, characterized in that, The qualified product refers to one with an EPI ≥ 60 and a detection value of not less than 3 ng / mL for P9 protein, Amuc_1100 protein, and Amuc_1409 protein; if the detection value of any protein is less than 3 ng / mL, even if the EPI ≥ 60, it should be judged as unqualified and downgraded or identified as an unqualified product.