Method for detecting multiple functions of akkermansia muciniphila based on liquid chip technology

Abstract

The present application relates to a liquid chip technology-based multiple-effect protein joint detection method for mucinophilic Akkermansia muciniphila. The method uses different coded microspheres coated with P9 protein, Amuc_1100 protein and Amuc_1409 protein specific antibodies to realize the synchronous detection of the three core functional proteins of AKK bacteria. The detection flux is 96 samples / batch, and the detection time is 3 hours. The judgment standard is: according to the detection results of the three proteins, calculate EPI, EPI >= 80 and each protein >= 3 ng / mL for excellent product, 60-80 for qualified product. The present application is suitable for comprehensive evaluation of AKK bacteria product quality.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for the joint detection of multiple functional proteins of Akkermansia myxophilus based on liquid phase chip technology. Background Technology

[0002] Liquid-phase chip technology, based on microspheres with different fluorescent codes, can simultaneously detect multiple target molecules in the same reaction system, offering advantages such as high throughput, high sensitivity, and low sample volume. Existing methods for detecting functional proteins in AKK bacteria are mostly single-indicator detection methods (such as detecting P9 protein or Amuc_1100 protein alone), lacking technical solutions for simultaneous detection of multiple indicators, thus failing to meet the needs of comprehensive product quality evaluation. Summary of the Invention

[0003] ① Problem to be solved: To provide a high-throughput joint detection method that can simultaneously detect the three core functional proteins of AKK bacteria (P9, Amuc_1100, Amuc_1409), and provide technical support for the comprehensive evaluation of product quality.

[0004] ② Technical solution: This invention provides a method for the simultaneous detection of multiple functional proteins of AKK bacteria based on liquid phase chip technology. Different encoded microspheres coated with specific antibodies for P9 protein, Amuc_1100 protein and Amuc_1409 protein are used to achieve the simultaneous detection of three core functional proteins. The detection throughput is 96 samples / batch and the detection time is 3 hours.

[0005] ③Beneficial effects: Single sampling and simultaneous detection of three proteins, resulting in high detection efficiency; small sample volume, suitable for analysis of precious samples; high throughput (96 samples / batch), suitable for large-scale product quality screening. Detailed Implementation

[0006] Example 1: Construction of a Liquid Chip (1) Three different encoded MagPlex microspheres (coded #25, #45, #65) were used for the detection of P9, Amuc_1100 and Amuc_1409 proteins, respectively.

[0007] (2) Microsphere activation: Take 1.25×10 6 Add 80 μL of activation buffer (0.1 M 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. Incubate at room temperature in the dark with shaking for 20 minutes, then wash twice with PBS.

[0008] (3) Antibody conjugation: Add 2 μg of capture antibody (specific antibodies corresponding to the three proteins respectively) to the activated microspheres, add PBS to 500 μL, incubate at room temperature in the dark with shaking for 2 hours, and wash twice with PBS.

[0009] (4) Blocking: Add 1 mL of blocking solution (PBS containing 1% BSA), incubate at room temperature in the dark with shaking for 30 minutes, wash and resuspend in 150 μL PBS, and store at 4℃.

[0010] Example 2: Joint Inspection Method Equal volumes of the three conjugated antibody microspheres (approximately 500 microspheres / μL of each) were mixed. 50 μL of the mixed microspheres was added to a 96-well filter plate, followed by 50 μL of pretreated sample. The plate was incubated at room temperature in the dark with shaking for 1 hour. The plate was washed three times with washing buffer. 50 μL of a biotinylated detection antibody mixture (a mixture of the three detection antibodies, each at a concentration of 1 μg / mL) was added, and the plate was incubated at room temperature in the dark with shaking for 45 minutes. After three washes, 50 μL of PE-labeled streptavidin (2 μg / mL) was added, and the plate was incubated at room temperature in the dark for 30 minutes. After three washes, the plate was resuspended in 100 μL of PBS and analyzed using a liquid chromatography-array analyzer. At least 100 events were collected from each microsphere cluster, and the quantitative results of the three proteins were output simultaneously.

[0011] Example 3: Sample Testing and Result Determination 3.1 Sample preparation Take the AKK bacteria sample (lyophilized powder, fermentation broth, or finished product) and process it as follows: (1) Lyophilized powder sample: Accurately weigh 100 mg of lyophilized powder, add 1 mL of sample extraction solution (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, and 1x protease inhibitor mixture), vortex to mix, extract at room temperature for 30 minutes, centrifuge at 10,000 g for 10 minutes at 4°C, and take the supernatant as the test solution.

[0012] (2) Fermentation broth sample: Take 1 mL of fermentation broth, centrifuge at 10,000 g for 10 minutes at 4°C, and take the supernatant as the test solution.

[0013] (3) End products (capsule contents, solid beverages, etc.): Take a sample equivalent to 100mg of the original sample, add 1mL of sample extract, vortex to mix, centrifuge at 10,000g for 10 minutes at 4°C, and take the supernatant as the test solution.

[0014] 3.2 Testing Operation The detection was performed according to the method in Example 2. The instrument automatically identified the microsphere code (to determine the type of protein) and the intensity of the PE fluorescence signal (to determine the protein content).

[0015] 3.3 Result Calculation (1) Record the measured concentrations (ng / mL) of P9 protein, Amuc_1100 protein and Amuc_1409 protein respectively.

[0016] (2) Calculate the Efficacy Protein Index (EPI) using the following formula: EPI = 0.4 × (CP9 / SP9) × 100 + 0.35 × (CAmuc1100 / SAmuc1100) ×100 + 0.25 × (CAmuc1409 / SAmuc1409) × 100 CP9, CAmuc1100, and CAmuc1409 represent the measured concentrations (ng / mL) of the three proteins; SP9, SAmuc1100, and SAmuc1409 represent the target reference concentrations (ng / mL) of the corresponding protein standards. Weighting coefficients (0.4, 0.35, and 0.25) were determined based on the functional importance of each protein.

[0017] 3.4 Standards for Passing and Failure Validity determination: The R² value of the standard curve should be ≥0.990, and the recovery rate of the internal standard for each protein detection should be between 80% and 120%; otherwise, the batch of experiments is invalid.

[0018] Sample judgment: Grade S (Superior): EPI ≥ 80, and the measured concentrations of all three proteins are ≥ 3 ng / mL.

[0019] Grade A (Qualified): 60 ≤ EPI < 80, and the measured concentrations of all three proteins are ≥ 3 ng / mL.

[0020] Grade B (Basic Qualified Product): 40 ≤ EPI < 60, and the measured concentration of all three proteins is ≥ 3 ng / mL.

[0021] Non-compliant products (low expression of functional proteins): EPI is between 40 and 60, but the detection value of any protein is lower than 3 ng / mL.

[0022] Serious non-compliance: EPI < 40, or the detection value of any one of the three proteins is below the detection limit (0.1 ng / mL).

[0023] Completely unqualified: If the detection values ​​of two or more proteins in the sample are below the detection limit (0.1 ng / mL), it is judged as completely unqualified and will not pass.

Claims

1. A method for the joint detection of multifunctional proteins from Akkermansia muciniphila, characterized in that, Liquid-phase chip technology is used to simultaneously detect two or more of the P9 protein, Amuc_1100 protein, and Amuc_1409 protein.

2. The joint inspection method according to claim 1, characterized in that, The liquid-phase chip technology uses different encoded microspheres coated with specific antibodies against P9 protein, Amuc_1100 protein, and Amuc_1409 protein, respectively.

3. The joint inspection method according to claim 1, characterized in that, The detection was performed using a flow cytometer or a liquid-phase chip analyzer, with at least 100 events collected for each group of microspheres.

4. The joint inspection method according to claim 1, characterized in that, The method has a detection throughput of 96 samples per batch and a detection time of 3 hours.

5. The application of the joint inspection method according to any one of claims 1 to 4 in the comprehensive quality evaluation of AKK bacteria products.