Monoclonal antibody recognizing amuc_1100 protein of akkermansia muciniphila and elisa test kit

Abstract

The present application relates to the monoclonal antibody of the identification of mucinophilic akkermansia Amuc_1100 protein and its ELISA detection kit. The monoclonal antibody is obtained by screening cell fusion after immunizing mice with recombinant Amuc_1100 protein as immunogen. The ELISA detection kit provided by the present application can realize the quantitative detection of AKK bacteria Amuc_1100 protein, and the linear range is 1-1000 ng / mL. The actual measured concentration of Amuc_1100 protein is greater than or equal to 3 ng / mL, which is qualified. It is suitable for product quality control.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody that identifies the Amuc_1100 protein of Akkermansia myxophilus and its ELISA detection kit. Background Technology

[0002] Amuc_1100 is one of the most abundant functional proteins expressed on the outer membrane of *AKK* bacteria, exerting metabolic and immunomodulatory functions through the Toll-like receptor 2 (TLR2) signaling pathway. Literature (e.g., Otman N. et al., Plos ONE, 2017) has confirmed that Amuc_1100 protein retains its biological activity in pasteurized *AKK* bacteria. While Muen Biotech has already established a presence in the mass spectrometry detection of Amuc_1100 protein, its immunological detection capabilities remain unexplored. This invention provides an ELISA detection method for Amuc_1100 protein based on monoclonal antibodies, complementing mass spectrometry techniques. Summary of the Invention

[0003] ① Problem to be solved: To provide an ELISA detection method for Amuc_1100 protein based on monoclonal antibodies to fill the gap in the field of immunological detection.

[0004] ② Technical Solution: This invention provides a monoclonal antibody that specifically binds to the Amuc_1100 protein and an ELISA detection kit based on this antibody. The monoclonal antibody is obtained by immunizing mice with recombinant Amuc_1100 protein as an immunogen followed by cell fusion screening. The ELISA detection kit is a double-antibody sandwich ELISA kit, comprising a capture antibody and a detection antibody.

[0005] ③ Beneficial effects: This method provides the first ELISA detection method for Amuc_1100 protein based on monoclonal antibodies; it is simple to operate, has high throughput, and is suitable for industrial-scale batch detection; it complements mass spectrometry and meets the needs of different application scenarios. Detailed Implementation

[0006] Example 1: Preparation of Amuc_1100 protein monoclonal antibody (1) The recombinant Amuc_1100 protein (molecular weight approximately 31.7 kDa) was diluted to 1 mg / mL with PBS and emulsified with an equal volume of Freund's complete adjuvant. 6-8 week old Balb / c female mice were injected subcutaneously at multiple points on their backs, with each mouse receiving 100 μg of Amuc_1100 protein.

[0007] (2) Immunization schedule: On days 14 and 28 after the initial immunization, booster immunizations were performed using Amuc_1100 protein emulsified with Freund's incomplete adjuvant. Blood was collected from the tail vein on day 35, and serum titers were detected by ELISA. The mice with the highest titers were used for cell fusion.

[0008] (3) Cell fusion and screening: Spleen cells from immunized mice were mixed with SP2 / 0 myeloma cells at a ratio of 5:1 and fused using PEG1450. After fusion, the cells were screened using HAT medium, and the supernatant of the hybridoma cells was used for ELISA screening. Positive clones were subcloned using the limiting dilution method for three consecutive times to obtain hybridoma cell lines that stably secrete monoclonal antibodies.

[0009] (4) Antibody production and purification: Hybridoma cells were injected into the peritoneal cavity of mice to induce ascites. The ascites was collected and purified by Protein G affinity chromatography to obtain purified monoclonal antibodies.

[0010] Example 2: Establishment of a double-antibody sandwich ELISA detection method (1) Antibody pairing screening: The best antibody pair was screened by checkerboard titration to determine the capture antibody C2 (2 μg / mL) and the detection antibody D5-HRP (1:5000 dilution).

[0011] (2) Establishment of standard curve: The Amuc_1100 protein standard was serially diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0 ng / mL, and ELISA was performed under optimized conditions. The four-parameter Logistic curve was fitted, R²>0.995, and the linear range was 1-1000 ng / mL.

[0012] 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 and add 1 mL of lysis buffer (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton) Mix X-100, 0.5% SDS, and 1x protease inhibitor (mixture), vortex to mix, incubate on ice for 30 minutes, sonicate (200W power, 2s on, 2s off, 3 minutes in total), centrifuge at 12,000g at 4°C for 20 minutes, and use the supernatant as the test solution.

[0013] (2) Fermentation broth sample: Take 1 mL of fermentation broth, centrifuge at 12,000 g for 20 minutes at 4°C, and take the supernatant as the test solution.

[0014] (3) End products (capsule contents, solid beverages, etc.): Take a sample equivalent to 100mg of the original sample, add 1mL of lysis buffer, process it according to the method of lyophilized powder sample, and take the supernatant as the test solution.

[0015] 3.2 Testing Operation (1) Add the sample extract to an ELISA plate coated with capture antibody C2 (100 μL / well) and incubate at 37°C for 1 hour.

[0016] (2) After washing 3 times, add HRP-labeled detection antibody D5 (100 μL / well, 1:5000 dilution) and incubate at 37°C for 30 minutes.

[0017] (3) After washing 3 times, add TMB colorimetric solution (100 μL / well) and incubate at 37°C in the dark for 15 minutes.

[0018] (4) Add stop solution (50 μL / well) and immediately read the OD450 value on the microplate reader.

[0019] 3.3 Result Calculation and Judgment (1) Plot a standard curve with the logarithm of the concentration of the standard as the abscissa and the OD450 value as the ordinate, fit a four-parameter Logistic equation, and R² should be ≥0.995.

[0020] (2) Substitute the OD450 value of the sample into the standard curve equation to calculate the measured concentration (ng / mL) of Amuc_1100 protein in the sample.

[0021] 3.4 Standards for Passing and Failure Validity determination: The standard curve R² should be ≥0.995; the OD450 value of the positive control should be within the linear range of the standard curve; the OD450 value of the negative control should be below the detection limit (<0.1 ng / mL). Otherwise, the batch of experiments is invalid.

[0022] Sample judgment: Qualified products (normal Amuc_1100 protein expression): must simultaneously meet the following requirements: (1) The measured concentration of the sample is within the linear range of the standard curve; (2) The calculated concentration of Amuc_1100 protein is ≥ 3 ng / mL (i.e. not less than 3 times the lower limit of the standard curve).

[0023] Non-compliant sample (low Amuc_1100 protein expression): The measured concentration of the sample is within the linear range of the standard curve, but the calculated measured concentration of Amuc_1100 protein is < 3 ng / mL.

[0024] Seriously unqualified (Amuc_1100 protein not detected): The sample OD450 value is lower than the detection limit of the standard curve (<0.1ng / mL).

[0025] Completely unqualified: If the test sample is found to have both Amuc_1100 protein and P9 protein or Amuc_1409 protein in a "seriously unqualified" state, it will be judged as completely unqualified and will not pass.

Claims

1. A monoclonal antibody that specifically binds to the Amuc_1100 protein of Akkermansia muciniphila.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody was obtained by cell fusion screening after immunizing mice with recombinant Amuc_1100 protein as an immunogen.

3. An ELISA detection kit for detecting Amuc_1100 protein in AKK bacteria, characterized in that, It contains the monoclonal antibody as described in claim 1 or 2.

4. The ELISA detection kit according to claim 3, characterized in that, The kit is a double-antibody sandwich ELISA kit, containing a capture antibody and a detection antibody, which respectively recognize different antigenic epitopes of the Amuc_1100 protein.

5. The ELISA detection kit according to claim 3, characterized in that, The kit also includes the Amuc_1100 protein standard.

6. The application of the monoclonal antibody or ELISA detection kit according to any one of claims 1 to 5 in the quantitative detection of Amuc_1100 protein in AKK bacteria or in product quality control.