Specific monoclonal antibody of AKK bacterial functional protein Amuc_1100, kit, detection method and application thereof

Abstract

The application belongs to the field of biological detection, and particularly relates to a specific monoclonal antibody of an AKK bacterial efficacy protein Amuc_1100, a kit, a detection method and application thereof. The antibody pair is 3E10 and 2F7 monoclonal antibodies, the heavy chain and light chain variable region amino acid sequences of 3E10 are SEQ ID NO. 2 and SEQ ID NO. 4, and the heavy chain and light chain variable region amino acid sequences of 2F7 are SEQ ID NO. 6 and SEQ ID NO. 8. The antibody pair has high specificity and high affinity, a colloidal gold rapid detection kit and a double antibody sandwich ELISA quantitative detection kit based on the antibody pair realize rapid screening and accurate quantification of the Amuc_1100 protein, have good linearity, high precision and excellent accuracy, and can be used for detection of the protein in mucoid Akkermansia and quality control and activity evaluation of related probiotic preparations, thereby providing key technical support for related basic research and industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a specific monoclonal antibody, kit, detection method, and application of the AKK bacterium functional protein Amuc_1100. Background Technology

[0002] Amuc_1100 protein, a core functional protein of Akkermansia muciniphila (AKK), plays a crucial role in physiological processes such as intestinal microenvironment homeostasis regulation and host metabolic balance maintenance. Its research has become a key focus in microbiology, clinical medicine, and bioengineering. With the deepening research into the probiotic mechanisms of AKK, the functional value of Amuc_1100 protein is being continuously explored. This not only provides key targets for elucidating the interaction between the gut microbiota and the host but also lays the foundation for the development of related functional products. Therefore, the demand for efficient preparation and accurate detection technologies for this protein is increasingly urgent.

[0003] In terms of protein preparation technology, the acquisition of existing microbial functional proteins mainly relies on extraction from natural strains or preparation using recombinant expression systems. For the Amuc_1100 protein, the culture conditions of natural AKK bacteria are harsh, the growth cycle is long, and the natural expression level of this protein in these strains is extremely low. Direct extraction from natural strains is not only complex and time-consuming, but also presents problems such as high extraction costs and difficulty in controlling purity, failing to meet the demands of subsequent experiments and applications for high-purity, large-scale protein production. While conventional prokaryotic expression systems are widely used in the preparation of various recombinant proteins, the expression of Amuc_1100 protein often faces specific challenges, such as low soluble protein expression ratios, easy formation of inclusion bodies in the expression product, and easy loss of activity during purification. This results in recombinant proteins that fail to meet the requirements of subsequent experiments such as antibody preparation and functional verification in terms of concentration, purity, and activity, hindering the advancement of related research.

[0004] In the field of detection technology, existing protein detection methods such as Western blotting and conventional ELISA have significant limitations when applied to the detection of Amuc_1100 protein. Western blotting is cumbersome, has a long detection cycle, and poor quantitative accuracy, making it only suitable for qualitative analysis of small samples and unable to meet the needs of rapid quantitative detection of large batches of samples. Conventional ELISA methods lack highly specific and high-affinity antibodies specific to Amuc_1100 protein, making them prone to cross-reaction with other gut microbiota proteins or other proteins in biological samples, resulting in insufficient detection sensitivity and poor specificity, making it difficult to achieve accurate quantification and specific identification of Amuc_1100 protein in samples. In addition, the application of mainstream high-efficiency immunoassay technologies such as colloidal gold immunochromatography and double-antibody sandwich ELISA in the detection of Amuc_1100 protein has not yet formed a standardized protocol. The core antibody pairing and screening lacks systematic optimization, and key parameters of the detection system (such as antibody coating concentration, reaction temperature and time, buffer formulation, sample processing conditions, etc.) have not been clearly and reasonably set, making it difficult to guarantee the reliability, stability, and repeatability of related detection technologies.

[0005] These technological bottlenecks not only severely hinder in-depth basic research on the physiological functions and mechanisms of action of the Amuc_1100 protein, but also result in a lack of effective technical means for activity evaluation and quality control of functional products based on this protein (such as AKK probiotic preparations and related biopharmaceutical products) during the research and development process, thus limiting their industrialization and market application. Therefore, developing a complete technical system encompassing efficient recombinant expression, high-purity purification, specific monoclonal antibody preparation, and standardized detection methods for the Amuc_1100 protein can not only fill the current gap in specialized research tools for this protein and provide key technical support for elucidating its physiological functions and mechanisms of action, but also provide accurate and efficient detection solutions for the research, production, and quality monitoring of AKK-related functional products. This will promote the industrial application of microbial functional proteins in biopharmaceuticals, health products, and clinical diagnostics, possessing significant academic value, clinical significance, and market application prospects. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides an optimal pairing of Amuc_1100 protein-specific monoclonal antibodies 3E10 and 2F7, as well as a colloidal gold rapid detection system and a dual-antibody sandwich ELISA quantitative detection method based on these paired antibodies, enabling highly efficient and specific detection of Amuc_1100 protein.

[0007] The technical solution of this invention is as follows:

[0008] On one hand, the present invention provides a specific monoclonal antibody pair for the active protein Amuc_1100 of AKK bacteria, consisting of a 3E10 monoclonal antibody and a 2F7 monoclonal antibody; the amino acid sequence of the heavy chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region of the 2F7 monoclonal antibody is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0009] In another aspect, the present invention provides a nucleic acid molecule encoding the aforementioned specific monoclonal antibody pair, wherein the nucleotide sequence encoding the heavy chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.3;

[0010] The nucleotide sequence encoding the heavy chain variable region of the 2F7 monoclonal antibody is shown in SEQ ID NO.5, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.7.

[0011] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0012] In another aspect, the present invention provides a host cell comprising the aforementioned specific monoclonal antibody pair or nucleic acid molecule or expression vector.

[0013] In another aspect, the present invention provides a kit for detecting Amuc_1100 protein, comprising the aforementioned specific monoclonal antibody pair or nucleic acid molecule or expression vector or host cell.

[0014] Specifically, the kit is a colloidal gold detection kit; the kit also includes one or more of the following: sample extraction solution, sample pad treatment solution, and reconstitution solution.

[0015] Specifically, the kit is a double-antibody sandwich ELISA detection kit; the kit also includes one or more of the following: enzyme-labeled secondary antibody, chromogenic solution, stop solution, washing solution, blocking solution, and coating buffer.

[0016] In another aspect, the present invention provides a method for detecting Amuc_1100 protein, using the aforementioned kit.

[0017] Specifically, the colloidal gold detection method includes the following steps:

[0018] Sample pretreatment: Take the sample, add sample dilution buffer, mix well, and prepare the test solution;

[0019] Sample addition test: Place the colloidal gold test strip horizontally, add the test solution into the sample well, start timing immediately, and incubate at room temperature;

[0020] Result interpretation: Observe the color development of the T line and C line on the test strip within 15 minutes. Color development of the C line indicates a valid detection, color development of the T line indicates a positive Amuc_1100 protein test, and no color development of the T line indicates a negative result. Observation results after 15 minutes are invalid.

[0021] Specifically, the quantitative detection method using a double-antibody sandwich ELISA includes the following steps:

[0022] ELISA plate coating: Dilute 3E10 monoclonal antibody with coating buffer, incubate, discard coating buffer, wash with washing buffer, add blocking buffer, block, discard blocking buffer and dry.

[0023] Incubation of Standards and Samples: Dilute the Amuc_1100 protein standard to a gradient concentration, dilute the sample proportionally, add standard / blank control / sample to be tested to each well, seal the membrane and react;

[0024] Antibody incubation: Dilute the horseradish peroxidase-labeled 2F7 monoclonal antibody with dilution buffer, seal the membrane, react, discard the solution, and wash with washing buffer.

[0025] Color development and termination: Add color development solution, seal the film and incubate in the dark, then add stop solution to terminate the reaction;

[0026] Quantitative calculation: Read the OD value at 450nm wavelength on the microplate reader, fit a linear regression equation with the Amuc_1100 standard concentration as the x-axis and the calibrated OD value as the y-axis, and substitute the calibrated OD value of the sample to calculate the Amuc_1100 protein concentration in the sample; the calibrated OD value is the sample / standard OD at 450nm value minus the absorbance value of the blank control well.

[0027] In another aspect, the present invention provides the application of the aforementioned specific monoclonal antibody pair, nucleic acid molecule, expression vector, host cell, or kit in the detection of Amuc_1100 protein in Akkermansia myxoma.

[0028] In another aspect, the present invention provides the application of the aforementioned specific monoclonal antibody pairs, nucleic acid molecules, expression vectors, host cells, or kits in the quality control and activity evaluation of Akkermansia muciniformis probiotic preparations.

[0029] The beneficial effects of this invention are as follows:

[0030] The 3E10 / 2F7 paired antibody obtained in this invention has high specificity and high affinity. Two detection methods based on this antibody have achieved accurate quantification and rapid screening of Amuc_1100 protein, respectively. The methods are simple to operate and the results are reliable, filling the gap in specialized detection tools for this protein and providing key technical support for related basic research and product quality control. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of the nH-Amuc_1100-pet28a prokaryotic expression vector.

[0032] Figure 2 This is a standard curve for detecting protein concentration using the BCA method.

[0033] Figure 3 The graph shows the antiserum titer detection curve after mice were immunized with Amuc_1100 protein; Ab-con in the graph represents the antibody concentration, and lgAb-con represents the logarithm of the antibody concentration.

[0034] Figure 4 The figure shows the response curve of a double-antibody sandwich ELISA using paired antibodies 3E10 / 2F7; Ab-con in the figure represents the antibody concentration, and lgAb-con represents the logarithm of the antibody concentration.

[0035] Figure 5 This is a diagram showing the preparation of a standard curve for detecting Amuc_1100 protein using a double-antibody sandwich ELISA.

[0036] Figure 6 This is a standard curve fitting diagram for the detection of Amuc_1100 protein using a double-antibody sandwich ELISA based on 3E10 / 2F7 paired antibodies.

[0037] Figure 7 This is a graph showing the results of specificity verification for colloidal gold detection.

[0038] Figure 8 The image shows the results of repeatability verification for colloidal gold detection.

[0039] Figure 9 This is a graph showing the results of the parallelism verification of colloidal gold detection. Detailed Implementation

[0040] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1: Prokaryotic Expression and Purification of Amuc_1100

[0042] 1.1 Preparation of main reagents

[0043] (1) LB liquid culture medium: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, autoclave at 121℃ for 30min, and store at 4℃.

[0044] (2) 20% 10× glucose solution: 200g / L glucose, used after filtration and sterilization.

[0045] (3) 0.2M IPTG inducer: IPTG 0.1g / mL, filtered and sterilized, then dispensed and stored at -20℃.

[0046] (4) Kanamycin: Kanamycin powder (kanamycin sulfate) 50 mg / mL, filtered and sterilized, then dispensed and stored at -20 ℃.

[0047] (5) 5×SDS-Running Buffer: 15.15 g / L tris(hydroxymethyl)aminomethane, 72 g / L glycine, 5 g / L sodium dodecyl sulfate, stored at room temperature. Dilute with ddH2O to 1×SDS-Running Buffer and use for polyacrylamide gel electrophoresis.

[0048] (6) Coomassie Brilliant Blue Decolorizing Agent: 100 mL methanol, 100 mL glacial acetic acid, ddH2O to a final volume of 1000 mL, store at room temperature.

[0049] (7) 10×PBS: 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate and 0.24 g of potassium dihydrogen phosphate in 800 mL of ddH2O, adjust the pH to 7.4 with HCl and bring the volume to 1L, sterilize at 121 °C for 30 min, and store at room temperature.

[0050] 1.2 Construction of expression vector

[0051] The nH-Amuc_1100-pet28a plasmid was synthesized by Suzhou Genewiz, and DH5a and BL21(DE3) were purchased from Shanghai Sangon Biotech.

[0052] Plasmid transformation of E. coli BL21(DE3): Take 2 μL of nH-Amuc_1100-pet28a plasmid ( Figure 1Mix the bacterial culture with BL21(DE3) competent cells (thawed on ice beforehand) and incubate on ice for 5 min to promote DNA uptake. Heat shock the mixture at 42°C for 90 s, then immediately transfer it to ice and cool for 10 min. Add 900 μL of LB liquid culture medium and incubate at 37°C on a shaker for 1 h. Spread the thawed bacterial culture onto plates containing kanamycin resistance and incubate overnight at 37°C. Pick well-grown colonies from the overnight incubation plates, inoculate with LB liquid medium containing kanamycin resistance, and incubate overnight at 37°C. Send a small amount of the bacterial culture for sequencing analysis by Genewiz.

[0053] 1.3 Preservation of Expression Strains

[0054] For the bacterial strain that has been correctly sequenced, take 500 μL of overnight culture and add 500 μL of 50% glycerol, mix well, and store at -80℃.

[0055] 1.4 Induced expression of recombinant Amuc_1100

[0056] The bacterial strain was inoculated into LB medium containing kanamycin resistance and activated overnight at 37 °C. The overnight culture was then inoculated into LB medium at a rate of 1% (v / v) and incubated at 220 r / min for 2-3 h at 37 °C until the OD of the culture was reached. 600 When the cells reached a growth rate of 0.4-0.6, IPTG was added to a final concentration of 1 mM, and expression was induced at 37°C for 3-5 hours. After induction, the cells were collected by high-speed centrifugation at 5000 rpm at 4°C, the supernatant was discarded, the cells were resuspended in ddH2O, and the cells were collected again at 5000 rpm. This process was repeated three times. The collected cells were stored at -20°C for later use.

[0057] 1.5 SDS-PAGE Detection Results

[0058] The bacterial cells induced by 1.4 were resuspended in lysis buffer, homogenized by high-pressure homogenizer, centrifuged at 12000 r / min for 10 min, and the supernatant and precipitate were collected for gel analysis.

[0059] (1) Rubber preparation

[0060] The ingredients are shown in Table 1:

[0061] Table 1 Rubber Compound Composition

[0062]

[0063] (2) Injection of glue

[0064] Fix two 1.5 mm thick glass plates onto the apparatus frame. Prepare a 12% separating gel according to (1), mix well, and add the separating gel between the two glass plates, avoiding air bubbles, until it reaches 2 / 3 of the glass plate. Gently add 2 mL of anhydrous ethanol on top of the solution to seal the surface of the separating gel. Let it stand at room temperature for 40 min. After separation and solidification, pour out the anhydrous ethanol and aspirate dry. Prepare a 5% stacking gel according to (1), mix well, pour it onto the top layer of the separating gel, and quickly insert the comb. Let it stand at room temperature for 40 min. After the stacking gel polymerizes, carefully remove the comb. Fix the glass plates in the electrophoresis tank and add 1×SDS-PAGE buffer.

[0065] (3) Sample loading

[0066] Take the sample, add protein loading buffer, mix well, and incubate in a 95 ℃ metal bath for 5 min. After returning to room temperature, load 20 μL of sample into each well.

[0067] (4) Electrophoresis

[0068] Turn on the power, 80 V, 30 min, then 120 V to continue electrophoresis until the bromophenol blue reaches the bottom of the separating gel.

[0069] (5) Coomassie brilliant blue staining

[0070] After electrophoresis, remove the gel and carefully place it in a plastic box. Wash three times with ddH2O for 5 minutes each time. Add Coomassie Brilliant Blue staining solution and shake on a horizontal shaker for 40 minutes.

[0071] (6) Decolorization

[0072] The staining solution was recovered, the gel was rinsed with ddH2O, and the destaining solution was poured in and the gel was shaken overnight on a horizontal shaker. The images were then observed in an imaging system.

[0073] 1.6 Purification of Recombinant Amuc_1100

[0074] (1) Regeneration of the Ni affinity column: Connect the Ni affinity column to a peristaltic pump and flush with ddH2O for 5 column volumes. Then flush with EDTA solution for 5 column volumes, followed by ddH2O for 5 column volumes. Flush with 0.2 M NaOH solution for 5 column volumes, followed by ddH2O for 10 column volumes. Pack the affinity column with NiSO4 solution, flush with 5 column volumes, and then flush with ddH2O for 5 column volumes. The above are the steps for regenerating the Ni affinity column.

[0075] (2) Preparation of purified samples: The results of SDS-PAGE electrophoresis confirmed that the recombinant Amuc_1100 was partially soluble. The bacterial culture expressing recombinant Amuc_1100 was resuspended in low concentration imidazole buffer, homogenized under high pressure, centrifuged at 12000 r / min for 10 min, and the supernatant was collected.

[0076] (3) Equilibration of Ni affinity column: Connect the Ni affinity column to the peristaltic pump and wash for 5 column volumes with low concentration imidazole salt buffer.

[0077] (4) Sample loading: The Ni affinity column is connected to the peristaltic pump, and the protein lysis buffer is slowly and constantly passed through the Ni affinity column at 4°C.

[0078] (5) Washing: Use a low concentration of imidazole buffer (10 mM imidazole) to flush the Ni affinity column at a rate of 2-5 mL / min for about 5 column volumes.

[0079] (6) Elution of target protein Amuc_1100: Elution was performed in stages using imidazole buffer containing 50, 100, 200, 300 and 400 mM respectively, at a flow rate of 2~5 mL / min. Elution peaks were collected at each stage, and each concentration of imidazole buffer was passed through 2 column volumes.

[0080] (7) Rinse the Ni affinity column with ddH2O for 5 column volumes, and then rinse the Ni affinity column with 20% ethanol for 3 column volumes. The flow rate is 2-5 mL / min. The column is always kept in a low temperature environment.

[0081] (8) Ultrafiltration concentration: The collected protein samples were analyzed by SDS-PAGE to determine the molecular weight and purity of the fusion protein. The high-purity protein obtained by SDS-PAGE was then concentrated.

[0082] 1.7 Protein Concentration Detection

[0083] The BCA method for quantitative detection of protein concentration follows these steps:

[0084] (1) Preparation of BCA working solution: Based on the calculated total amount of BCA, prepare the working solution by mixing reagent A and reagent B in a ratio of 50:1 and place it on ice.

[0085] (2) Preparation of standard products: Prepare protein standard products according to Table 2.

[0086] Table 2 Protein Standards

[0087]

[0088] (3) Protein concentration determination: Add 20 μL of standard and diluted sample to a 96-well plate, add 200 μL of BCA working solution to each well, mix well and incubate at 37℃ for 30 min, measure absorbance at 562 nm using an ELISA reader, plot a standard curve (see Table 3), and calculate protein concentration.

[0089] Table 3. Protein standard concentrations and average OD values

[0090]

[0091] (4) Protein standard curve

[0092] See protein standard curve Figure 2 .

[0093] Example 2: Preparation and Screening of Monoclonal Antibodies

[0094] In this experiment, Amuc_1100 expressed in prokaryotes was used as an antigen to immunize 6-8 week old female BALB / c mice and prepare monoclonal antibodies. Finally, 10 monoclonal antibodies with good reactivity and specificity were screened.

[0095] 2.1 Cells and Laboratory Animals

[0096] HEK293T and SP2 / 0 myeloma cells were purchased and preserved by our company; BALB / c female mice (SPF, 6-8 weeks old) were purchased from an animal experimental breeding farm and raised in a sterile room.

[0097] 2.2 Preparation of main reagents

[0098] (1) Hybridoma cell cryopreservation solution: 90 mL fetal bovine serum and 10 mL dimethyl sulfoxide were mixed evenly, aseptically dispensed, and stored at 4°C.

[0099] (2) Ascites purification / washing buffer: Weigh 8.766 g NaCl and 2.839 g Na2HPO4, place them in a 1L beaker, add about 800 mL of deionized water, adjust the pH to 7.0, stir to dissolve, add deionized water to make up to 1L of the solution, and store at room temperature.

[0100] (3) Ascites purification elution buffer: Weigh 7.506 g g of glycine, place it in a 1L beaker, add about 800 mL of deionized water, adjust the pH to 3.0, stir to dissolve, add deionized water to make up to 1L of solution, and store at room temperature.

[0101] (4) Ascites purification and neutralization buffer: Weigh 157.6g Tris-HCl, place it in a 1L beaker, add about 800 mL of deionized water, adjust the pH to 8.5, stir to dissolve, add deionized water to make up to 1L, and store at room temperature.

[0102] 2.3 Mouse Immunization

[0103] 50 μg of purified Amuc_1100 recombinant protein was used as the antigen, mixed with an equal volume of CFA adjuvant, emulsified, and administered subcutaneously at multiple sites to 6-8 week old female Balb / C mice at a dose of 50 μg / mouse. A total of three immunizations were administered, with a two-week interval between each. Serum titers were measured two weeks later. Myeloma cell SP2 / 0 fusion was initiated if the titer was greater than 1:1,000,000. A booster immunization of 50 μg of Amuc_1100 recombinant protein was administered intraperitoneally three days before fusion. Figure 3 (Table 4) shows that the titer of the immune antiserum is >2,000,000.

[0104] Table 4. Results of antiserum titer detection after Amuc_1100 protein immunization in mice.

[0105]

[0106] Note: In the table, Ab-con represents the antibody concentration (μg / mL), and lgAb-con represents the logarithmic value of the antibody concentration.

[0107] 2.4 Cell Fusion

[0108] (1) Preparation of SP2 / 0 myeloma cells

[0109] 7-10 days before cell fusion, SP 2 / 0 cells stored in liquid nitrogen were removed and rapidly thawed in a preheated 37°C water bath. Cells were then revived and cultured for 2-3 passages using HyGro hybridoma cell serum-free medium. Cells of uniform size, with clear cell boundaries and free of impurities in the culture medium were selected for expansion to two flasks of T75 cells. Before cell fusion, cells were aspirated from the cells into 50 mL centrifuge tubes using sterile PBS.

[0110] (2) Preparation of feeder cells

[0111] Negative Balb / c female mice were euthanized by cervical dislocation and immersed in 75% alcohol for 10 min for disinfection. They were then transferred to a biosafety cabinet for further procedures. The mice were fixed using a dissecting board, and the abdominal skin was cut open with pre-sterilized scissors, extending down to the peritoneum. 10 mL of HAT culture medium was injected into the peritoneal cavity using a sterile syringe. The peritoneal cavity was then compressed, and the syringe was repeatedly aspirated to extract macrophages into 180 mL of culture medium. This step was repeated twice. The entire procedure was performed under aseptic conditions.

[0112] (3) Preparation of immune spleen cells

[0113] Mice that had completed immunization and had high serum titers were selected and euthanized by euthanasia via orbital bleeding. The blood was collected and centrifuged at 3000 rpm for 10 min, and the supernatant was collected as positive serum. The mice were then disinfected by immersing them in 75% alcohol for 5 min. The spleen was removed using the same procedure as for feeder cells; it was visibly enlarged and swollen. The spleen was placed on a cell strainer and gently ground with a grinding rod. The spleen cells were then resuspended in serum-free culture medium, and the suspension was collected in a 50 mL centrifuge tube. The tube was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded.

[0114] (4) Cell fusion

[0115] In a 50 mL centrifuge tube, the pre-treated SP2 / 0 myeloma cells and spleen lymphocytes were mixed at a ratio of 1:10. The mixture was centrifuged at 1000 rpm for 10 min to collect the cells. The supernatant was discarded, and the bottom of the tube was gently tapped to loosen the cell pellet. Using a sterile disposable dropper, 1 mL of preheated 37°C 50% PEG was slowly added to the centrifuge tube over 1 min (slow rotation of the centrifuge tube enhances the fusion effect). After adding the PEG, the tube was placed in a 37°C water bath for 1 min. Then, at the next 1 min, 2 min, 3 min, 4 min, and 5 min, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of serum-free culture medium were added sequentially to a final volume of 30 mL to terminate PEG-induced cell fusion. The tubes were centrifuged at 1000 rpm for 10 min. Discard the supernatant; add an appropriate amount of HAT medium to the centrifuge tube, add the resuspended and mixed cells to the serum-free medium with feeder cells added above, and then add 200 μL to each well of a 96-well cell culture plate; place the 96-well plate in a 37°C CO2 incubator for further culture; observe cell growth 5-7 days after fusion, discard 100 μL of medium and add fresh HAT medium, discard the original medium and replace it with HT medium 36-48 h after the first medium change, wait until the cell density of the fused cells is about 20% and then replace it with HT medium again, and then 24-36 h later, aspirate the cell supernatant for antibody detection.

[0116] 2.5 Screening of positive hybridoma cells

[0117] Positive hybridoma cells were screened using an indirect ELISA method. ELISA plates coated with Amuc_1100 protein were prepared in advance. 96-well plates and ELISA plates were labeled for easy selection of positive wells. In a biosafety cabinet, 50 μL of supernatant from the 96-well cell plate was added to the prepared ELISA plate and incubated at 37°C for 1 h. The plate was washed 5 times, and HRP goat anti-mouse antibody diluted 1:5000 with PBS (containing 1% BSA) was added. The plate was incubated for 1 h, washed 5 times, and 100 μL of TMB was added to each well for 15 min of color development. Then, 50 μL of stop solution was added. Unconverged SP 2 / 0 cell supernatant was used as a negative control, 1:500 diluted positive serum as a positive control, and PBS solution as a blank control. A P / N ratio ≥ 2.1 was used as the positive cutoff value.

[0118] 2.6 Subcloning of hybridoma cells

[0119] Subcloning of hybridoma cells was performed using the limiting dilution method: Positive hybridoma cells with good cell condition and relatively homogeneous cell communities were selected. Cells were resuspended in 1 mL of serum-free medium containing HT and counted. After counting, 200 cells were added to 10 mL of medium containing HT for reselection and mixing. 200 μL of the cell suspension was added to 24 wells (rows A and B) of a 96-well cell culture plate, approximately 4 cells per well. 5 mL of medium was added to the cell suspension, and 200 μL was added to rows C and D, approximately 2 cells per well. 5 mL of medium was added to each well, and 200 μL was added to rows E to H, approximately 1 cell per well. Each plate was labeled, and cell condition was observed periodically. The cells were incubated at 37°C in a 5% CO2 incubator for 5-7 days. The hybridoma cell supernatant was collected for ELISA testing, and cell lines with an OD value greater than 2.5 were selected. After three subcloning processes, ten monoclonal antibodies were obtained, named 4A6, 5A8, 3E10, 7C3, 6D5, 2F7, 3F11, 10F2, 2H9, and 4G8. Hybridoma cells with 100% positivity were expanded and cultured, then cryopreserved in liquid nitrogen for future use.

[0120] 2.7 Preparation and purification of monoclonal antibody-containing ascites fluid

[0121] Before preparing ascites fluid, paraffin injection is performed into the peritoneal cavity of mice to create a suitable environment for antibody cell growth. Cells can be injected one week after paraffin injection. About one week after cell injection, the mice's abdomen will show obvious swelling, at which point ascites fluid needs to be collected; otherwise, the mice will die. The collected ascites fluid needs to be centrifuged, and the supernatant is collected and stored at -80℃ for later use. Some ascites fluid can be mixed with an equal volume of glycerol and stored at -20℃ for short-term storage and immediate experimental use. The mouse ascites fluid is centrifuged at 13,400×g at 4℃ for 10 min, the supernatant is collected and filtered through a 0.45 μm filter membrane, and purified using a Protein A column to obtain monoclonal antibodies 4A6, 5A8, 3E10, 7C3, 6D5, 2F7, 3F11, 10F2, 2H9, and 4G8.

[0122] 2.8 Identification of the purified monoclonal antibody subtype

[0123] Purified Amuc_1100 protein was used as the antigen to coat the plate (1 μg / mL, 100 μL / well) and incubated overnight at 4°C. The plate was washed once with 1×PBST. Blocking was performed with 2% BSA (250 μL / well) at room temperature for 1 h. After washing three times with 1×PBST, 100 pL / well of diluted 1 μg / mL monoclonal antibody was added to each well. The negative control was SP2 / 0 cell culture supernatant. The plates were incubated at 37°C for 35 min. After washing five times with 1×PBST, 100 μL / well of diluted HRP goat anti-mouse IgG antibody (1:10000) was added and incubated at 37°C for 35 min. After washing five times with 1×PBST, 100 pL / well of TMB chromogenic solution was added and incubated at room temperature for 5 min. Finally, 100 pL / well of stop solution was added to terminate the reaction. OD was measured using a microplate reader within 30 min. 450 Value. (sample well OD) 450 Value - Blank Hole OD 450 Value) / (Negative pore OD) 450 Value - Blank Hole OD 450 A value > 2.1, i.e., S / N ≥ 2.1, is considered positive. Subtype identification was performed using a mouse monoclonal antibody typing kit.

[0124] Table 5. Identification of Monoclonal Antibody Subtypes

[0125]

[0126] The supernatant of 10 positive hybridoma cells was used as the primary antibody. The monoclonal antibody subtypes were identified according to the mouse monoclonal subtype identification kit. The results are shown in Table 5. 4A6, 3E10, 6D5, 3F11, and 2H9 belong to IgG1, 5A8 and 10F2 belong to IgG2a, and 7C3, 2F7, and 4G8 belong to IgG2b. The light chains of all of them are Kappa chains.

[0127] 2.9 Pairing screening of monoclonal antibodies against Amuc_1100 protein

[0128] A double-antibody sandwich ELISA method was used to screen for paired antibodies capable of detecting the Amuc_1100 antigen. Ten purified monoclonal antibodies against Amuc_1100 protein were non-site-specifically biotin-labeled at a biotin to antibody molar ratio of 20:1. The biotin-labeled antibodies served as the paired detection antibodies. Ten unlabeled monoclonal antibodies were coated at 2 μg / mL onto polyvinyl chloride (PVC) plates as coating antibodies. Amuc_1100 protein was added at 1 μg / mL and in 3-fold serial dilutions to ELISA plates coated with the ten different antibodies. After one hour of binding, each ELISA plate was supplemented with nine biotin-labeled detection antibodies (2 μg / mL, excluding the coating antibody), and SA-HRP was used for color development. The specific procedures are as follows:

[0129] (1) Dilute the coating antibody with 20mM PB, pH7.4 coating solution to 1μg / mL, add 100μL to each well of a 96-well microplate (i.e., 100ng / well), and incubate overnight at 4℃.

[0130] (2) Remove the liquid from the wells, wash each well three times with 300 μL of PBST washing buffer, add 300 μL of blocking buffer (5% skim milk powder, prepared with PBST), and incubate at 37°C for 1 h. Add 100 μL of Amuc_1100 recombinant antigen sample diluted 2-fold to each well and incubate at 37°C for 1 h.

[0131] (3) Remove the liquid from the wells and wash each well three times with 300 μL of washing buffer. Dilute the detection antibody to 1 μg / mL with 20 mM PB, pH 7.4, and add 100 μL of detection antibody to each well.

[0132] (4) Remove the liquid from the wells, wash each well three times with 300 μL of washing buffer, add 100 μL of HRP-labeled goat anti-mouse antibody (1:5000, diluted with PBST), and incubate at 37°C for 30 min.

[0133] (5) Remove the liquid from the wells, wash each well 5 times with 300 μL of washing buffer, add 100 μL of colorimetric solution, and develop the color at 37°C for 10 min. Then add 50 μL of 2 mol / L H2SO4 stop solution. Within 20 min after adding the stop solution, read the OD value using a microplate reader. 450 Numerical value.

[0134] The obtained monoclonal antibodies were paired up and used as coating and detection antibodies, respectively. A double-sandwich ELISA method was used to detect diluted Amuc_1100 protein recombinant antigen samples. Finally, the pair of paired antibodies 3E10 and 2F7, which showed the strongest pairing and signal, were selected. The detection results of paired antibodies 3E10 and 2F7 are shown below. Figure 4 As shown in Table 6.

[0135] Table 6. Detection response data of double-antibody sandwich ELISA with paired antibodies 3E10 and 2F7.

[0136]

[0137] Note: In the table, Ab-con represents the antibody concentration (μg / mL), and lgAb-con represents the logarithmic value of the antibody concentration.

[0138] Hybridoma cell lines 3E10 and 2F7 were cultured to a cell density of 1E+07 cells / mL and sent for hybridoma monoclonal antibody sequencing (Suzhou Genewiz Biotechnology Co., Ltd.). Gene sequences encoding signal peptides, variable regions, and constant regions of the antibody heavy and light chains were synthesized and constructed into mammalian cell expression vectors. Any commercially available mammalian cell expression vector, such as pTT5 or pCDNA3.1, could be used. Recombinant plasmids were transfected into HEK293 mammalian cells, secreting and expressing 3E10 and 2F7 antibodies. The cell expression supernatant was purified by protein A affinity to obtain Amuc_1100 protein-specific recombinant antibodies 3E10 and 2F7. These were used for antibody characterization and ELISA kit development.

[0139] Sequencing results of antibody 3E10 and 2F7 amino acid sequences:

[0140] (1) Results of the variable region of the 3E10 heavy chain

[0141] Base sequence (SEQ ID NO.1):

[0142] Caggtgcagctgaagcagtctggggctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggcttttggctacaccttcactacctatccaatagaatggatgaagcagaatcatgggaagagcctagagtggattggaaattttcatccttacaatgatgatgctaagtacaatgaaaaattcaagggcaaggccaaattgactgtagaaaaatcctctaccacagtcttcttggagctcagccgattaacatctgatgactctgctgtttattactgtgcaagggggggctccaatgggacggattactatggtatggactactggggtcaaggaacctcagtcaccgtctcctca。

[0143] Amino acid sequence (SEQ ID NO.2):

[0144] QVQLKQSGAELVKPGASVKMSCKAFGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDAKYNEKFKGKAKLTVEKSSTTVFLELSRLTSDDSAVYYCARGGSNGTDYYGMDYWGQGTSVTVSS。

[0145] (2)Results of the variable region of the light chain of 3E10

[0146] Base sequence (SEQ ID NO.3):

[0147] Gacattgtgatgacccagtctccttcctccctagctgtgtcagttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagtatcaatcaaaacaactacttggcctggtaccagcagaagccagggcagtctcctaaattgctgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagtgtgaaggctgaagacctggcagtttattactgtcagcaatattatagctatccgctcacgttcggtactgggaccaagctggagctgaaa。

[0148] Amino acid sequence (SEQ ID NO.4):

[0149] DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSINQNNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGTGTKLELK。

[0150] (3) Results of the variable region of the 2F7 heavy chain

[0151] Base sequence (SEQ ID NO.5):

[0152] Gaggtgaagctggtggagtctggaggaggcttggtgcaacctggaggatccatgaaactctcctgtgttgcctctggattcactttcaataacttctggatgaactgggtccgccagtctccagagaaggggcttgaatgggttgctgaaattagattgaaatctgataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgattccaaaagtagtgtctacctgcaaatgaacaacttaagagctgaagacactggcatttattactgtaaccggggttttatttactggggccaagggactctggtcactgtctctgca。

[0153] Amino acid sequence (SEQ ID NO.6):

[0154] EVKLVESGGGLVQPGGSMKLSCVASGFTFNNFWMNWVRQSPEKGLEWVAEIRLKSDNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCNRGFIYWGQGTLVTVSA.

[0155] (4) Results of the variable region of 2F7 light chain

[0156] Base sequence (SEQ ID NO.7):

[0157] Gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcatc caatctagaatctgggatcccagccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggatgctgcaacctattactgtcagcaaagtgatgaggacccgtacacgttcggaggggggaccaagctggcaataaaa.

[0158] Amino acid sequence (SEQ ID NO.8):

[0159] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSDEDPYTFGGGTKLAIK.

[0160] Example 3: Detection of Amuc_1100 protein with colloidal gold

[0161] 3.1 Preparation of Colloidal Gold

[0162] (1) Envelopment conditions

[0163] NC membrane: Tianren Membrane Industry, model 140T;

[0164] 3E10 antibody streak concentration: 1.5 mg / mL;

[0165] Streak dilution: final concentration 2% sucrose + 10mM PBS buffer, pH 7.40;

[0166] 3E10 antibody streaking spray volume: 1.0 μL / cm.

[0167] (2) Marking conditions

[0168] Colloidal gold: chloroauric acid from Sinopharm; gold solution concentration: 0.04%; the mass ratio of gold particles: chloroauric acid: trisodium citrate is 1:1.3. (Colloidal gold particles are approximately 30nm).

[0169] pH setting: Add 5 μL of 0.2 mol / L potassium carbonate to 1 mL of colloidal gold to adjust the pH to approximately 6.7.

[0170] Antibody dosage: Add 20 μg of 2F7 antibody to 1 mL of colloidal gold solution and label for 10 min. Add 30 μL of 10% BSA to 1 mL of colloidal gold solution and block for 30 min.

[0171] Reconstitute after centrifugation: Reconstitute by double the original amount (add 1 mL of reconstituted solution after centrifuging 1 mL of gold solution).

[0172] Reconstitution solution formulation: 20mM Tris + 0.5% BSA (final concentration) + 5% sucrose (final concentration) + 0.5% Tween 20 (final concentration). Hydrochloric acid is used to adjust the pH of the reconstitution solution to 8.50-8.60.

[0173] Table 7 Reconstituted Solution Formulation

[0174]

[0175] Gold plating after reconstitution: The gold-labeled antibody, which has been reconstituted at the original concentration, is directly plated onto the 8964 binding pad.

[0176] (3) Components of sample extract

[0177] The sample extraction solution consisted of 10 mM PB + 0.5% Triton 100 + 0.5% sodium caseinate + 0.9% sodium chloride. NaOH was used to adjust the pH of the reconstitution solution to 7.6-8.0.

[0178] Table 8 Sample Extract Formulation

[0179]

[0180] (4) Sample pad treatment conditions

[0181] Sample pad treatment solution composition: 10mM Tris+ blocking agent, hydrochloric acid is used to adjust the pH of the reconstitution solution to 7.6-8.0, and the amount added should be adjusted as needed.

[0182] Table 9 Sample Pad Treatment Solution Formulation

[0183]

[0184] Hydrochloric acid is used to adjust the pH of the sample pad treatment solution to 7.6-8.0.

[0185] 3.2 Detection methods and result analysis

[0186] 3.2.1 Experimental Objective

[0187] Complete the accuracy, specificity, repeatability, and parallelism validation of colloidal gold.

[0188] 3.2.2 Test Materials

[0189] a. Core testing reagent: Amuc_1100 colloidal gold test strip

[0190] b. Supporting consumables:

[0191] Disposable sterile dropper (100μL / drop, no risk of cross-contamination);

[0192] Sample dilution buffer (provided by the manufacturer, containing Tris-HCl buffer, sodium chloride, preservative, pH 7.2±0.2, to ensure sample antigen stability and solubility);

[0193] Sterile centrifuge tubes (size: 1.5mL, used for sample dilution and mixing).

[0194] c. Environment and Instruments:

[0195] Testing environment: Ambient temperature constant temperature room (20-30℃, relative humidity 40%-60%, no direct sunlight, no airflow interference);

[0196] Auxiliary tools: timer, disposable gloves (powder-free latex material to avoid contaminating the sample).

[0197] 3.2.3 Sample Information

[0198] Sample information is shown in Table 10:

[0199] Table 10 Sample Information

[0200]

[0201] 3.2.4 Specific Steps

[0202] a. Sample pretreatment (operation time: 30 minutes before detection):

[0203] Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences).

[0204] Take 0.01g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 990μL of sample dilution buffer;

[0205] Use a pipette to repeatedly blow and aspirate 5-10 times to ensure the sample is fully dissolved and mixed, to prepare a 1:100 diluted test solution, and then gradually dilute it 10-fold.

[0206] b. Sample addition operation (single sample operation time: 1 minute):

[0207] Take out the Amuc_1100 colloidal gold test strip, place it horizontally on a clean lab bench, and label the sample number (to avoid confusion).

[0208] Wear disposable gloves, use a dropper to draw up the test solution, add 100 μL into the sample well of the test strip, and ensure that the liquid flows in completely without overflowing.

[0209] Start the timer immediately and record the start time.

[0210] c. Incubation and observation (incubation time: 15 minutes):

[0211] Keep the test strip horizontal during incubation and avoid moving, tilting or touching the sample well;

[0212] Within 15 minutes, observe the color development of the T and C lines on the test strip under natural light (avoid strong light), and record whether color development is present and the intensity of the color (light red / dark red, if there is a difference, please indicate it);

[0213] Results are invalid after 15 minutes (to avoid false negatives caused by excessive diffusion of the chromatography solvent).

[0214] 3.2.5 Experimental Results

[0215] (1) Specificity

[0216] The results are as follows Figure 7 As shown, only positive samples A and D showed bands in the T region, indicating that colloidal gold has strong specificity for detecting AKK-active Amuc_1100 protein.

[0217] (2) Repeatability

[0218] 100 μL of the AKK PROBIO supernatant was added to each well of the test strip, ensuring complete flow without spillage. Results are as follows: Figure 8As shown, there was no significant color difference in the T-region bands in three parallel detections, indicating that the colloidal gold has good repeatability in detecting AKK-active Amuc_1100 protein.

[0219] (3) Parallelism

[0220] Dilute the protein concentration of the standard to 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, and 0.1 ng / mL, respectively. Take 100 μL of each concentration and add it to the sample well of the test strip, ensuring that the liquid flows in completely without overflowing.

[0221] The results are as follows Figure 9 As shown, the color of the T-region band gradually fades until it disappears as the protein concentration decreases, indicating that the colloidal gold has good parallelism in detecting AKK-active Amuc_1100 protein.

[0222] In summary, this validation covered the three core performance indicators of specificity, repeatability, and parallelism. All validation results were satisfactory, indicating that this Amuc_1100 protein colloidal gold can be effectively used for the qualitative detection of active Amuc_1100 protein in AKK PROBIO.

[0223] Example 4: Detection of Amuc_1100 protein using a double-antibody sandwich ELISA

[0224] 4.1 Horseradish peroxidase-labeled antibody

[0225] (1) Take 10 μL of horseradish peroxidase solution into a clean 0.5 mL centrifuge tube.

[0226] (2) Take 10 mg of 2F7 antibody and mix it thoroughly with horseradish peroxidase by pipetting.

[0227] (3) Add 1 μL of periodate. The initiator needs to be thoroughly mixed with the solution.

[0228] (4) React at 37℃ for 1-2 hours or at 4℃ overnight.

[0229] (5) After the reaction is complete, add 3 μL of sodium cyanoborohydride and store the labeled enzyme in a 4°C refrigerator.

[0230] 4.2 Coating antibodies onto ELISA plates

[0231] (1) Dilute the 3E10 antibody to 5 μg / mL with coating buffer, coat 100 μL per well, and let stand at 4℃ for 16 h.

[0232] (2) Discard the coating buffer, add washing buffer and wash 3 times, 300 μL per well, let stand for 5 min, and pat dry on absorbent paper.

[0233] (3) Blocking: Add 200 μL of blocking buffer to each well and block at 37°C for 1 h; discard the blocking solution and dry at 37°C for 30 min.

[0234] 4.3 Test Methods

[0235] (1) Preparation of standard: Prepare and use immediately. Dilute the standard (3200 ng / mL) to 320 ng / mL with diluent, and then serially dilute (dilution factor: 2 times) to prepare the standard, such as... Figure 5 .

[0236] (2) Sample incubation: Add 100 μL of standard / blank (diluent) / sample to each well, seal with membrane, and react at 37℃ for 1 h.

[0237] (3) Washing: After incubation, discard the liquid in the wells, dilute the washing buffer with deionized water 20 times, wash 3 times (250 μL / well), and pat dry; (Note: If there are crystals in the washing buffer, it should be placed at room temperature until the crystals are completely dissolved before use.)

[0238] (4) Incubation of enzyme-labeled antibody: Prepare fresh and use immediately. Dilute the enzyme-labeled antibody 20 times with diluent, add 100 μL of working concentration of enzyme-labeled antibody to each well, seal the membrane, and react at 37°C for 1 h.

[0239] (5) Washing the plate: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250 μL / well), and pat dry.

[0240] (6) Color development: Prepare and use immediately. Mix equal volumes of color development solutions A and B, add 100 μL to each well, seal with a membrane, and incubate at 37°C in the dark for 15 min.

[0241] (7) Termination: Add 100 μL of termination solution to each well to terminate the process, and then take the reading.

[0242] (8) Reading: Place the ELISA plate into the ELISA reader and read the OD value at a wavelength of 450 nm. The measurement should be completed within 20 minutes after termination.

[0243] 4.4 Results Analysis

[0244] (1) Calculation of absorbance value:

[0245] The light absorption calibration value for each standard or sample is: OD 450 nm - Absorbance value of blank control well

[0246] (2) Fitting the standard curve:

[0247] Using the concentration of Amuc_1100 standard (X, ng / mL) as the x-axis and the corresponding calibration OD value (Y, ΔOD standard) as the y-axis, a standard curve was fitted using univariate linear regression, yielding the linear regression equation: Y = aX + b

[0248] In the formula:

[0249] Y—Calibration OD value of standard sample, unitless;

[0250] a—Slope of the standard curve, AU·mL / ng;

[0251] b—Standard curve intercept, unitless;

[0252] X—Amuc_1100 standard concentration, ng / mL. (3) Calculation of the baseline concentration of Amuc_1100 protein in the sample.

[0253] Based on the standard curve regression equation, substitute the calibrated absorbance value of the sample (ΔODsample) into the equation to calculate the baseline concentration after sample dilution. Then multiply this by the sample pretreatment dilution factor to obtain the Amuc_1100 protein mass concentration of the sample. The calculation formula is: Xmass = (… OD-like b) / a×D

[0254] In the formula:

[0255] X-quality—Sample Amuc_1100 protein concentration, ng / g;

[0256] OD sample — Sample calibration absorbance value, unitless;

[0257] a— Slope of the standard curve, AU g / ng;

[0258] b— Standard curve intercept, unitless;

[0259] D—Dilution factor during sample pretreatment, unitless.

[0260] (4) Protein quantification conversion based on Avogadro's constant

[0261] Using Avogadro's constant as the core, the protein molar concentration of Amuc_1100 is converted into molar concentration and molecular number concentration to achieve precise protein quantification. The molar mass of Amuc_1100 protein is denoted as M (unit: g / mol, calculated according to the amino acid sequence; if it is a known constant, it is directly substituted into the calculation).

[0262] (5) Conversion of molar concentration

[0263] Convert the mass concentration to mol / L using the following formula:

[0264] X_matter = X_mass × 10 9M

[0265] In the formula:

[0266] X_substance — The molar concentration of protein in sample Amuc_1100, in mol / g;

[0267] X-quality—Sample Amuc_1100 protein concentration, ng / g;

[0268] 10 9 — Unit conversion factor (ng to g);

[0269] M—Amuc_1100 represents the molar mass of protein, in g / mol.

[0270] (6) If the OD value of the sample exceeds the OD value of the highest point of the standard curve, the sample needs to be diluted and measured again.

[0271] Table 11

[0272]

[0273] result Figure 6 As shown.

[0274] Example 5: Performance Testing of the Reagent Kit

[0275] 5.1 Core Preparations Before Verification

[0276] 5.1.1 Experimental Materials and Reagents

[0277] Amuc_1100 protein ELISA kit to be validated: contains a coated plate, Amuc_1100 protein standard (positive control), detection antibody, enzyme-labeled secondary antibody, substrate solution, stop solution, and washing solution;

[0278] Specified samples: AKK PROBIO, Lactobacillus A, Bifidobacterium B, Cocci C;

[0279] Blank matrix: PBS buffer;

[0280] Auxiliary consumables: sterile centrifuge tubes, pipette tips, ELISA plate sealing film, etc.

[0281] 5.1.2 Instruments and Environment

[0282] Core instruments: Microplate reader (pre-calibrated, detection wavelength 450 nm), constant temperature incubator (37℃), pipettes (10 μL-1000 μL);

[0283] Test environment: temperature 25±2℃, humidity 40%-60%, no direct sunlight, no vibration, and avoid cross-contamination.

[0284] 5.1.3 Sample Preprocessing

[0285] Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences).

[0286] Take 0.1g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 900μL of sample dilution buffer.

[0287] Use a pipette to repeatedly pipette and aspirate to ensure the sample is fully dissolved and mixed, preparing a 1:10 dilution for testing.

[0288] Preparation of standards: Prepare fresh before use. Dilute the standard (3200 ng / mL) to 320 ng / mL with PBS buffer, then serially dilute (dilution factor: 2 times) to prepare the standard.

[0289] Add 100 μL of the test solution, blank (PBS buffer), and standard to the well, seal with a membrane, and react at 37°C for 1 hour.

[0290] Washing: After incubation, discard the liquid in the wells, dilute the washing buffer 20 times with deionized water, and wash 3 times (250 μL / well). Pat dry. (Note: If there are crystals in the washing buffer, let it stand at room temperature until the crystals are completely dissolved before use.)

[0291] Enzyme-labeled antibody incubation: Prepare fresh and use immediately. Dilute the enzyme-labeled antibody 20 times with PBS buffer, add 100 μL of working concentration enzyme-labeled antibody to each well, seal the membrane, and react at 37°C for 1 hour.

[0292] Washing: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250 μL / well), and pat dry.

[0293] Color development: Prepare fresh before use. Mix equal volumes of color development solution A (100 mL citrate-phosphate buffer (pH 5.0) + 15 mg TMB) and solution B (100 mL distilled water + 100 μL 30% H2O2. Usage: mix 1:1 before use). Add 100 μL to each well, seal with a membrane, and incubate at 37°C in the dark for 15 min.

[0294] Termination: Add 100 μL of stop solution to each well to terminate the process, and then take the reading.

[0295] Reading: Place the ELISA plate into the microplate reader and read the OD value at a wavelength of 450 nm. The measurement should be completed within 20 minutes after termination.

[0296] 5.2 Core Performance Verification Items and Specific Methods

[0297] This protocol focuses on the suitability of the kit for specified microbial samples, with particular emphasis on validating linear range, precision, accuracy, and specificity.

[0298] 5.2.1 Linearity Range Verification

[0299] (1) Experimental design

[0300] Preparation of standard curve: The Amuc_1100 protein standard (positive control) was diluted to 11 concentration gradients using the diluent provided with the kit: 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 60, 80, and 160 ng / mL.

[0301] AKK PROBIO sample gradient: Take the pretreated AKK PROBIO test solution and perform serial dilutions (1:1, 1:2, 1:4, 1:8, 1:16) with diluent to cover the concentration range of the standard curve;

[0302] Detection procedure: Add sample according to the kit instructions (100 μL per well), incubate at 37℃ for 60 min → wash 3 times → add enzyme-labeled antibody → incubate for 60 min → wash → add substrate for color development for 15 min → add stop solution → read the value using the microplate reader; set up 3 replicates for each concentration, and set up a blank control (add diluent only).

[0303] (2) Judgment indicators

[0304] Linear range: Plot a linear standard curve with the standard concentration as the x-axis (X) and the OD value as the y-axis (Y). The correlation coefficient R² ≥ 0.98 is required. The OD value of the AKK PROBIO diluted sample must fall within the linear range, and the deviation between the measured concentration and the theoretical diluted concentration must be ≤ 15%.

[0305] 5.2.2 Precision verification (repeatability + reproducibility)

[0306] Objective: To verify the stability of the kit in detecting Amuc_1100 protein in AKK PROBIO under the same / different conditions.

[0307] (1) Intra-batch repeatability

[0308] Experimental design: AKK PROBIO samples of low, medium and high concentrations were selected, and tested simultaneously by the same operator on the same microplate according to the instructions. Each concentration was set up with 10 replicates.

[0309] Judgment criteria: Calculate the relative standard deviation (RSD) of OD values ​​for each concentration. Formula: RSD (%) = (SD / mean) × 100%. The requirement is that the RSD within the batch is ≤ 10%.

[0310] (2) Batch-to-batch reproducibility

[0311] Experimental design: AKK PROBIO samples of the above three concentrations were selected, and three different batches of the test kit were used. The tests were completed by two different operators on three different days. Each concentration and each batch had three replicates.

[0312] Judgment criteria: Calculate the total RSD of OD values ​​of the same concentration samples among the three batches, and require that the batch-to-batch RSD be ≤15%.

[0313] 5.2.3 Accuracy

[0314] (1) Experimental design

[0315] AKK PROBIO samples with baseline concentrations determined by the kit were selected as baseline samples. Two spiking gradients were set up, and low and medium concentrations of Amuc_1100 protein standard were added to equal volumes of baseline samples, respectively. Parallel controls (unspecified) were also set up for baseline samples. Each sample and spiking gradient was configured with 3 replicates. Sample pretreatment and detection operations were completed according to the kit instructions. The OD values ​​of each well were recorded and the corresponding concentrations were calculated.

[0316] (2) Judgment indicators

[0317] The required recovery rate for Amuc_1100 protein detection is between 80% and 110% to be considered accurate.

[0318] 5.2.4 Specificity

[0319] (1) Experimental design

[0320] Lactobacillus A, Bifidobacterium B, and Cocci C were selected as interference samples. AKK PROBIO samples (target samples), Amuc_1100 protein standard (30 ng / mL, positive control), and diluent (blank matrix, negative control) were also prepared. All samples were pretreated using the same method, with each sample in triplicate. The entire detection process was completed according to the kit instructions. The OD450nm value of each well was recorded, and the mean of the three replicates was calculated.

[0321] (2) Judgment indicators

[0322] ① For interfering samples: The t-test was used to analyze the difference between the mean OD of each interfering sample in 3 replicates and the mean OD of the blank matrix. P>0.05 indicates that there is no statistical difference between the interfering sample and the blank matrix, and the kit does not cross-react with non-target bacteria related proteins.

[0323] ② For the target sample and positive control: the deviation between the detected concentration and the theoretical concentration of the AKK PROBIO sample is ≤15%, and the Amuc_1100 protein standard shows normal color development (OD value conforms to the concentration range corresponding to the standard curve), indicating that the kit has good specific recognition ability for the target protein;

[0324] ③ If both of the above indicators are met, the kit is deemed to be of qualified specificity.

[0325] 5.3 Verification Results of Each Project

[0326] 5.3.1 Linear Range

[0327] Table 12 Linear Range

[0328]

[0329] 5.3.2 Precision

[0330] Table 13 Intra-batch Precision

[0331]

[0332] Table 14 Inter-batch precision

[0333]

[0334] 5.3.3 Accuracy

[0335] Table 15 Accuracy

[0336]

[0337] 5.3.4 Specificity

[0338] Table 16 Specificity

[0339]

[0340] In summary, this validation covered four core performance indicators: linear range, precision, accuracy, and specificity. The validation results for all projects met the criteria specified in the protocol, with the correlation coefficient R of the linear regression equation being [value missing]. 2The RSDs reached 0.995, with both intra- and inter-assay RSDs well below the limits. The spiked recoveries were between 85% and 90%. In the specificity assay, there was no significant difference between interfering samples and the blank control. This indicates that the Amuc_1100 protein ELISA kit exhibits good linearity, strong detection stability, accurate and reliable results, and outstanding specificity. It can be effectively used for the detection of Amuc_1100 protein in AKK PROBIO, meeting the requirements for research-grade kits and suitable for subsequent sample testing.

[0341] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A specific monoclonal antibody pair against the active protein Amuc_1100 of *AKK* bacteria, characterized in that, It is composed of 3E10 monoclonal antibody and 2F7 monoclonal antibody; the amino acid sequence of the heavy chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region of the 2F7 monoclonal antibody is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

2. A nucleic acid molecule encoding the specific monoclonal antibody pair of claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID NO.

3. The nucleotide sequence encoding the heavy chain variable region of the 2F7 monoclonal antibody is shown in SEQ ID NO.5, and the nucleotide sequence encoding the light chain variable region of the 2F7 monoclonal antibody is shown in SEQ ID NO.

7.

3. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. A kit for detecting Amuc_1100 protein, characterized in that, It comprises the specific monoclonal antibody pair as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit is a colloidal gold detection kit; the kit also includes sample extraction solution, sample pad treatment solution and reconstitution solution.

7. The reagent kit according to claim 5, characterized in that, The kit is a double-antibody sandwich ELISA detection kit; the kit also includes enzyme-labeled secondary antibody, chromogenic solution, stop solution, washing solution, blocking solution and coating buffer.

8. A method for detecting Amuc_1100 protein for non-disease diagnostic purposes, characterized in that, The detection was performed using the kit described in any one of claims 5-7.

9. The use of the specific monoclonal antibody of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the host cell of claim 4 in the preparation of a kit for detecting Amuc_1100 protein in Akkermansia myxoma.

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