Specific monoclonal antibody for AKK bacterium active protein P9, detection kit and application of specific monoclonal antibody

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a specific monoclonal antibody for AKK bacterium active protein P9, a detection kit and application of the specific monoclonal antibody. The antibody pair is 7G4 and 8B2, the amino acid sequence of a heavy chain variable region of the 7G4 is SEQ ID NO.2, and the amino acid sequence of a light chain variable region of the 7G4 is SEQ ID NO.4; the amino acid sequence of the 8B2 heavy chain variable region is SEQ ID NO.6, and the amino acid sequence of the 8B2 light chain variable region is SEQ ID NO.8. The antibody pair has strong specificity and high affinity, and can accurately recognize different antigen epitopes of the P9 protein. A colloidal gold detection and double-antibody sandwich ELISA detection system constructed based on the kit adapts to a complex matrix and various process treatment samples, is stable and reliable in detection, has qualitative and quantitative capabilities, can be used for scenes of P9 protein detection, fermentation monitoring, quality control and the like, and solves the problems of insufficient specificity, poor adaptability and the like in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a specific monoclonal antibody for the active protein P9 of AKK bacteria, a detection kit, and their applications. Background Technology

[0002] As a key functional protein in the metabolic processes of microorganisms such as AKK PROBIO strain, the expression level and activity of P9 protein are directly related to the metabolic state of the strain, the stability of the fermentation process, and the quality level of the final biological product. It has irreplaceable detection value in the fields of microbial fermentation process monitoring, biological product quality control, and probiotic product efficacy evaluation.

[0003] Currently, detection methods for P9 protein are mainly divided into two categories: general protein quantification methods and immunoassay techniques. General protein quantification methods, represented by the BCA method and the Bradford method, can only measure the total protein content of the sample and cannot specifically distinguish P9 protein from other proteins. In complex matrix samples such as fermentation broth and bacterial powder, they are easily interfered with by components such as polysaccharides and metabolic byproducts, resulting in poor detection specificity and significant quantitative deviation, making it difficult to meet the needs of accurate detection. Immunoassay techniques mostly use monoclonal or polyclonal antibodies to construct systems, but they generally suffer from problems such as insufficient antibody specificity, single antigenic epitope recognition, and poor pairing combination efficacy. Some antibodies only bind to non-critical epitopes of P9 protein, are easily affected by cross-reactions with homologous proteins, and the binding affinity of paired antibodies is low, resulting in weak detection signals and insufficient sensitivity, making it difficult to detect P9 protein in low-concentration samples. Meanwhile, existing detection methods are limited in their applicability, with most only applicable to purified P9 protein standards. They are poorly adapted to complex samples that have undergone high-temperature, high-pressure steam inactivation, pH adjustment, and oxidation treatment. Furthermore, the activity of P9 protein itself is easily affected by environmental factors such as temperature, pH, and oxidants, further exacerbating the instability of detection results. Ultimately, this leads to four core problems in P9 protein detection: insufficient specificity, weak environmental tolerance, inadequate performance of paired antibodies, and difficulty in eliminating matrix interference. In some scenarios, the recovery rate is insufficient, which not only limits its industrial-scale application but also fails to provide accurate data for fermentation process optimization and biological product quality control.

[0004] Therefore, developing highly specific paired antibodies and supporting detection systems for P9 protein to address the core pain points of existing technologies, such as insufficient specificity, poor adaptability, and matrix interference, is of great practical significance for improving the controllability of microbial fermentation processes and ensuring the quality stability and safety of biological products. It can also provide a useful reference for the development of detection technologies for related functional proteins. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a pair of monoclonal antibodies (7G4 and 8B2) that specifically recognize the P9 protein. This antibody pair exhibits strong binding specificity and high affinity, enabling precise identification of different antigenic epitopes of the P9 protein. Furthermore, based on this antibody pair, a colloidal gold detection system and a double-antibody sandwich ELISA detection system have been established, effectively addressing the needs for P9 protein detection under complex matrix samples and different processing conditions.

[0006] The technical solution of this invention is as follows:

[0007] On the one hand, the present invention provides a monoclonal antibody pair for the detection of AKK bacteria functional protein P9, comprising a first monoclonal antibody and a second monoclonal antibody; The heavy chain CDR sequence of the first monoclonal antibody is shown in SEQ ID NO.9-SEQ ID NO.11, and the light chain CDR sequence is shown in SEQ ID NO.12-SEQ ID NO.14; The heavy chain CDR sequence of the second monoclonal antibody is shown in SEQ ID NO.15-SEQ ID NO.17, and the light chain CDR sequence is shown in SEQ ID NO.18-SEQ ID NO.20.

[0008] Specifically, the amino acid sequence of the heavy chain variable region of the first monoclonal antibody is SEQ ID NO.2, and the amino acid sequence of the light chain variable region is SEQ ID NO.4; the amino acid sequence of the heavy chain variable region of the second monoclonal antibody is SEQ ID NO.6, and the amino acid sequence of the light chain variable region is SEQ ID NO.8.

[0009] In another aspect, the present invention provides a nucleic acid molecule encoding the aforementioned monoclonal antibody pair, wherein the nucleotide sequence encoding the variable region of the heavy chain of the first monoclonal antibody is SEQ ID NO.1, the nucleotide sequence encoding the variable region of the light chain of the first monoclonal antibody is SEQ ID NO.3, the nucleotide sequence encoding the variable region of the heavy chain of the second monoclonal antibody is SEQ ID NO.5, and the nucleotide sequence encoding the variable region of the light chain of the second monoclonal antibody is SEQ ID NO.7.

[0010] In another aspect, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecules.

[0011] In another aspect, the present invention provides an engineered cell containing the aforementioned nucleic acid molecules or recombinant expression vectors.

[0012] In another aspect, the present invention provides a kit for detecting the AKK bacterium functional protein P9, comprising the aforementioned monoclonal antibody pair or nucleic acid molecule or recombinant expression vector or engineered cell.

[0013] Specifically, the kits include, but are not limited to: colloidal gold detection kits or double antibody sandwich ELISA kits.

[0014] Preferably, the colloidal gold detection kit includes a solid-phase carrier coated with a first monoclonal antibody, a colloidal gold binding pad labeled with a second monoclonal antibody, and a sample extraction solution.

[0015] Preferably, the double-antibody sandwich ELISA kit includes a solid-phase carrier coated with a first monoclonal antibody, an enzyme-labeled second monoclonal antibody, a chromogenic solution, a stop solution, and a washing solution.

[0016] In another aspect, the present invention provides the application of the aforementioned monoclonal antibody pair, nucleic acid molecule, recombinant expression vector, engineered cell, or kit in the detection of AKK bacterium functional protein P9.

[0017] The beneficial effects of this invention are as follows: (1) The monoclonal antibodies 7G4 and 8B2 of the present invention have high specificity and high affinity, and can accurately identify different antigenic epitopes of P9 protein, effectively avoiding cross-reaction and solving the problem of insufficient performance of existing antibodies.

[0018] (2) Based on the antibody pair, the colloidal gold and double antibody sandwich ELISA detection system is adapted to complex matrices and various process samples, breaking through the limitations of traditional detection scenarios.

[0019] (3) The two systems complement each other, possessing both rapid qualitative and precise quantitative capabilities, good repeatability and stability, and strong resistance to matrix interference, providing efficient technical support for fermentation monitoring, quality control, etc. Attached Figure Description

[0020] Figure 1 This is a graph showing the results of specificity verification for colloidal gold detection.

[0021] Figure 2 The image shows the results of repeatability verification for colloidal gold detection.

[0022] Figure 3 The figure shows the results of the parallelism verification of colloidal gold detection.

[0023] Figure 4 A schematic diagram showing the setup of a serial dilution gradient for P9 protein standards.

[0024] Figure 5 This is a schematic diagram of the standard curve of P9 protein concentration-OD value. Detailed Implementation

[0025] 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.

[0026] Example 1 1.1 Antibody 7G4 and 8B2 hybridoma cell lines 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 7G4 and 8B2 antibodies. The cell expression supernatant was purified by protein A affinity to obtain P9 protein-specific recombinant antibodies 7G4 and 8B2. These were used for antibody characterization and ELISA kit development.

[0027] Sequencing results of antibody 7G4 and 8B2 amino acid sequences: (1) Results of the variable region of the 7G4 heavy chain Base sequence (SEQ ID NO.1): gaggtccagctgcaacaatctggacctgtactggtgaagcctggggcctcagtgaagatttcctgtaaagcttctggctacgcattcagtacctcttggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacggatttatcctggagatggagatactaactacaa tgggaagttcaagggcaaggccacactgactgcagacaaatcctccagcacagcctacatgcacctcagcagcctgacatctgtggactctgaggtctatttctgtgcaagatgggggggtggttatccttggtatggtatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0028] Amino acid sequence (SEQ ID NO.2): EVQLQQSGPVLVKPGASVKISCKASGYAFSTSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMHLSSLTSVDSEVYFCARWGGGYPWYGMDYWGQGTSVTVSS.

[0029] Table 1 Heavy Chain CDR Sequences

[0030] (2) Results of the variable region of 7G4 light chain Base sequence (SEQ ID NO.3): gatatccagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctaccacacatcaagatt acattcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccactttttgccaacagagtaatacgcttccgtggacgttcggtggaggcaccaagctggaaatcaaa.

[0031] Amino acid sequence (SEQ ID NO.4): DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSNTLPWTTFGGGTKLEIK.

[0032] Table 2 Light chain CDR sequences

[0033] (3) Results of the variable region of the 8B2 heavy chain Base sequence (SEQ ID NO.5): gaggtccagctgcaacagtctggagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcagtaactactggctaggttgggtaaagcagaggcctggacatggacttgagtggattggagatatttaccctggaagtggtaatacttactacaatgagaagttcaagggcaaagtcacactgactgcagacaattcctcgaacacagcctatatgcagctcagtagcctgacgtctgaggactctgctgtctatttctgtgcaagcgggggattacgacgtaggatttactatcctatggactactggggtcaaggaacctcagtcaccgtctcctca。

[0034] Amino acid sequence (SEQ ID NO.6): EVQLQQSGAELVRPGTSVKISCKASGYAFSNYWLGWVKQRPGHGLEWIGDIYPGSGNTYYNEKFKGKVTLTADNSSNTAYMQLSSLTSEDSAVYFCASGGLRRRIYYPMDYWGQGTSVTVSS。

[0035] Table 3 Heavy chain CDR sequences

[0036] (4)Results of the variable region of the 8B2 light chain Base sequence (SEQ ID NO.7): gacattgtgatgacccagtctcaaaaattcatgtccacatctgtaggagacagggtcagcgtcacctgccaggccagtcagagtgtgggtactaatgtagcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcttcctaccg gttcagtggagttcctgatcgcttcacaggcagtggatctgggacagaattcactctcaccatcagcaatgtgcagtctgaagacttggcagagtttttctgtcagcaatctaacagctatccgtggacgttcggtggaggcaccaagctggaaatcaaa.

[0037] Amino acid sequence (SEQ ID NO.8): DIVMTQSQKFMSTSVGDRVSVTCQASQSVGTNVAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTEFTLTISNVQSEDLAEFFCQQSNSYPWTFGGGTKLEIK.

[0038] Table 4 Light chain CDR sequences

[0039] Example 2 Colloidal Gold Detection 2.1 Preparation of Colloidal Gold (1) Envelopment conditions NC membrane: Tianren Membrane Industry, model 140T; 7G4 antibody streak concentration: 1.5 mg / mL; Streak dilution: final concentration 2% sucrose + 10mM PBS buffer, pH 7.40; 7G4 antibody streaking spray volume: 1.0 μL / cm.

[0040] (2) Marking conditions 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 about 30nm).

[0041] 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.

[0042] Antibody dosage: Add 20 μg of 8B2 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.

[0043] Reconstitute after centrifugation: Reconstitute by double the original amount (add 1 mL of reconstituted solution after centrifuging 1 mL of gold solution).

[0044] 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.

[0045] Table 5 Reconstituted Solution Formulation

[0046] Gold plating after reconstitution: The gold-labeled antibody, which has been reconstituted at the original concentration, is directly plated onto the 8964 binding pad.

[0047] (3) Components of sample extract 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.

[0048] Table 6 Sample Extract Formulation

[0049] (4) Sample pad treatment conditions 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.

[0050] Table 7 Sample Pad Treatment Solution Formulation

[0051] Hydrochloric acid is used to adjust the pH of the sample pad treatment solution to 7.6-8.0.

[0052] 2.2 Detection methods and result analysis 2.2.1 Experimental Objective Complete the verification of the accuracy, specificity, repeatability, and parallelism of colloidal gold.

[0053] 2.2.2 Test Materials a. Core testing reagent: P9 colloidal gold test strip.

[0054] b. Supporting consumables: Disposable sterile dropper (100μL / drop, no risk of cross-contamination); 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); Sterile centrifuge tubes (size: 1.5mL, used for sample dilution and mixing).

[0055] c. Environment and Instruments: Testing environment: Room temperature constant temperature room (20-30℃, relative humidity 40%-60%, no direct sunlight, no airflow interference); Auxiliary tools: timer, disposable gloves (powder-free latex material to avoid contaminating the sample).

[0056] 2.2.3 Sample Information Sample information is shown in Table 8: Table 8 Sample Information

[0057] 2.2.4 Specific Steps a. Sample pretreatment (operation time: 30 minutes before detection): Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences). Take 0.01g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 990μL of sample dilution buffer; 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.

[0058] b. Sample addition procedure (single sample operation time: 1 minute): Take out the P9 colloidal gold test strip, place it horizontally on a clean lab bench, and label the sample number (to avoid confusion). 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. Start the timer immediately and record the start time.

[0059] c. Incubation and observation (incubation time: 15 minutes): Keep the test strip horizontal during incubation and avoid moving, tilting or touching the sample well; 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); Results are invalid after 15 minutes (to avoid false negatives caused by excessive diffusion of the chromatography solvent).

[0060] 2.2.5 Experimental Results (1) Specificity The results are as follows Figure 1 As shown, only positive samples 15 and 16 showed a band in the T region, indicating that colloidal gold has strong specificity for detecting AKK active P9 protein.

[0061] (2) Repeatability 100 μL of AKK PROBIO supernatant was added to each well of the test strip, ensuring complete flow without spillage. Results are as follows: Figure 2 As 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 P9 protein.

[0062] (3) Parallelism Dilute the protein concentration of the standard to 110 ng / mL, 55 ng / mL, 22 ng / mL, 11 ng / mL, 5.5 ng / mL, 2.75 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.

[0063] The results are as follows Figure 3 As shown, the color of the T-region band gradually fades until it disappears as the protein concentration decreases, indicating that colloidal gold has good parallelism in detecting AKK active P9 protein.

[0064] In summary, this validation covered the three core performance indicators of specificity, repeatability, and parallelism. All validation results were satisfactory, indicating that this P9 protein colloidal gold can be effectively used for the qualitative detection of active P9 protein in AKK PROBIO.

[0065] Example 3: Detection of P9 protein using a double-antibody sandwich ELISA 3.1 Horseradish peroxidase-labeled antibody (1) Take 10 μL of horseradish peroxidase solution into a clean 0.5 mL centrifuge tube.

[0066] (2) Take 10 mg of 8B2 antibody and mix it thoroughly with horseradish peroxidase by pipetting.

[0067] (3) Add 1 μL of periodate. The periodate needs to be thoroughly mixed with the solution.

[0068] (4) React at 37℃ for 1-2 hours or at 4℃ overnight.

[0069] (5) After the reaction is complete, add 3 μL of sodium cyanoborohydride and store the labeled enzyme in a 4°C refrigerator.

[0070] 3.2 Coating of enzyme-labeled plates with antibodies (1) Dilute 7G4 antibody to 5 μg / mL with coating buffer, coat 100 μL per well, and let stand at 4℃ for 16 h.

[0071] (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.

[0072] (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.

[0073] 3.3 Test Methods (1) Preparation of standard: Prepare fresh and use immediately. Dilute the standard (3200ng / mL) to 320ng / mL with PBS buffer, and then serially dilute (dilution factor: 2 times) to prepare the standard.

[0074] (2) Sample incubation: Add 100 μL of standard / blank (diluent) / sample to each well, seal with membrane, and react at 37℃ for 1 h.

[0075] (3) Washing: After incubation, discard the liquid in the wells, dilute the washing buffer with deionized water 20 times, wash 3 times (250uL / 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).

[0076] (4) Incubation of enzyme-labeled antibody: Prepare fresh and use immediately. Dilute the enzyme-labeled antibody 20 times with PBS buffer, add 100 μL of working concentration of enzyme-labeled antibody to each well, seal the membrane, and react at 37°C for 1 h.

[0077] (5) Washing the plate: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250uL / well), and pat dry.

[0078] (6) 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). After mixing, add 100 μL to each well, seal with a membrane, and incubate at 37°C in the dark for 15 min.

[0079] (7) Termination: Add 100 μL of termination solution to each well to terminate the process, and then take the reading.

[0080] (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.

[0081] 3.4 Results Analysis (1) Calculation of absorbance value: The light absorption calibration value for each standard or sample is: OD450nm - absorbance value of the blank control well.

[0082] (2) Plot a standard curve with the concentration of the standard sample as the x-axis (X) and the light absorption calibration value of the standard sample as the y-axis (Y), and fit the equation using a four-parameter Logistic mathematical model: Y=((AD) / (1+(X / C)^B))+D Substitute the sample OD value (OD450nm - absorbance value of blank control well) into the formula to calculate the content of P9 protein in the sample, paying attention to the dilution factor.

[0083] (3) If the OD value of the sample to be tested exceeds the OD value of the highest point of the standard curve, the sample needs to be diluted and retested.

[0084] Table 9

[0085] result Figure 4 As shown.

[0086] Example 4: Reagent Kit Performance Testing 4.1 Core Preparations Before Verification 4.1.1 Experimental Materials and Reagents P9 protein ELISA kit to be validated: contains a coating plate, P9 protein standard (positive control), detection antibody, enzyme-labeled secondary antibody, substrate solution, stop solution, and washing solution; Specified samples: AKK PROBIO, Lactobacillus A, Bifidobacterium B, Cocci C; Blank matrix: PBS buffer; Auxiliary consumables: sterile centrifuge tubes, pipette tips, ELISA plate sealing film, etc.

[0087] 4.1.2 Instruments and Environment Core instruments: Microplate reader (pre-calibrated, detection wavelength 450 nm), constant temperature incubator (37℃), pipettes (10 μL-1000 μL); Test environment: temperature 25±2℃, humidity 40%-60%, no direct sunlight, no vibration, and avoid cross-contamination.

[0088] 4.1.3 Sample Preprocessing Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences). Take 0.1g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 900μL of sample dilution buffer; Use a pipette to repeatedly blow and aspirate to ensure the sample is fully dissolved and mixed, preparing a 1:10 diluted test solution; Preparation of standards: Prepare fresh before use. Dilute the standard (3200 ng / mL) to 320 ng / mL with PBS buffer, and then serially dilute (dilution factor: 2 times) to prepare the standard. 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.

[0089] Washing: After incubation, discard the liquid in the wells, dilute the washing buffer 20 times with deionized water, and wash 3 times (250 uL / 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.)

[0090] 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.

[0091] Washing: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250uL / well), and pat dry.

[0092] 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.

[0093] Termination: Add 100 μL of stop solution to each well to terminate the process, and then take the reading.

[0094] 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.

[0095] 4.2 Core Performance Verification Items and Specific Methods This protocol focuses on the suitability of the kit for specified microbial samples, with particular emphasis on validating linear range, precision, accuracy, and specificity.

[0096] 4.2.1 Linearity Range Verification (1) Experimental design Preparation of standard curve: The P9 protein standard (positive control) was diluted to nine concentrations using the reagent kit diluent: 0, 1.25, 2.5, 5, 10, 20, 40, 60, and 80 ng / mL. AKK PROBIO sample gradient: Take the pretreated AKK PROBIO test solution and perform a series of dilutions (1:1, 1:2, 1:4, 1:8, 1:16) to cover the concentration range of the standard curve; 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).

[0097] (2) Judgment Indicators Linear range: Plot a linear standard curve with the concentration of the standard (X) on the x-axis and the OD value on the y-axis, and require the correlation coefficient R. 2 ≥0.99; 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%.

[0098] 4.2.2 Precision verification (repeatability + reproducibility) Objective: To verify the stability of the kit in detecting P9 protein in AKK PROBIO under the same / different conditions.

[0099] (1) Intra-batch repeatability 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. 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%.

[0100] (2) Batch-to-batch reproducibility 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. 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%.

[0101] 4.3 Verification Results of Each Project 4.3.1 Linear Range Table 10 Linear Range

[0102] 5.3.2 Precision Table 11 Intra-batch Precision

[0103] Table 12 Inter-batch precision

[0104] 4.3.3 Accuracy Table 13 Accuracy

[0105] 4.3.4 Specificity Table 14 Specificity

[0106] 4.4 Conclusion This validation covered four core performance indicators: linear range, precision, accuracy, and specificity. The validation results for all items met the criteria specified in the protocol, with the correlation coefficient R of the linear regression equation being [value missing]. 2 The RSDs reached 0.991, with both intra- and inter-assay RSDs well below the limits. The spiked recoveries ranged from 88.75% to 90.22%. In the specificity assay, there was no significant difference between interfering samples and the blank control. This indicates that the P9 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 P9 protein in AKK PROBIO, meets the requirements for research-grade kits, and can be used for subsequent sample testing.

[0107] 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 monoclonal antibody pair for detecting AKK microbial efficacy protein P9, characterized in that, comprising a first monoclonal antibody and a second monoclonal antibody; the heavy chain CDR sequences of the first monoclonal antibody are as shown in SEQ ID NO. 9-SEQ ID NO. 11, and the light chain CDR sequences are as shown in SEQ ID NO. 12-SEQ ID NO. 14; the heavy chain CDR sequences of the second monoclonal antibody are as shown in SEQ ID NO. 15-SEQ ID NO. 17, and the light chain CDR sequences are as shown in SEQ ID NO. 18-SEQ ID NO.

20.

2. The monoclonal antibody pair according to claim 1, characterized in that, the heavy chain variable region amino acid sequence of the first monoclonal antibody is SEQ ID NO. 2, and the light chain variable region amino acid sequence is SEQ ID NO. 4; the heavy chain variable region amino acid sequence of the second monoclonal antibody is SEQ ID NO. 6, and the light chain variable region amino acid sequence is SEQ ID NO.

8.

3. A nucleic acid molecule encoding the monoclonal antibody pair of claim 1 or 2, characterized in that, the nucleotide sequence encoding the heavy chain variable region of the first monoclonal antibody is SEQ ID NO. 1, and the nucleotide sequence encoding the light chain variable region of the first monoclonal antibody is SEQ ID NO. 3; the nucleotide sequence encoding the heavy chain variable region of the second monoclonal antibody is SEQ ID NO. 5, and the nucleotide sequence encoding the light chain variable region of the second monoclonal antibody is SEQ ID NO.

7.

4. A recombinant expression vector containing the nucleic acid molecule of claim 3.

5. An engineered cell containing the recombinant expression vector of claim 4.

6. A kit for detecting AKK microbial efficacy protein P9, characterized in that, comprising the monoclonal antibody pair of any one of claims 1-2, or the nucleic acid molecule of claim 3, or the recombinant expression vector of claim 4, or the engineered cell of claim 5.

7. The kit of claim 6, wherein The kit is a colloidal gold detection kit or a double antibody sandwich ELISA kit.

8. The kit of claim 7, wherein The colloidal gold detection kit comprises a solid phase carrier coated with the first monoclonal antibody, a colloidal gold conjugated pad labeled with the second monoclonal antibody, and a sample extraction solution.

9. The kit of claim 7, wherein The double antibody sandwich ELISA kit comprises a solid phase carrier coated with the first monoclonal antibody, an enzyme-labeled second monoclonal antibody, a color developing solution, a stop solution, and a washing solution.

10. Use of the monoclonal antibody pair of any one of claims 1-2, or the nucleic acid molecule of claim 3, or the recombinant expression vector of claim 4, or the engineered cell of claim 5, or the kit of any one of claims 6-9 in detecting the efficacy protein P9 of AKK bacteria.

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