Specific monoclonal antibody for AKK active protein P9, detection kit and application thereof

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a specific monoclonal antibody for the active protein P9 of *AKK* bacteria, a detection kit, and their applications. The antibody pair consists of 7G4 and 8B2. The amino acid sequence of the heavy chain variable region of 7G4 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 8B2 is SEQ ID NO.6, and the amino acid sequence of the light chain variable region is SEQ ID NO.8. This antibody pair exhibits high specificity and affinity, accurately identifying different antigenic epitopes of the P9 protein. Based on this, a colloidal gold detection and dual-antibody sandwich ELISA detection system is constructed, adaptable to complex matrices and samples processed using various techniques. The detection is stable and reliable, possessing both qualitative and quantitative capabilities. It can be used for P9 protein detection, fermentation monitoring, quality control, and other scenarios, solving the problems of insufficient specificity and poor adaptability in existing technologies.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a specific monoclonal antibody for AKK bacterial active protein P9, a detection kit and application thereof. BACKGROUND

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

[0003] At present, the detection methods for P9 protein mainly include general protein quantification methods and immunodetection techniques: the general protein quantification methods represented by BCA method and Bradford method can only determine the total protein content of the sample, and cannot specifically distinguish P9 protein from impurities, and are easily interfered by polysaccharides, metabolic byproducts and other components in complex matrix samples such as fermentation broth and bacterial powder, resulting in poor detection specificity, significant quantitative deviation and difficulty in meeting the precise detection requirements; the immunodetection techniques mostly use monoclonal antibodies or polyclonal antibodies to construct systems, but generally have problems such as insufficient antibody specificity, single antigen epitope recognition, poor performance of paired combinations, and the like, some antibodies only bind to non-key epitopes of P9 protein, are easily affected by cross-reactions of homologous proteins, and have low binding affinity of paired antibodies, weak detection signal and insufficient sensitivity, which makes it difficult to detect P9 protein in low-concentration samples. At the same time, the existing detection methods are limited in application scenarios, most of which are only suitable for purified P9 protein standards, and have poor adaptability to complex samples after processes such as high-temperature high-pressure steam sterilization, pH adjustment and oxidation treatment, while the activity of P9 protein itself is easily affected by environmental factors such as temperature, pH and oxidizing agents, which further aggravates the instability of the detection results, ultimately leading to the four core problems of P9 protein detection, i.e., insufficient specificity, weak environmental tolerance, performance deficiency of paired antibodies and difficulty in eliminating matrix interference, and insufficient recovery in some scenarios, which not only limits its industrial scale application, but also cannot provide accurate data for fermentation process optimization and biological product quality control.

[0004] Therefore, developing high-specificity paired antibodies for P9 protein and supporting detection systems to solve the core problems of insufficient specificity, poor adaptability and matrix interference in the prior art has important practical significance for improving the controllability of microbial fermentation process, ensuring the quality stability and safety of biological products, and also provides a beneficial reference for the development of detection technology for related functional proteins. SUMMARY

[0005] In view of the above problems, the application provides a pair of monoclonal antibodies (7G4 and 8B2) which specifically recognize P9 protein, the antibodies have high binding specificity and high affinity, and can accurately recognize different antigen epitopes of P9 protein, and a colloidal gold detection and double antibody sandwich ELISA detection system are established based on the antibodies, so that the detection requirements of P9 protein under different process conditions and in complex matrix samples can be effectively met.

[0006] The technical scheme of the application is as follows:

[0007] In one aspect, the application provides a pair of monoclonal antibodies for detecting AKK bacterial efficacy protein P9, comprising a first monoclonal antibody and a second monoclonal antibody.

[0008] 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.

[0009] 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.

[0010] Specifically, 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.

[0011] In another aspect, the application provides a nucleic acid molecule encoding the aforementioned pair of monoclonal antibodies, the nucleotide sequence encoding the heavy chain variable region of the first monoclonal antibody is SEQ ID NO. 1, 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.

[0012] In another aspect, the application provides a recombinant expression vector containing the aforementioned nucleic acid molecule.

[0013] In another aspect, the application provides an engineering cell containing the aforementioned nucleic acid molecule or recombinant expression vector.

[0014] In another aspect, the application provides a kit for detecting AKK bacterial efficacy protein P9, comprising the aforementioned pair of monoclonal antibodies or nucleic acid molecule or recombinant expression vector or engineering cell.

[0015] Specifically, the kit includes but is not limited to a colloidal gold detection kit or a double antibody sandwich ELISA kit.

[0016] Preferably, the colloidal gold detection kit includes a solid phase carrier coated with a first monoclonal antibody, a colloidal gold conjugated pad labeled with a second monoclonal antibody, and a sample extraction solution.

[0017] 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 color developing solution, a termination solution, and a washing solution.

[0018] In another aspect, the present application provides the use of the aforementioned monoclonal antibody pair, nucleic acid molecule, recombinant expression vector, engineered cell, or kit in detecting the efficacy protein P9 of AKK bacteria.

[0019] The present application has the following advantages:

[0020] (1) The monoclonal antibodies 7G4 and 8B2 of the present application have high specificity and affinity, and can accurately recognize different antigen epitopes of P9 protein, effectively avoiding cross-reactions, and solving the problem of insufficient performance of existing antibodies.

[0021] (2) The colloidal gold and double antibody sandwich ELISA detection systems based on the antibody pair are suitable for complex matrix and various process samples, and break through the limitations of traditional detection scenarios.

[0022] (3) The two systems are complementary in function, with rapid qualitative and accurate quantitative ability, good repeatability and stability, and strong resistance to matrix interference, providing efficient technical support for fermentation monitoring, quality control, etc. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure is a colloidal gold detection specificity verification result graph.

[0024] Figure 2 Figure is a colloidal gold detection repeatability verification result graph.

[0025] Figure 3 Figure is a colloidal gold detection parallelism verification result graph.

[0026] Figure 4 Figure is a P9 protein standard product series dilution gradient setting schematic diagram.

[0027] Figure 5 Figure is a P9 protein concentration-OD value standard curve schematic diagram. DETAILED DESCRIPTION

[0028] The application will be further clarified by the following examples. The examples are only a part of the application and are not used to limit the application. The experimental methods used in the following examples are conventional experiments. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0029] Example 1

[0030] 1.1 Antibody

[0031] The 7G4 and 8B2 hybridoma cell lines were cultured to a cell density of 1E+07 cell / mL, and were sent for hybridoma monoclonal antibody sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.). The antibody heavy chain and light chain gene sequences encoding the signal peptide, variable region and constant region were synthesized and constructed into mammalian cell expression vectors, such as pTT5, pCDNA3.1, etc. The recombinant plasmid was transfected into HEK293 mammalian cells to express the 7G4 and 8B2 antibodies. The cell expression supernatant was purified by protein A affinity to obtain the P9 protein specific recombinant antibodies 7G4 and 8B2. The antibodies were used for antibody characterization and Elisa kit development.

[0032] Antibody 7G4 and 8B2 amino acid sequence sequencing results:

[0033] (1) 7G4 heavy chain variable region results

[0034] Base sequence (SEQ ID NO. 1):

[0035] gaggtccagctgcaacaatctggacctgtactggtgaagcctggggcctcagtgaagatttcctgtaaagcttctggctacgcattcagtacctcttggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacggatttatcctggagatggagatactaactacaatgggaagttcaagggcaaggccacactgactgcagacaaatcctccagcacagcctacatgcacctcagcagcctgacatctgtggactctgaggtctatttctgtgcaagatgggggggtggttatccttggtatggtatggactactggggtcaaggaacctcagtcaccgtctcctca.

[0036] Amino acid sequence (SEQ ID NO. 2):

[0037] EVQLQQSGPVLVKPGASVKISCKASGYAFSTSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMHLSSLTSVDSEVYFCARWGGGYPWYGMDYWGQGTSVTVSS.

[0038] Table 1 Heavy Chain CDR Sequences

[0039]

[0040] (2) Results of the variable region of 7G4 light chain

[0041] Base sequence (SEQ ID NO.3):

[0042] gatatccagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctaccacacatcaagatt acattcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccactttttgccaacagagtaatacgcttccgtggacgttcggtggaggcaccaagctggaaatcaaa.

[0043] Amino acid sequence (SEQ ID NO.4):

[0044] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSNTLPWTTFGGGTKLEIK.

[0045] Table 2 Light chain CDR sequences

[0046]

[0047] (3) Results of the variable region of the 8B2 heavy chain

[0048] Base sequence (SEQ ID NO.5):

[0049] gaggtccagctgcaacagtctggagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcagtaactactggctaggttgggtaaagcagaggcctggacatggacttgagtggattggagatatttaccctggaagtggtaatacttactacaatgagaagttcaagggcaaagtcacactgactgcagacaattcctcgaacacagcctatatgcagctcagtagcctgacgtctgaggactctgctgtctatttctgtgcaagcgggggattacgacgtaggatttactatcctatggactactggggtcaaggaacctcagtcaccgtctcctca。

[0050] Amino acid sequence (SEQ ID NO.6):

[0051] EVQLQQSGAELVRPGTSVKISCKASGYAFSNYWLGWVKQRPGHGLEWIGDIYPGSGNTYYNEKFKGKVTLTADNSSNTAYMQLSSLTSEDSAVYFCASGGLRRRIYYPMDYWGQGTSVTVSS。

[0052] Table 3 Heavy chain CDR sequences

[0053]

[0054] (4) Results of the variable region of the 8B2 light chain

[0055] Base sequence (SEQ ID NO.7):

[0056] gacattgtgatgacccagtctcaaaaattcatgtccacatctgtaggagacagggtcagcgtcacctgccaggccagtcagagtgtgggtactaatgtagcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcttcctaccg gttcagtggagttcctgatcgcttcacaggcagtggatctgggacagaattcactctcaccatcagcaatgtgcagtctgaagacttggcagagtttttctgtcagcaatctaacagctatccgtggacgttcggtggaggcaccaagctggaaatcaaa.

[0057] Amino acid sequence (SEQ ID NO.8):

[0058] DIVMTQSQKFMSTSVGDRVSVTCQASQSVGTNVAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTEFTLTISNVQSEDLAEFFCQQSNSYPWTFGGGTKLEIK.

[0059] Table 4 Light chain CDR sequences

[0060]

[0061] Example 2 Colloidal Gold Detection

[0062] 2.1 Preparation of Colloidal Gold

[0063] (1) Envelopment conditions

[0064] NC membrane: Tianren Membrane Industry, model 140T;

[0065] 7G4 antibody streak concentration: 1.5 mg / mL;

[0066] Streak dilution: final concentration 2% sucrose + 10mM PBS buffer, pH 7.40;

[0067] 7G4 antibody streaking spray volume: 1.0 μL / cm.

[0068] (2) Marking conditions

[0069] 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).

[0070] 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.

[0071] 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.

[0072] Reconstitute after centrifugation: Reconstitute by double the original amount (add 1 mL of reconstituted solution after centrifuging 1 mL of gold solution).

[0073] 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.

[0074] Table 5 Reconstituted Solution Formulation

[0075]

[0076] Gold plating after reconstitution: The gold-labeled antibody, which has been reconstituted at the original concentration, is directly plated onto the 8964 binding pad.

[0077] (3) Components of sample extract

[0078] 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.

[0079] Table 6 Sample Extract Formulation

[0080]

[0081] (4) Sample pad treatment conditions

[0082] 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.

[0083] Table 7 Sample Pad Treatment Solution Formulation

[0084]

[0085] Hydrochloric acid is used to adjust the pH of the sample pad treatment solution to 7.6-8.0.

[0086] 2.2 Detection methods and result analysis

[0087] 2.2.1 Experimental Objective

[0088] Complete the verification of the accuracy, specificity, repeatability, and parallelism of colloidal gold.

[0089] 2.2.2 Test Materials

[0090] a. Core testing reagent: P9 colloidal gold test strip.

[0091] b. Supporting consumables:

[0092] Disposable sterile dropper (100μL / drop, no risk of cross-contamination);

[0093] 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);

[0094] Sterile centrifuge tubes (size: 1.5mL, used for sample dilution and mixing).

[0095] c. Environment and Instruments:

[0096] Testing environment: Room temperature constant temperature room (20-30℃, relative humidity 40%-60%, no direct sunlight, no airflow interference);

[0097] Auxiliary tools: timer, disposable gloves (powder-free latex material to avoid contaminating the sample).

[0098] 2.2.3 Sample Information

[0099] Sample information is shown in Table 8:

[0100] Table 8 Sample Information

[0101]

[0102] 2.2.4 Specific Steps

[0103] a. Sample pretreatment (operation time: 30 minutes before detection):

[0104] Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences).

[0105] Take 0.01g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 990μL of sample dilution buffer;

[0106] 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.

[0107] b. Sample addition procedure (single sample operation time: 1 minute):

[0108] Take out the P9 colloidal gold test strip, place it horizontally on a clean lab bench, and label the sample number (to avoid confusion).

[0109] 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.

[0110] Start the timer immediately and record the start time.

[0111] c. Incubation and observation (incubation time: 15 minutes):

[0112] Keep the test strip horizontal during incubation and avoid moving, tilting or touching the sample well;

[0113] 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);

[0114] Results are invalid after 15 minutes (to avoid false negatives caused by excessive diffusion of the chromatography solvent).

[0115] 2.2.5 Experimental Results

[0116] (1) Specificity

[0117] 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.

[0118] (2) Repeatability

[0119] 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.

[0120] (3) Parallelism

[0121] 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.

[0122] 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.

[0123] 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.

[0124] Example 3: Detection of P9 protein using a double-antibody sandwich ELISA

[0125] 3.1 Horseradish peroxidase-labeled antibody

[0126] (1) Take 10 μL of horseradish peroxidase solution into a clean 0.5 mL centrifuge tube.

[0127] (2) Take 10 mg of 8B2 antibody and mix it thoroughly with horseradish peroxidase by pipetting.

[0128] (3) Add 1 μL of periodate. The periodate needs to be thoroughly mixed with the solution.

[0129] (4) React at 37℃ for 1-2 hours or at 4℃ overnight.

[0130] (5) After the reaction is complete, add 3 μL of sodium cyanoborohydride and store the labeled enzyme in a 4°C refrigerator.

[0131] 3.2 Coating of enzyme-labeled plates with antibodies

[0132] (1) Dilute 7G4 antibody to 5 μg / mL with coating buffer, coat 100 μL per well, and let stand at 4℃ for 16 h.

[0133] (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.

[0134] (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.

[0135] 3.3 Test Methods

[0136] (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.

[0137] (2) Sample incubation: Add 100 μL of standard / blank (diluent) / sample to each well, seal with membrane, and react at 37℃ for 1 h.

[0138] (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).

[0139] (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.

[0140] (5) Washing the plate: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250uL / well), and pat dry.

[0141] (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.

[0142] (7) Termination: Add 100 μL of termination solution to each well to terminate the process, and then take the reading.

[0143] (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.

[0144] 3.4 Results Analysis

[0145] (1) Calculation of absorbance value:

[0146] The light absorption calibration value for each standard or sample is: OD450nm - absorbance value of the blank control well.

[0147] (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:

[0148] Y=((AD) / (1+(X / C)^B))+D

[0149] 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.

[0150] (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.

[0151] Table 9

[0152]

[0153] result Figure 4 As shown.

[0154] Example 4: Reagent Kit Performance Testing

[0155] 4.1 Core Preparations Before Verification

[0156] 4.1.1 Experimental Materials and Reagents

[0157] 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;

[0158] Specified samples: AKK PROBIO, Lactobacillus A, Bifidobacterium B, Cocci C;

[0159] Blank matrix: PBS buffer;

[0160] Auxiliary consumables: sterile centrifuge tubes, pipette tips, ELISA plate sealing film, etc.

[0161] 4.1.2 Instruments and Environment

[0162] Core instruments: Microplate reader (pre-calibrated, detection wavelength 450 nm), constant temperature incubator (37℃), pipettes (10 μL-1000 μL);

[0163] Test environment: temperature 25±2℃, humidity 40%-60%, no direct sunlight, no vibration, and avoid cross-contamination.

[0164] 4.1.3 Sample Preprocessing

[0165] Remove all samples and allow them to equilibrate at room temperature for 30 minutes (to avoid changes in sample solubility due to temperature differences).

[0166] Take 0.1g of each sample and add it to a 1.5mL sterile centrifuge tube, then add 900μL of sample dilution buffer;

[0167] Use a pipette to repeatedly blow and aspirate to ensure the sample is fully dissolved and mixed, preparing a 1:10 diluted test solution;

[0168] 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.

[0169] 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.

[0170] 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.)

[0171] 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.

[0172] Washing: Same as step (3). After incubation, discard the liquid in the wells, wash 3 times (250uL / well), and pat dry.

[0173] 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.

[0174] Termination: Add 100 μL of stop solution to each well to terminate the process, and then take the reading.

[0175] 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.

[0176] 4.2 Core Performance Verification Items and Specific Methods

[0177] This protocol focuses on the suitability of the kit for specified microbial samples, with particular emphasis on validating linear range, precision, accuracy, and specificity.

[0178] 4.2.1 Linearity Range Verification

[0179] (1) Experimental design

[0180] 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.

[0181] 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;

[0182] 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).

[0183] (2) Judgment indicators

[0184] 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%.

[0185] 4.2.2 Precision verification (repeatability + reproducibility)

[0186] Objective: To verify the stability of the kit in detecting P9 protein in AKK PROBIO under the same / different conditions.

[0187] (1) Intra-batch repeatability

[0188] 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.

[0189] 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%.

[0190] (2) Batch-to-batch reproducibility

[0191] 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.

[0192] 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%.

[0193] 4.3 Verification Results of Each Project

[0194] 4.3.1 Linear Range

[0195] Table 10 Linear Range

[0196]

[0197] 5.3.2 Precision

[0198] Table 11 Intra-batch Precision

[0199]

[0200] Table 12 Inter-batch precision

[0201]

[0202] 4.3.3 Accuracy

[0203] Table 13 Accuracy

[0204]

[0205] 4.3.4 Specificity

[0206] Table 14 Specificity

[0207]

[0208] 4.4 Conclusion

[0209] 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.

[0210] 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 the functional protein P9 of AKK bacteria, characterized in that, Including first-line monoclonal antibodies and second-line monoclonal antibodies; 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.

2. The monoclonal antibody pair according to claim 1, characterized in that, 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.

3. A nucleic acid molecule encoding the monoclonal antibody pair of claim 1 or 2, characterized in that, 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.

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 the functional protein P9 in AKK bacteria, characterized in that, It comprises the monoclonal antibody pair according to any one of claims 1-2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the engineered cell according to claim 5.

7. The reagent kit according to claim 6, characterized in that, The kit is a colloidal gold detection kit or a double-antibody sandwich ELISA kit.

8. The reagent kit according to claim 7, characterized in that, 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.

9. The reagent kit according to claim 7, characterized in that, 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.

10. The use of any monoclonal antibody pair of claim 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 AKK bacterium functional protein P9.

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