Antibody and application thereof in detection of pepsin

Abstract

The invention relates to the technical field of biology, and particularly discloses an antibody and application thereof in pepsin detection. A CDR1 sequence, a CDR2 sequence and a CDR3 sequence of a heavy chain variable region of the antibody or the antigen binding fragment of the antibody are respectively as shown in SEQ ID NO: 2-4; a CDR1 sequence, a CDR2 sequence and a CDR3 sequence of the light chain variable region are respectively shown as SEQ ID NO: 6-8; or the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of the heavy chain variable region are respectively as shown in SEQ ID NO: 10 to SEQ ID NO: 12; and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 14-16. The antibody provided by the invention can be used for effectively realizing pepsin detection through a double-antibody sandwich method, and is good in linear range and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an antibody and an application thereof in detecting pepsin. Background Art

[0002] Pepsin is a digestive protease that breaks down proteins in food into small peptide fragments. Pepsinogen is secreted by the chief cells of the gastric mucosa in the stomach, and pepsinogen is stimulated by gastric acid or pepsin to form pepsin.

[0003] Pepsin detection can be used to monitor and diagnose various gastrointestinal diseases and evaluate gastric mucosal function. Several methods are available for pepsin detection, one of which is fluorescent immunochromatography.

[0004] Fluorescent immunochromatography is a novel membrane detection technology based on antigen-antibody specific immune reactions. This technology uses a strip of fiber chromatography material fixed with a test line (coated antibody or coated antigen) and a quality control line (antibody) as the stationary phase, a test fluid as the mobile phase, and fluorescently labeled antibodies or antigens fixed to the connection pad. Capillary action allows the analyte to move on the chromatography strip. For large molecular antigens with multiple antigenic determinants (proteins, viruses, pathogenic bacteria, etc.), a "sandwich" double antibody sandwich immunochromatography method is usually used. That is, the analyte first binds to the fluorescent labeled antibody under the action of the mobile phase, and then binds to the coated antibody when it reaches the test line to form a double antibody sandwich.

[0005] However, currently known antibodies have low titers, poor purification, poor compatibility with immunofluorescence chromatography detection technology, narrow linear range, low accuracy, and poor anti-interference properties, resulting in poor test results. Therefore, further research on fluorescent immunochromatographic detection of pepsin is still necessary. Summary of the Invention

[0006] One of the purposes of the present invention is to provide an antibody pair that can be used to effectively detect pepsin and its application in fluorescent immunochromatography platform technology.

[0007] The present invention provides an antibody or antigen-binding fragment thereof, wherein the CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 3, and the CDR3 sequence is shown in SEQ ID NO: 4; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 6, the CDR2 sequence is shown in SEQ ID NO: 7, and the CDR3 sequence is shown in SEQ ID NO: 8; Alternatively, the CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO: 10, the CDR2 sequence is shown in SEQ ID NO: 11, and the CDR3 sequence is shown in SEQ ID NO: 12; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 14, the CDR2 sequence is shown in SEQ ID NO: 15, and the CDR3 sequence is shown in SEQ ID NO: 16.

[0008] The antibody or antigen-binding fragment thereof of the present invention, wherein the heavy chain variable region sequence is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 5; Alternatively, the heavy chain variable region sequence is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO: 13.

[0009] The antibodies of the present invention also include constant regions known in the art.

[0010] The present invention also provides an antibody pair, comprising a first antibody and a second antibody; The CDR1 sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 3, and the CDR3 sequence is shown in SEQ ID NO: 4; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 6, the CDR2 sequence is shown in SEQ ID NO: 7, and the CDR3 sequence is shown in SEQ ID NO: 8; The CDR1 sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO: 10, the CDR2 sequence is shown in SEQ ID NO: 11, and the CDR3 sequence is shown in SEQ ID NO: 12; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 14, the CDR2 sequence is shown in SEQ ID NO: 15, and the CDR3 sequence is shown in SEQ ID NO: 16.

[0011] In the antibody pair of the present invention, the heavy chain variable region sequence of the first antibody is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 5; The heavy chain variable region sequence of the second antibody is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO: 13.

[0012] The present invention uses pepsin as an immune antigen injected into mice, allowing the mice's immune system to generate multiple pepsin antibodies. Ascites is extracted from the mice and purified to obtain multiple corresponding monoclonal antibodies. The different antibodies are cross-paired in various permutations and combinations, and screened using an in vitro fluorescence immunochromatography platform. Ultimately, an antibody pair, specifically a coated antibody and a labeled antibody, was selected that optimally detects pepsin levels. This antibody pair can be used in the in vitro detection platform to detect pepsin levels in humans.

[0013] The present invention also provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0014] The present invention also provides a biological material containing the above nucleic acid molecule, wherein the biological material is a vector, an expression cassette or a host cell.

[0015] The present invention also provides use of the above-mentioned antibody or antigen-binding fragment thereof, antibody pair, nucleic acid molecule or biological material in preparing a reagent or kit for detecting pepsin.

[0016] The present invention also provides a reagent or kit for detecting pepsin, which comprises the above-mentioned antibody or antigen-binding fragment thereof or antibody pair.

[0017] The present invention also provides a method for detecting pepsin for purposes other than disease diagnosis or treatment, which utilizes the above-mentioned body or its antigen-binding fragment, antibody pair, or reagent or kit for detecting pepsin for detection.

[0018] In the method of the present invention, the first antibody is used as the coating antibody and the second antibody is used as the labeling antibody for detection; the first antibody and the second antibody are as described above.

[0019] As a specific embodiment, the first antibody and the second antibody of the present invention can be used for immunochromatographic detection. Specifically, the first antibody is used as the coating antibody and is set at the detection line. The second antibody is used as the labeled antibody and is coupled with the signal material and is set on the labeled membrane. The detection of pepsin is achieved by the double antibody sandwich of the first antibody and the second antibody.

[0020] The beneficial effects of the present invention are at least: The antibody pair of the present invention is applied to the immunochromatographic technology to detect pepsin, which has better linearity, wider linear range, higher signal value, and thus better accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the linear fitting result of pepsin detection using the antibody pair of the present invention.

[0022] Figure 2This is the linearity result of the commercially available antibody and the antibody of the present invention in detecting pepsin in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0025] The meanings of some abbreviations in this invention are as follows: HAT: selective culture medium, McAb: monoclonal antibody, BALB / C: albino laboratory mouse, ip: intraperitoneal injection, iv: intravenous injection, rpm: centrifugation speed.

[0026] Example 1 This example provides a method for preparing a pepsin monoclonal antibody, which is specifically prepared by ascites extraction. The specific preparation process of the mouse pepsin monoclonal antibody is as follows: 1. Animal selection and immunization 1. Animal selection: Purebred BALB / C mice.

[0027] The first immunization is 1-50 μg of antigen plus Freund's complete adjuvant injected subcutaneously at multiple points (usually 0.8-1 ml, 0.2 ml / point); the second immunization 3 weeks later, the dose is the same as the first immunization, plus Freund's incomplete adjuvant injected subcutaneously; the third immunization 3 weeks later, the dose is the same as the first immunization, without adjuvant, ip (blood is collected 5-7 days later to measure its titer); the booster immunization 2-3 weeks later, the dose is 100-400 μg, iv (intravenous injection); the spleen is taken out for fusion 3 days later.

[0028] (2) Cell fusion 1. Preparation before cell fusion (1) Selection of myeloma cell lines: Myeloma cells should be of the same strain as the immunized animal, so that the hybrid fusion rate is high and it is also convenient to produce a large number of McAbs in the abdominal cavity of mice of the same strain after inoculation of hybridomas.

[0029] (2) Feeder cells: In tissue culture, single or a few scattered cells are not easy to grow and reproduce. If other living cells are added, the growth and reproduction of these cells can be promoted. The added cells are called feeder cells. Commonly used feeder cells are: mouse peritoneal macrophages (more commonly used), mouse spleen cells or thymus cells. The amount of feeder cells is generally 2×10 4 or 2×10 5 cells / well.

[0030] 2. Steps of cell fusion (1) Preparation of feeder cell layer: Mouse peritoneal macrophages are generally used.

[0031] Mice of the same strain as the immunized mice (BALB / C mice, 6-10 weeks) were selected and killed by cervical dislocation. The mice were immersed in 75% alcohol for 3-5 minutes. The skin was cut open with sterile scissors to expose the peritoneum. 5-6 ml of pre-cooled culture medium was injected with a sterile syringe (puncture of the intestine was strictly prohibited). The cells were rinsed repeatedly, and the rinsing fluid was aspirated and placed in a 10 ml centrifuge tube. After separation at 1200 rpm for 5-6 minutes, the cells were suspended in culture medium containing 20% ​​fetal calf serum (FCS) and the cell count was adjusted to 1×10 5 / ml, add to 96-well plate, 100μl / well, and culture in a 37℃ CO2 incubator.

[0032] (2) Preparation of immune spleen cells Three days after the last booster immunization, the mice were killed by pulling the neck, and the spleen was removed aseptically and washed once with culture medium. The spleen was crushed, passed through a cell sieve, centrifuged, and the cells were washed twice with culture medium, counted, and 10 8 The spleen lymphocyte suspension was set aside.

[0033] (3) Preparation of myeloma cells Take logarithmically growing myeloma cells, centrifuge them, wash them twice with serum-free culture medium, count them, and obtain 10 7 The cells are ready for use.

[0034] (4) Integration ① Mix myeloma cells and spleen cells at a ratio of 1:10, wash once with serum-free incomplete culture medium in a 50ml centrifuge tube, centrifuge at 1200rpm for 8min, discard the supernatant, aspirate the remaining liquid with a pipette, and gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly.

[0035] ② Add 1 ml of 45% PEG (molecular weight 4000) solution pre-warmed at 37°C within 90 seconds, shaking gently. Incubate in a 37°C water bath for 90 seconds.

[0036] ③Add incomplete culture medium preheated at 37℃ to terminate the action of PEG, and add 1ml, 2ml, 3ml, 4ml, 5ml and 6ml every 2 minutes.

[0037] ④ Centrifuge, 800rpm, 6min.

[0038] ⑤ Fill the supernatant and resuspend in HAT selection culture medium containing 20% ​​calf serum.

[0039] ⑥ Add the above cells to a 96-well plate with a feeder cell layer, add 100 μl to each well, and inoculate 10 96-well plates.

[0040] ⑦ Place the culture plate in a 37°C, 5% CO2 incubator.

[0041] (III) Hybridoma cell selection and antibody detection 1. HAT selection of hybridoma cells After PEG treatment, spleen cells and myeloma cells form a mixture of multiple cells. Only hybridoma cells formed by spleen cells and bone marrow cells are meaningful. When cultured in HAT selective medium, myeloma cells cannot grow and reproduce because they lack thymidine kinase or hypoxanthine guanine ribosyltransferase. However, hybridoma cells possess these two enzymes and can grow and reproduce in HAT selective medium.

[0042] Within 1-2 days of HAT selection culture, a large number of tumor cells will die. After 3-4 days, the tumor cells disappear, and the hybrid cells form small colonies. After 7-10 days of HAT selection culture, the culture medium should be switched to HT culture medium. After another 2 weeks, the medium should be switched to standard culture medium. During this selection culture period, when the hybridoma cells cover 1 / 10 of the well bottom area, specific antibody testing can be started to screen for the desired hybridoma cell line. During the selection culture period, half of the culture medium should be replaced every 2-3 days.

[0043] 2. Antibody detection The method for detecting antibodies should be selected according to the nature of the antigen and the type of antibody. Generally, a rapid, simple, specific and sensitive method is used as the principle. The present invention uses an enzyme-linked immunosorbent assay (ELISA) to initially screen for immunopepsin antibodies.

[0044] (IV) Cloning of hybridomas Hybridoma cloning generally involves cloning antibody-positive wells. The principle of cloning is to clone antibody-positive hybridoma clones as soon as possible. Otherwise, antibody-secreting cells will be suppressed by non-antibody-secreting cells. This is because non-antibody-secreting cells grow faster than antibody-secreting cells, and competition between the two will result in the loss of antibody-secreting cells. The cloning method used in this invention is the limiting dilution method.

[0045] Limiting dilution cloning: (1) Prepare the feeder cell layer one day before cloning (see the cell fusion step above for the method).

[0046] (2) Gently blow dry the hybridoma cells to be cloned from the culture wells and count them.

[0047] (3) Adjust the cell density to 3-10 cells / ml.

[0048] (4) Take the cell culture plate with the feeder cell layer prepared the day before and add 100 μl of diluted cells to each well. Incubate in a 37°C, 5% CO2 incubator.

[0049] (5) Change the medium on the 7th day, and then every 2 to 3 days.

[0050] (6) Cell clone formation can be seen in 8 to 9 days, and antibody activity can be detected in time.

[0051] (7) Transfer the cells from the positive wells to a 24-well plate for expansion culture.

[0052] (8) Each clone should be frozen as soon as possible.

[0053] (V) Preparation of mouse ascites Select BABL / c female mice over 10 weeks old and inject 0.5 mL of paraffin into the peritoneal cavity of each mouse. One week later, inoculate the cells. 2 days before injection, subculture the hybridoma cells with fresh culture medium to ensure that they are in the logarithmic growth phase. Inoculate each mouse with 0.5 mL of 1×10 6 7-10 days after the injection of hybridoma cells, ascites formed in the mice and was collected.

[0054] (VI) Antibody purification 1. Purification of antibodies using ammonium octanoate sulfate High concentrations of salt ions in a protein solution can compete with the protein for water molecules, thereby destroying the hydration membrane on the protein surface, reducing its solubility, and causing it to precipitate from the solution. Different proteins have different solubilities, so different concentrations of salt solutions can be used to precipitate different proteins. This method is called salting out.

[0055] 1. Reagent preparation 0.06M acetate buffer (pH=4.8); 0.1M PBS buffer (pH=7.2-7.4); 2M HCl solution; 2M NaOH solution.

[0056] 2. Purification steps (1) Remove ascites at -20°C and thaw at room temperature for 30 minutes (or thaw at 4°C overnight); (2) Ascites was filtered through a 0.45 μm filter to initially remove impurities, fat, and cell debris; (3) Centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant, and discard the precipitate. (4) Accurately measure the volume of serum ascites, mix thoroughly with 3 volumes of acetate buffer, and adjust the pH to 4.5-4.8 with 2M HCl; (5) Slowly add n-octanoic acid to a final concentration of 33 μL / mL ascites under magnetic stirring. Mix at room temperature for 30 minutes and then let stand at 4°C for more than 2 hours. (6) Centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant, and discard the precipitate. (7) Filter the supernatant with a 0.45 μm filter, accurately measure the volume (Vsupernatant), add 1 / 10 volume of 0.1 M PBS (VPBS), adjust the pH to 7.4 with 2 M NaOH, and record the volume of NaOH used (VNaOH); (8) Add ammonium sulfate solid to the supernatant in an ice bath with magnetic stirring to a final concentration of 0.277 g / mL within 30 min and let stand at 4°C overnight. (9) Centrifuge at 12000 rpm for 30 min at 4°C, discard the supernatant, and collect the precipitate; (10) Dissolve the precipitate in 0.01 M PBS (pH = 7.4) with a volume of 1 / 10 of the original ascites fluid, filter with a 0.22 μm filter membrane, and set aside.

[0057] 2. Purification of Protein A by Affinity Chromatography Protein A can specifically bind to the Fc region of antibody IgG molecules. Protein A can be coupled to agarose gel as a ligand. When antiserum flows through it, specific IgG binds to the ligand, while other miscellaneous proteins pass through.

[0058] 1. Reagent preparation (1) Binding / washing solution: 0.15M NaCl, 20mM Na2HPO4 (PH = 7.0, 2L) 17.55g NaCl, 14.325g Na2HPO4 4· 12H2O, dissolve and adjust pH to 7.0, and make up to 2L; (2) Elution Buffer: 0.1M glycine (pH = 3.0, 500ml) Dissolve 3.7525 g of glycine, adjust the pH to 3.0, and make up to 500 ml. (3) Neutralizing solution: 1 M Tris-HCl, pH 8.0, homemade in the laboratory; (4) 0.01M PBS pH = 7.2-7.4, homemade in the laboratory; (5) 20% ethanol: 20 ml ethanol + 80 ml ultrapure water.

[0059] 2. Purification process (1) Dilute the above sample with 5 times the volume of buffer to ensure that the sample solution has appropriate ionic strength and pH value; (2) Take out the Protein A Beads from 4°C and equilibrate them with 2 column volumes of ddH2O; then equilibrate them with 2 column volumes of combined buffer to keep the filler in the same buffer system as the target protein, thereby protecting the protein. (3) Add the diluted sample to the equilibrated Protein A Beads at a moderate flow rate. (4) Wash with 5 column volumes of washing buffer to remove non-specifically adsorbed proteins; (5) Connect the nucleic acid protein detector, adjust the wavelength to 280nm, and wash with the wash buffer until the numbers on the display no longer change, then start elution; (6) Use 2 column volumes of elution buffer and collect the eluate, i.e., the target protein. To obtain a higher purity antibody, start collecting the eluate when the display value reaches 0.500, and stop collecting when it is lower than 0.500; (7) Add 1 M Tris-HCl for neutralization. Add 1 ml of neutralizing solution to 5 ml of eluate. (8) The eluate was aspirated into a dialysis bag, placed in 0.01 M PBS, and dialyzed overnight at 4°C; (9) The Protein A Beads column was equilibrated with 2 column volumes of washing buffer, then equilibrated with 2 column volumes of ddH2O, and finally equilibrated with 2 column volumes of 20% ethanol. The column was then stored in an equal volume of 20% ethanol at 4°C to prevent bacterial contamination of the filler. (10) Remove the dialysis bag and concentrate it. Use PEG20000 to concentrate the sample to 1-2 ml. Measure the concentration with BCA and store at -20°C. Record the concentration and volume. When storing, label the antibody name, concentration, purification date, purification method, etc.

[0060] Example 2 After animal immunization, antibody screening, and antibody purification, a total of six pepsin antibodies were obtained, numbered 1#, 2#, 3#, 4#, 5#, and 6#. Next, they were paired according to the permutations and combinations, and immunofluorescence labeling and coating were performed, resulting in a total of 30 combinations. By examining the reaction signal value, linearity, and detection range, it was determined that the optimal antibody pair for the fluorescent immunochromatographic platform to detect pepsin was 2# and 6#.

[0061] Specifically, all antibodies were paired and applied to the immunofluorescence chromatography platform to examine the linear coefficient and detection range. The results are shown in Table 1, and the examination method is shown in Example 3. After comparison, the linear advantages of pairing 2# and 6# were the most obvious, and the detection linear coefficients and linear ranges of other antibody pairs were not much different.

[0062] Table 1 Linear correlation coefficients R of different antibody pairs 2 contrast By analyzing the above data, four antibody combinations with relatively good linearity were selected for investigation within the detection range. The results are shown in Table 2. Samples containing varying concentrations of pepsin were prepared (0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, and 5 ng / mL). Testing was primarily conducted at the lowest detectable concentration. Performance requirements: For samples with concentrations less than 1 ng / mL, the absolute deviation should be ≤ ±0.2 ng / mL; for samples ≥1 ng / mL, the relative deviation should be <10%. "-" indicates non-detection due to low concentration.

[0063] Table 2 After the above screening, it was finally determined that the optimal antibody pair for detecting pepsin on the fluorescent immunochromatography platform was 2# and 6#.

[0064] Antibody sequence determination: The heavy and light chain CDR regions of antibodies are important amino acid sequence regions for recognizing and binding to their respective antigens. Several conservative amino acid substitutions within the CDR region amino acid sequence generally do not alter the protein's structure. Therefore, amino acid substitutions in the variable region sequence may still confer binding properties to the corresponding antigen. Total RNA from hybridoma cell lines was extracted using the Trizol method. PCR amplification was performed using primers specific to the heavy and light chains of the corresponding antibodies. The resulting RNA was expressed in E. coli using a vector, and positive results were selected for sequencing. The determined sequences were compared with the CDR region sequences (variable regions) of mouse monoclonal antibodies using the BLAST website (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0065] The final amino acid sequence of the heavy chain variable region of antibody 2# is: KGLEWYAESVKGRFTISRDDCAASGFTVESGGGEKGLEWCAASGFGSGYSFIDNWIASDTAMYYCARRTWDGSLKGQRISISCSVQSGAEVKKPGESLKISCKGSGYSFI (SEQ ID No: 1) Among them, CDR1 sequence (positions 24-31): SGFTVESG (SEQ ID No: 2); CDR2 sequence (positions 48-57): GYSFIDNWIA (SEQ ID No: 3); CDR3 sequence (positions 95-102): GESLKISC (SEQ ID No: 4).

[0066] The amino acid sequence of the light chain variable region of antibody 2# is: MHWNQQYRASKSDCYGGGSKSGTSASVRQSISISCSGSLEWMGIIHRYSPSFQTQSPASLAQQKPINAAYLQWNSLAPSWKSGGGFYDEQGAVFFYEETRGFE (SEQ ID No: 5).

[0067] Among them, CDR1 sequence (positions 18-27): GSKSGTSASV (SEQ ID No: 6); CDR2 sequence (positions 42-46): MGIIH (SEQ ID No: 7); CDR3 sequence (positions 87-94): YDEQGAVF (SEQ ID No: 8).

[0068] The amino acid sequence of the heavy chain variable region of antibody #6 is: ADKSINAAYLQWSGLQSEDESGAPGQRDSKSSIVLTQSASGAPGQRSNERPSGVPDRKLSCAYHHLPGTAPRQSEDEADSGVPDRDNWIAWVMSADKSINAAYELTVL (SEQ ID No: 9).

[0069] CDR1 sequence (positions 17-23): SEDESGA (SEQ ID No: 10); CDR2 sequence (positions 41-45): GAPGQ (SEQ ID No: 11); CDR3 sequence (positions 91-100): WVMSADKSIN (SEQ ID No: 12).

[0070] 6# Antibody light chain variable region amino acid sequence: PEKGLEWPGKGLEWMGIIHPVKKPGESLKISCKGSGYSFIDNWIAWVRGGGQVQLVQSGAEVKKPGESLKISCKGSGYSVFGGGTELTVLRVDCYGGPCYPG (SEQ ID No: 13).

[0071] Among them, CDR1 sequence (positions 22-32): KKPGESLKISC (SEQ ID No: 14); CDR2 sequence (positions 47-53): VRGGGQV (SEQ ID No: 15); CDR3 sequence (positions 89-96): VLRVDCYG (SEQ ID No: 16).

[0072] Example 3 Antibodies 2# and 6# of Example 2 were applied to a fluorescent immunochromatographic platform to perform pepsin detection, as follows: 1. Preparation of fluorescent immunochromatographic test strips The test strips consist of a sample membrane, a marker membrane, a test membrane, and a driver membrane. The test membrane includes test lines and quality control lines. The sample and marker membranes are both made of fiberglass, the test membrane is made of nitrocellulose, and the driver membrane is made of cellulose.

[0073] (1) Fluorescent microsphere labeling / labeling membrane preparation The labeled membrane was treated by dip-coating with 10 mM PBS (pH 7.6) containing 0.01% Tween 20. The treated membrane was allowed to air dry at room temperature for 24 hours. The microspheres to be labeled were washed with 0.05 M MES solution. Centrifugation was performed, and the supernatant was removed. The labeled sites on the microspheres were activated with EDC and NHS solutions, and the above-mentioned antibody #6 (detection / labeling antibody, final concentration 0.5 mg / mL) was added for labeling. The labeled membrane was gently mixed for 2 hours at room temperature. Centrifugation was performed, the supernatant was removed, and BSA solution was added. The membrane was blocked by gentle mixing at room temperature for 1 hour. The BSA concentration was 2 mg / mL. Centrifugation was performed, the supernatant was removed, and 0.1 M TRIS solution was added for storage, resulting in a final antibody concentration of 0.5 mg / mL. The labeled microsphere solution was sprayed onto the surface of the labeled membrane. When spraying the solution containing the detection antibody / fluorescent microsphere conjugate, a 0.1% concentration of 10 mM PBS (pH 7.6) was used as the solvent, which contained a final concentration of 10% trehalose, 0.1% BSA, and 0.1% Tween 20. The sprayed labeled membrane was dried at 37°C for 2 hours.

[0074] (2) Preparation of detection membrane (antibody coating) The test line on the test membrane was sprayed with the aforementioned antibody 2#, where the concentration of antibody 2# was 0.6 mg / mL and the solvent was 10 mM PBS (pH 7.6), which also contained BSA at a final concentration of 0.1% and 0.5% methanol. The spray volume was 0.08 μL / cm 2 After spraying, dry at room temperature for 72 hours. The spray solution at the quality control line is a fixed solution containing goat anti-mouse IgG (Sigma, M6898), where the concentration of goat anti-mouse IgG is 1.0 mg / mL and the solvent is 10 mM PBS (pH 7.6), which also contains BSA with a final concentration of 0.1% and 0.5% methanol. The spray volume is 0.01 μL / cm 2 After spraying, dry at 37℃ for 2h.

[0075] (3) Assembly Driving membrane: The driving membrane is made of filter paper and dried at 100°C for 24 hours before use. The sample membrane, marker membrane, test membrane, and driving membrane are assembled by overlapping the sample membrane (15 mm long), marker membrane (10 mm long), test membrane (25 mm long), and drive membrane (30 mm long) in this order (the overlap width between the sample membrane and marker membrane, and between the marker membrane and test membrane is 2 mm; the overlap width between the test membrane and the drive membrane is 4 mm). These membranes are then attached to a PVC substrate to form a large plate. The test line is approximately 8 mm from the edge of the marker membrane closest to the test line, and the control line is approximately 10 mm from the edge of the drive membrane closest to the control line. The assembled plate is cut into narrow strips 4.0 mm wide to prepare test strips.

[0076] 2. Pepsin detection The test strips were used to test pepsin samples at concentrations of 5 ng / mL, 50 ng / mL, 150 ng / mL, 300 ng / mL, and 600 ng / mL. The samples were diluted to the target concentrations from a fixed-concentration pepsin solution. The diluent consisted of 0.1 M Tris solution and 0.1% Tween-20. The sample was added, and after 12 minutes of chromatography, fluorescence detection was performed. Detection wavelength: 610 nm.

[0077] The concentration values ​​were linearly fitted with the T / C values ​​of the test strips. The results are shown in Table 3 and Figure 1 Observe the linear correlation coefficient R 2 =0.997.

[0078] Table 3 Example 4 This example further compares the detection ranges of the aforementioned pepsin antibodies 2# and 6# with those of commercially available antibody pairs: Medix, labeling antibody item number: HM1134, coating antibody item number: HM1136.

[0079] Experimental plan: Prepare the commercially available antibodies into immunofluorescence test strips according to the above steps of Example 3, and then compare the test strips prepared with the antibodies screened by the present invention. Prepare a standard curve by testing pepsin samples with concentrations of 5ng / mL, 50ng / mL, 150ng / mL, 300ng / mL, and 600ng / mL, and detect specific values ​​to evaluate the detection range. Performance requirements: For samples with a detection concentration less than 1ng / mL, the absolute deviation is ≤±0.2ng / mL, and for samples ≥1ng / mL, the relative deviation is <10%. "-" indicates that it was not detected due to low concentration. Linear results are shown in Figure 2 The test results of samples with specific concentrations are shown in Table 4.

[0080] Table 4 After analyzing the above test results, the following conclusions can be drawn: 1. The antibody pair screened by the present invention can detect 0.1ng / mL, which meets the requirements, while the commercially available antibody pair cannot detect this concentration. 2. For the detection of low, medium and high concentration samples, the test results of the reagent strips prepared by the antibody pair screened by the present invention all meet the requirements, while the results of the commercially available antibody pairs at three concentrations of 0.1ng / mL, 800ng / mL and 1000ng / mL do not meet the requirements. This shows that the antibody pairs 6# and 2# screened by the present invention have a wider detection range (0.1-1000ng / mL) when used in the fluorescent immunochromatography detection platform, with small deviation and better accuracy.

[0081] Example 5 According to the description of Example 3, three batches of fluorescent immunochromatographic test strips were prepared continuously using three batches of antibody pairs and applied to the fluorescent immunochromatographic platform for detection experiments (see Example 3 for the method), and the inter-batch range of the antibody pairs was investigated. The results are shown in Table 5. Range R = (X max -X min ) / A, X is the test value, A is the average of three consecutive batches of test values. Performance index: range ≤10%.

[0082] Table 5 Analysis of the above data shows that the inter-batch ranges are all within the performance index requirements. The antibody pairs screened by the present invention meet the performance requirements.

[0083] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that: The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 3, and the CDR3 sequence is shown in SEQ ID NO: 4; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 6, the CDR2 sequence is shown in SEQ ID NO: 7, and the CDR3 sequence is shown in SEQ ID NO: 8; Alternatively, the CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO: 10, the CDR2 sequence is shown in SEQ ID NO: 11, and the CDR3 sequence is shown in SEQ ID NO: 12; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 14, the CDR2 sequence is shown in SEQ ID NO: 15, and the CDR3 sequence is shown in SEQ ID NO:

16.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region sequence is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 5; Alternatively, the heavy chain variable region sequence is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO:

13.

3. An antibody pair, characterized in that Including primary antibody and secondary antibody; The CDR1 sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 3, and the CDR3 sequence is shown in SEQ ID NO: 4; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 6, the CDR2 sequence is shown in SEQ ID NO: 7, and the CDR3 sequence is shown in SEQ ID NO: 8; The CDR1 sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO: 10, the CDR2 sequence is shown in SEQ ID NO: 11, and the CDR3 sequence is shown in SEQ ID NO: 12; the CDR1 sequence of the light chain variable region is shown in SEQ ID NO: 14, the CDR2 sequence is shown in SEQ ID NO: 15, and the CDR3 sequence is shown in SEQ ID NO:

16.

4. The antibody pair according to claim 3, characterized in that The heavy chain variable region sequence of the first antibody is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 5; The heavy chain variable region sequence of the second antibody is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO:

13.

5. A nucleic acid molecule, characterized in that Encodes the antibody or antigen-binding fragment thereof according to claim 1 or 2.

6. A biological material containing the nucleic acid molecule according to claim 5, wherein the biological material is a vector, an expression cassette or a host cell.

7. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2, the antibody pair according to claim 3 or 4, the nucleic acid molecule according to claim 5, or the biomaterial according to claim 6 in the preparation of a reagent or kit for detecting pepsin.

8. A reagent or kit for detecting pepsin, characterized in that: The method comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody pair according to claim 3 or 4.

9. A method for detecting pepsin for purposes other than disease diagnosis or treatment, characterized in that: Detection is performed using the antibody or antigen-binding fragment thereof according to claim 1 or 2, the antibody pair according to claim 3 or 4, or the reagent or kit for detecting pepsin according to claim 8.

10. The method according to claim 9, characterized in that The first antibody is used as the coating antibody and the second antibody is used as the labeling antibody for detection; the first antibody and the second antibody are as described in claim 3 or 4.

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