Anti-aeromonas hydrophila monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-aeromonas hydrophila monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of antibodies. When the monoclonal antibody or the antigen binding part of the monoclonal antibody is used for detecting aeromonas hydrophila, the sensitivity is high, and the specificity is strong. The colloidal gold test strip prepared from the monoclonal antibody 2D5 and the monoclonal antibody 8F3 can be used for detecting aeromonas hydrophila in a biological sample so as to judge whether a source host suffers from aeromonas hydrophila related diseases or not, and also has the advantages of high sensitivity and strong specificity, so that the colloidal gold test strip has high detection accuracy, and is suitable for popularization and application. When the method is used for detection, expensive instruments and equipment are not needed, and the method has huge clinical significance and wide application prospects.

Description

Technical Field

[0001] The present application relates to the technical field of antibodies, and particularly relates to a monoclonal antibody against Aeromonas hydrophila, a preparation method thereof, and an application thereof. Background Art

[0002] Aeromonas hydrophila ( Aeromonas hydrophila ) is distributed all over the world and widely exists in fresh water, brackish water, and domestic sewage. It is pathogenic to cultured and wild fish, shrimps, frogs, soft-shelled turtles and other organisms, and can further infect humans through the food chain, causing diarrhea and septicemia in humans. The Aeromonas hydrophila diseases caused by them in fish and other organisms are characterized by wide prevalence, rapid onset, high mortality, etc., often causing huge economic losses to aquaculture fisheries and seriously endangering the development of fisheries and other aquaculture industries.

[0003] The adhesiveness of Aeromonas hydrophila is related to components such as pili, flagella, and lipopolysaccharide "O" antigen (O-Ag). Flagella are speculated to be related to the adhesion and invasion of Aeromonas hydrophila on fish cell line cells. Flagella and lipopolysaccharide "O" antigen are related to the adhesion of Aeromonas hydrophila to human epithelial cells, and the genes flmA and flmB regulate the biosynthesis of lipopolysaccharide "O" antigen and the assembly of flagella. The main infection route of Aeromonas hydrophila to humans is the digestive tract, and it can also infect humans through damaged skin. Susceptible populations include children, the elderly, and immunocompromised individuals.

[0004] Monoclonal antibodies against Aeromonas hydrophila can provide technical support for the detection of Aeromonas hydrophila. However, there is a lack of monoclonal antibodies against Aeromonas hydrophila with high sensitivity and strong specificity in this field. Summary of the Invention

[0005] In order to solve at least one of the above technical problems, the technical solutions adopted in the present application are as follows.

[0006] In the first aspect of the present application, there is provided a monoclonal antibody 2D5 against Aeromonas hydrophila or an antigen-binding portion thereof, wherein the monoclonal antibody 2D5 or the antigen-binding portion thereof comprises: a heavy chain variable region, the amino acid sequences of its CDR-H1, CDR-H2, and CDR-H3 are respectively as shown in SEQ ID No. 3 to SEQ ID No. 5; and a light chain variable region, the amino acid sequences of its CDR-L1, CDR-L2, and CDR-L3 are respectively as shown in SEQ ID No. 8 to SEQ ID No. 10.

[0007] In some embodiments of the present application, the monoclonal antibody 2D5 or the antigen-binding portion thereof comprises: a heavy chain variable region, the amino acid sequence of which is as shown in SEQ ID No. 2; and The light chain variable region, the amino acid sequence of which is shown in SEQ ID No. 7.

[0008] In the present invention, the light chain constant region subtype of the monoclonal antibody 2D5 against Aeromonas hydrophila is of the Igκ type, and the heavy chain constant region subtype is of the IgG1 type.

[0009] In the present application, the antigen-binding portion refers to the antigen-binding fragment of an antibody and antibody mimetics, which generally includes at least a part of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The fragment of the antibody retains at least some of the binding specificities of the parent antibody. In some embodiments of the present invention, the antigen-binding portion is selected from any one of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, and scFv antibodies. Among them, A "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain.

[0010] A "Fab' fragment" contains a light chain and a part of a heavy chain that includes the VH domain, the CH1 domain, and a portion of the constant region between the CH1 and CH2 domains. An interchain disulfide bond is formed between the two heavy chains of two Fab' fragments to form an F(ab')2 fragment.

[0011] An "F(ab')2 fragment" contains two light chains and two parts of heavy chains that include the VH domain, the CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Therefore, an F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0012] An "Fv fragment" contains the variable regions from both the heavy chain and the light chain, but lacks the constant regions.

[0013] A "single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment that contains the VH and VL domains of an antibody, and these domains are contained in a single polypeptide chain.

[0014] The second aspect of the present application provides a gene encoding the monoclonal antibody 2DF against Aeromonas hydrophila or its antigen-binding portion as described in any one of the first aspect of the present application.

[0015] In some embodiments of the present application, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2DF is shown in SEQ ID No. 1.

[0016] In some embodiments of the present application, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2DF is shown in SEQ ID No. 6.

[0017] The third aspect of the present application provides the use of the monoclonal antibody 2D5 against Aeromonas hydrophila according to any one of the first aspects of the present invention or its antigen-binding portion in the preparation of a kit for detecting Aeromonas hydrophila in a biological sample.

[0018] In some embodiments of the present application, the kit further comprises another monoclonal antibody against Aeromonas hydrophila or its antigen-binding portion that has a different antigenic epitope from the antigenic epitope of the monoclonal antibody 2D5 against Aeromonas hydrophila or its antigen-binding portion.

[0019] In some specific embodiments of the present application, the another monoclonal antibody against Aeromonas hydrophila or its antigen-binding portion is the monoclonal antibody 8F3 or its antigen-binding portion, comprising: a heavy chain variable region, the amino acid sequences of its CDR-H1, CDR-H2, and CDR-H3 are respectively shown as SEQ ID No. 13 to SEQ ID No. 15; and a light chain variable region, the amino acid sequences of its CDR-L1, CDR-L2, and CDR-L3 are respectively shown as SEQ ID No. 18 to SEQ ID No. 20.

[0020] In some embodiments of the present application, the monoclonal antibody 8F3 or its antigen-binding portion comprises: a heavy chain variable region, the amino acid sequence of which is shown as SEQ ID No. 12; and a light chain variable region, the amino acid sequence of which is shown as SEQ ID No. 17.

[0021] The fourth aspect of the present application provides a kit comprising the monoclonal antibody 2D5 against Aeromonas hydrophila according to any one of the first aspects of the present invention or its antigen-binding portion.

[0022] In some embodiments of the present application, the kit further comprises another monoclonal antibody against Aeromonas hydrophila or its antigen-binding portion that has a different antigenic epitope from the antigenic epitope of the monoclonal antibody 2D5 against Aeromonas hydrophila or its antigen-binding portion.

[0023] In some specific embodiments of the present application, the another monoclonal antibody against Aeromonas hydrophila or its antigen-binding portion is the monoclonal antibody 8F3 or its antigen-binding portion, comprising: a heavy chain variable region, the amino acid sequences of its CDR-H1, CDR-H2, and CDR-H3 are respectively shown as SEQ ID No. 13 to SEQ ID No. 15; and a light chain variable region, the amino acid sequences of its CDR-L1, CDR-L2, and CDR-L3 are respectively shown as SEQ ID No. 18 to SEQ ID No. 20.

[0024] In some embodiments of the present application, the monoclonal antibody 8F3 or its antigen-binding portion comprises: A heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 12; and A light chain variable region having an amino acid sequence as shown in SEQ ID No. 17.

[0025] In some feasible embodiments of the present application, a colloidal gold test strip is prepared using the monoclonal antibody 2D5 or its antigen-binding portion and the monoclonal antibody 8F3 or its antigen-binding portion.

[0026] In some feasible embodiments of the present application, the colloidal gold test strip comprises a bottom plate, on which a sample pad, a colloidal gold pad, a nitrocellulose membrane, and a water-absorbing pad are sequentially fixed.

[0027] In some specific embodiments of the present application, one of ① the monoclonal antibody 2D5 or its antigen-binding portion and ② the monoclonal antibody 8F3 or its antigen-binding portion is used to prepare the colloidal gold pad, and the other of ① the monoclonal antibody 2D5 or its antigen-binding portion and ② the monoclonal antibody 8F3 or its antigen-binding portion is used to prepare the test line (T line).

[0028] In some feasible embodiments of the present application, the preparation method of the colloidal gold pad is as follows: Adjust the colloidal gold solution to pH 8.0 with 0.2M K2CO3 solution, add one of ① the monoclonal antibody 2D5 or its antigen-binding portion and ② the monoclonal antibody 8F3 or its antigen-binding portion to a final concentration of 10 μg / mL, and stir for 30 min; slowly add an appropriate amount of 10% BSA to make its final concentration 1%, and stir for 30 min; then centrifuge the solution at 1000 rpm at 4°C for 15 min, and take the supernatant; then centrifuge the supernatant at 12000 rpm at 4°C for 30 min, discard the supernatant, and dissolve and concentrate the precipitate with reconstitution solution; spray the concentrated colloidal gold complex onto the colloidal gold pad with a gold spraying and membrane scribing integrated machine, the gold spraying concentration is OD20, the spraying amount is 2 μL / cm, and dry naturally.

[0029] The test line (T line) and the quality control line (C line) are sprayed on the surface of the nitrocellulose membrane, and the test line is the other of ① the monoclonal antibody 2D5 or its antigen-binding portion and ② the monoclonal antibody 8F3 or its antigen-binding portion.

[0030] The present application also provides a monoclonal antibody 8F3 against Aeromonas hydrophila or its antigen-binding portion, and the monoclonal antibody 8F3 or its antigen-binding portion comprises: A heavy chain variable region, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID No. 13 to SEQ ID No. 15 respectively; and A light chain variable region, wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID No. 18 to SEQ ID No. 20 respectively.

[0031] In some embodiments of the present application, the monoclonal antibody 8F3 or its antigen-binding portion comprises: A heavy chain variable region, wherein the amino acid sequence is shown in SEQ ID No. 2; and A light chain variable region, wherein the amino acid sequence is shown in SEQ ID No. 7.

[0032] The present application also provides a gene encoding the monoclonal antibody 8F3 or its antigen-binding portion.

[0033] In some embodiments of the present application, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8F3 is shown in SEQ ID No. 11.

[0034] In some embodiments of the present application, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8F3 is shown in SEQ ID No. 16.

[0035] The present application also provides the use of the monoclonal antibody 2D5 or its antigen-binding portion described in ① and the monoclonal antibody 8F3 or its antigen-binding portion described in ② in the preparation of a kit for detecting Aeromonas hydrophila based on the sandwich ELISA method.

[0036] Compared with the prior art, the present application has the following beneficial effects: Using the monoclonal antibody or its antigen-binding portion of the present application to detect Aeromonas hydrophila has high sensitivity and strong specificity.

[0037] Using the monoclonal antibody 2D5 and the monoclonal antibody 8F3 of the present application to prepare a colloidal gold test strip can be used to detect Aeromonas hydrophila in biological samples, and then judge whether the source host has Aeromonas hydrophila-related diseases. It also has high sensitivity and strong specificity, thus having high detection accuracy. When used for detection, it does not require expensive instruments and equipment, and has great clinical significance and broad application prospects.

[0038] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present application, nor is it used to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] These and other objects, features, and advantages of the exemplary embodiments of the present application will become readily apparent by reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of the present application are shown by way of illustration and not limitation, wherein: Figure 1 Shows the results of the specific identification of the monoclonal antibody in Example 2 of the present application; Figure 2 Shows the results of detecting Aeromonas hydrophila at different concentrations with a colloidal gold test strip in Example 7 of the present application. Detailed implementation manners

[0040] Unless otherwise specified, implied from the context, or a convention of the prior art, all parts and percentages in this application are based on weight, and the testing and characterization methods used are synchronized with the filing date of this application. Where applicable, any patents, patent applications, or disclosures referred to in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0041] In order to make the technical problems, technical solutions, and beneficial effects solved by the present application more clearly understood, the following further describes the present application in detail with reference to the embodiments.

[0042] The following examples are used herein to demonstrate the preferred embodiments of the present application. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies discovered by the inventors that can be used to implement the present application, and thus can be regarded as the preferred solutions for implementing the present application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed here, and still obtain the same or similar results without departing from the spirit or scope of the present application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs. The materials cited herein and their citations will be incorporated by reference in the manner cited.

[0044] Those skilled in the art will recognize or, through routine experimentation, will be able to learn many equivalent technologies of many of the specific embodiments of the inventions described herein. These equivalents will be included in the claims.

[0045] In the following embodiments, the experimental methods are conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following embodiments are obtained from a conventional biochemical reagent store unless otherwise specified.

[0046] Example 1 Preparation of Monoclonal Antibody Against Aeromonas hydrophila 1. Preparation of Antigen Aeromonas hydrophila was inoculated on LB solid bacterial medium by the streak plate method and incubated in an incubator at 37°C in an inverted position. When colonies appeared on the medium, monoclonal colonies were picked and inoculated into LB liquid medium for amplification. After the liquid medium became significantly turbid, it was centrifuged at 3000 rpm for 20 minutes, the supernatant was discarded, 1% formaldehyde was added for overnight fixation, then centrifuged at 3000 rpm for 20 minutes again, the supernatant was discarded, and it was resuspended with physiological saline for counting and stored in a refrigerator at 4°C for mouse immunization.

[0047] 2. Preparation of Monoclonal Antibody Against Aeromonas hydrophila Five 6-8-week-old female Balb / c mice were selected, and the formaldehyde-inactivated bacterial solution (1×10 8 cells / mL) was intraperitoneally injected into the mice at a dose of 0.5 mL / mouse. The mice were immunized again in the same way on the 7th, 14th, 21st, and 28th days, and spleen cells were taken on the 31st day for cell fusion.

[0048] 3. Cell Fusion After grinding and counting the mouse spleen cells, they were mixed with mouse myeloma cells sp2 / 0 at a ratio of 3:1 in a 50 mL centrifuge tube. After centrifugation at 1500 rpm for 3 min, the supernatant was aspirated completely, and the bottom of the tube was tapped to make the cells evenly distributed. The centrifuge tube was placed in a water bath preheated to 37°C, and 1 mL of cell fusion agent (50% PEG-1500) was slowly added within 1 min. After standing for 1 min, 40 mL of DMEM medium preheated to 37°C was gradually added to dilute the PEG and make it lose its effect. After centrifugation at 1500 rpm for 3 min, the supernatant was discarded. After resuspension with HAT medium, it was plated on a 96-well cell culture plate pre-added with feeder cells at a density of 10 5 spleen cells / well, 200 μL per well. It was cultured in a CO2 incubator at 5% (V / V) and 37°C.

[0049] 4. Screening of Positive Wells and Cell Cloning When the cells in the small wells grew to cover one-fourth of the bottom area of the well, the indirect ELISA method was used to aspirate the culture medium supernatant to screen for positive cell wells. Five rounds of cloning culture were carried out by the limited dilution method until the positive rate reached 100%.

[0050] Thus, pure hybridoma cells 2D5 and 8F3 were obtained.

[0051] 5. Preparation and purification of monoclonal antibody ascites Take Balb / c female mice at about 10 - 12 weeks old, intraperitoneally inject 0.3 mL of Freund's incomplete adjuvant. After 7 days, intraperitoneally inject 0.3 mL of cell suspension containing 10 6 hybridoma cells. It can be seen that the abdomen of the mice swells significantly 7 - 10 days after injection. After sacrificing the mice by cervical dislocation, take the ascites, centrifuge at 12000 rpm for 3 min, and collect the supernatant, which is the ascites - type monoclonal antibody (2D5 or 8F3). Purify the monoclonal antibody by Protein A column chromatography and store it at - 80 °C.

[0052] Example 2 Identification of monoclonal antibody specificity The detection targets are Aeromonas hydrophila, Aeromonas sobria, Salmonella, Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter sakazakii respectively. Dilute the above antigens to 1×10 6 cells / mL with the coating buffer, add 100 μL per well to the enzyme - linked immunosorbent assay (ELISA) plate. Use the coating buffer as the blank control, and detect the specificity of 2D5 and 8F3 antibodies by indirect ELISA. The primary antibodies are 2D5 and 8F3 monoclonal antibodies diluted 2000 times, and the enzyme - labeled secondary antibody GAM - HRP is diluted 10000 times.

[0053] The detection results are as Figure 1 shown. The antibody reacts strongly with Aeromonas hydrophila, but does not cross - react with other bacteria. Therefore, the antibody has strong species specificity and can be used to detect Aeromonas hydrophila, and further diagnose the human and animal diseases caused by Aeromonas hydrophila.

[0054] Example 3 Identification of monoclonal antibody epitopes To determine whether the obtained monoclonal antibodies 2D5 and 8F3 target different epitopes of Aeromonas hydrophila, the ELISA additive index (AI) method was used for verification. The specific operation steps are as follows: (1) Dilute Aeromonas hydrophila to 1×10 6 cells / mL with the coating buffer to coat a 96 - well ELISA plate, 100 μL / well, coat at 37 °C for 2 h, and discard the coating buffer; (2) Block with 5% skim milk powder at 37 °C for 2 h, and discard the blocking solution in the wells; (3) Add the two different monoclonal antibodies to be measured diluted 2000 times respectively, add 100 μL / well of each monoclonal antibody. At the same time, take 50 μL of each of the above monoclonal antibodies diluted 1000 times and add them to the same well. The final volume of the additive well is 100 μL, react at 37 °C for 1 h, wash 3 times with PBST washing solution (0.01 M PBS, 0.1% Tween20, pH 7.4), and discard the liquid; (4) Add 100 μL of HRP-labeled goat anti-mouse IgG antibody to each well, incubate at 37 °C for 1 h, wash 3 times with PBST, and discard the liquid. (5) Add 100 μL of TMB substrate solution to each well. After 15 min, add 2 M H2SO4 stop solution, and read the results with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0055] Calculate the additive index, where A1 and A2 are the final absorbance values of the wells with the addition of two different monoclonal antibodies, and A 1+2 is the absorbance value of the well with the addition of the mixed monoclonal antibodies. According to the additive index calculation formula: AI = (A 1+2 - A1) / A2 × 100% to calculate the result. If AI is greater than 10%, it indicates that the two monoclonal antibodies target different antigen epitopes and have an additive effect; if AI is less than 10%, it indicates that the antigen epitopes of the two monoclonal antibodies are adjacent or the same and there is no additive effect.

[0056] The results showed that AI = 25%, indicating that 2D5 and 8F3 respectively target different antigen epitopes of Aeromonas hydrophila.

[0057] Example 4 Detection of the type and subclass of monoclonal antibodies A mouse monoclonal antibody subclass identification kit was used to identify the monoclonal antibodies. Aeromonas hydrophila was diluted to 1 × 10 6 cells / mL with the coating solution, and 100 μL of it was added to each well of the enzyme-linked immunosorbent assay (ELISA) plate, repeated 16 wells, and incubated at 37 °C for 2 h. After discarding the coating solution, wash 3 times with PBST. Take 50 μL of the monoclonal antibody 2D5 or monoclonal antibody 8F3 diluted 2000 times and add it to each well of the ELISA plate, incubate at 37 °C for 1 h, wash 3 times with PBST, take 50 μL of each of the 8 enzyme-labeled antibodies for detecting the Ig type and subclass in the kit and add them to each well, set 3 replicates for each enzyme-labeled antibody, incubate at 37 °C for 30 min, wash 3 times with PBST, add TMB substrate chromogenic solution for color development, add 50 μL of 2 M H2SO4 to each well to terminate the color development, and then read the OD450nm value with an ELISA reader. The antibody Ig type and subclass corresponding to the positive well are the antibody type and subclass of this monoclonal antibody.

[0058] After detection, it was determined that the antibody types of monoclonal antibody 2D5 and monoclonal antibody 8F3 were of the IgG type, among which the light chain constant region subtype was of the Igκ type and the heavy chain constant region subtype was of the IgG1 type.

[0059] Example 5 Gene sequencing of hybridoma cells 2D5 and 8F3 The sequencing of the variable regions of the light and heavy chains of monoclonal antibodies 2D5 and 8F3 was completed by Anhui General Biology.

[0060] The coding nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 2D5 is shown as follows: CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGGTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTATCTACTGGATAGAGTGGTTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGATATTTTACCTGGAAGTGGTAGTACTCACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCATTCTAGATATATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATTTGAAGACTCTGCCGTCTATTACTGTGCAAGAACTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 1) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 2D5 is as follows: QVQLKQSGAEVMKPGASVKISCKATGYTFSIYWIEWLKQRPGHGLEWIGDILPGSGSTHYNEKFKGKATFILDISSNTAYMQLSSLTFEDSAVYYCARTFDYWGQGTTLTVSS (SEQ ID No. 2) The variable region sequence of the heavy chain of monoclonal antibody 2D5 was analyzed (Kabat) to obtain its CDR regions. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the variable region of the light chain of monoclonal antibody 2D5 are respectively: CDR-H1: IYWIE (SEQ ID No. 3) CDR-H2: DILPGSGSTHYNEKFKG (SEQ ID No. 4) CDR-H3: TFDY (SEQ ID No. 5) The coding nucleotide sequence of the variable region of the light chain of monoclonal antibody 2D5 is as follows: GACATTGTGCTGACCCAATCTCCAGTTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTGTGCACTGGTACCAGCAGAAACCAGGACAGTCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 6) The amino acid sequence of the variable region of the light chain of monoclonal antibody 2D5 is as follows: DIVLTQSPVSLAVSLGQRATISCRASESVDSYGNSFVHWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK (SEQ ID No. 7) The sequence of the variable region of the light chain of monoclonal antibody 2D5 was analyzed (Kabat) to obtain its CDR regions. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the variable region of the light chain of monoclonal antibody 2D5 are respectively: CDR-L1: RASESVDSYGNSFVH (SEQ ID No. 8) CDR-L2: LASNLES (SEQ ID No. 9) CDR-L3: QQNNEDPWT (SEQ ID No. 10) The coding nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 8F3 is as follows: GATGTGCAGCTGAAGCAGTCTGGAGCTGAGGTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTATCTACTGGATAGAGTGGTTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGATATTTTACCTGGAAGTGGTAGTACTCACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCATTCTAGATATATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATTTGAAGACTCTGCCGTCTATTACTGTGCAAGAACTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 11) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 8F3 is as follows: DVQLKQSGAEVMKPGASVKISCKATGYTFSIYWIEWLKQRPGHGLEWIGDILPGSGSTHYNEKFKGKATFILDISSNTAYMQLSSLTFEDSAVYYCARTFDYWGQGTTLTVSS (SEQ ID No. 12) The variable region sequence of the heavy chain of monoclonal antibody 8F3 was analyzed (Kabat) to obtain its CDR regions. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the variable region of the light chain of monoclonal antibody 8F3 are respectively: CDR-H1: IYWIE (SEQ ID No. 13) CDR-H2: DILPGSGSTHYNEKFKG (SEQ ID No. 14) CDR-H3: TFDY (SEQ ID No. 15) The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 8F3 is as follows: GACATTGTGCTGACCCAATCTCCAGCTTCCTTGGCTGTGTCTCTTGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTCTGGCAAGAGTTTTATGCACTGGTTCCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCAGCAGCCTATTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGAC (SEQ ID No. 16) The amino acid sequence of the light chain variable region of monoclonal antibody 8F3 is shown as follows: DIVLTQSPASLAVSLGQRATISCRASESVDSSGKSFMHWFQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAAAYYCQQNNEDPWTFGGGTKLEIR (SEQ ID No. 17) The sequence of the light chain variable region of monoclonal antibody 8F3 was analyzed (Kabat) to obtain its CDR regions. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of monoclonal antibody 8F3 are respectively: CDR-L1: RASESVDSSGKSFMH (SEQ ID No. 18) CDR-L2: LASNLES (SEQ ID No. 19) CDR-L3: QQNNEDPWT (SEQ ID No. 20) From the antibody variable region sequences and CDR sequences, it can be seen that the light chain variable region sequences of monoclonal antibody 2D5 and monoclonal antibody 8F3 are different, and the CDR sequences are also different, resulting in different antigen epitopes being targeted.

[0061] Example 6 Detection of Preparation of Colloidal Gold Test Strip for Aeromonas hydrophila 1. Preparation of Colloidal Gold Pad Labeled with Monoclonal Antibody 2D5 Colloidal gold labeling: Adjust the colloidal gold solution to pH 8.0 with 0.2M K2CO3 solution, add an appropriate amount of 200μg / mL monoclonal antibody 2D5 solution to a final concentration of 10μg / mL, and stir for 30 min; slowly add an appropriate amount of 10% BSA to a final concentration of 1%, and stir for 30 min; then centrifuge the solution at 1000 rpm at 4°C for 15 min, and take the supernatant; then centrifuge the supernatant at 12000 rpm at 4°C for 30 min, discard the supernatant, and dissolve and concentrate the precipitate with the reconstitution solution (sucrose 0.2 g / mL, Tris:BSA (2.42:10), pH 8.5). Spray the concentrated colloidal gold complex onto the colloidal gold pad with a gold spraying and membrane scribing machine, with a gold spraying concentration of OD20 and a spraying volume of 2 μL / cm, and air dry naturally.

[0062] 2. Spraying of the test line and control line on the nitrocellulose membrane Spray the test line (T) and control line (C) on the surface of the nitrocellulose membrane with a gold spraying and membrane scribing machine. The test line is monoclonal antibody 8F3, and the control line is goat anti-mouse IgG antibody; the working concentration of 8F3 is 0.5 mg / mL, and the working concentration of goat anti-mouse IgG antibody is 2.0 mg / mL. The spraying volume of both during membrane scribing is 0.7 μL / cm.

[0063] 3. Judgment of the test results of the colloidal gold test strip Absorb 100 μL of the diluted sample to be tested, and drop it into the sample addition hole of the horizontally placed colloidal gold test strip, and let it stand at room temperature for 5 - 10 min, and observe and judge the result with the naked eye. If red bands appear in both the control line and the test line, the sample to be tested contains Aeromonas hydrophila, and the result is positive; if a red band appears in the control line and no red band appears at the test line, there is no Aeromonas hydrophila in the sample to be tested, and the result is negative; if no red band appears in the control line, the test result of this test strip is invalid.

[0064] Example 7 Determination of the minimum detection sensitivity of the colloidal gold test strip Dilute Aeromonas hydrophila with PBST buffer to 1×10 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL. Absorb 100 μL of the diluted bacterial solution to be tested and drop it into the sample addition hole of the horizontally placed colloidal gold test strip, and observe the result after standing at room temperature for 5 - 10 minutes.

[0065] As Figure 2 shown, the results show that: the test strip can detect 1×10 6Aeromonas hydrophila at CFU / mL. The color change of the test line on the test strip is used to judge the bacterial content in the sample. If the test line of the test sample shows color, it indicates that the test sample contains Aeromonas hydrophila at 1×10 6 CFU / mL or more, and the darker the color, the higher the content.

[0066] Example 8 Specificity Test of Colloidal Gold Immunochromatographic Test Strip Aeromonas hydrophila, Aeromonas sobria, Salmonella, Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, Enterobacter cloacae, and Cronobacter sakazakii were respectively diluted to 1×10 9 CFU / ml and 1×10 7 CFU / ml with PBST buffer and detected with the test strip. The results are shown in Table 1.

[0067] Table 1 Detection Results of Specificity Test of Colloidal Gold Immunochromatographic Test Strip

[0068] Among them, "+" indicates that the T line shows color, indicating that the test strip has a specific reaction with the corresponding strain, and "-" indicates that the T line does not show color, indicating that the test strip has no specific reaction with the corresponding strain.

[0069] As can be seen from Table 1, the colloidal gold immunochromatographic test strip prepared with monoclonal antibody 2D5 and monoclonal antibody 8F3 only reacts with Aeromonas hydrophila and has no visible reaction with other experimental strains, which is consistent with the indirect ELISA detection results, indicating that the colloidal gold immunochromatographic test strip has good specificity.

[0070] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof, characterized in that: The monoclonal antibody 2D5 or an antigen-binding portion thereof comprises: A heavy chain variable region, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID No. 3 to SEQ ID No. 5, respectively; and The amino acid sequences of the light chain variable region, CDR-L1, CDR-L2, and CDR-L3, are shown in SEQ ID No. 8 to SEQ ID No. 10, respectively.

2. The anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof according to claim 1, characterized in that: The monoclonal antibody 2D5 or an antigen-binding portion thereof comprises: A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID No. 2; and The amino acid sequence of the light chain variable region is shown in SEQ ID No.

7.

3. The anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof according to claim 1 or 2, characterized in that: The antigen binding portion is selected from any one of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment and a scFv antibody.

4. A gene encoding the anti-Aeromonas hydrophila monoclonal antibody 2DF or an antigen-binding portion thereof according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 1; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.

6.

6. Use of the anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof according to any one of claims 1 to 3 in the preparation of a kit for detecting Aeromonas hydrophila in a biological sample.

7. The use according to claim 6, characterized in that: The kit also includes another anti-Aeromonas hydrophila monoclonal antibody or an antigen-binding portion thereof having an antigenic epitope different from that of the anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof.

8. The use according to claim 7, characterized in that The other anti-Aeromonas hydrophila monoclonal antibody or antigen-binding portion thereof is monoclonal antibody 8F3 or an antigen-binding portion thereof, comprising: A heavy chain variable region, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID No. 13 to SEQ ID No. 15, respectively; and The amino acid sequences of the light chain variable region, CDR-L1, CDR-L2, and CDR-L3, are shown in SEQ ID No. 18 to SEQ ID No. 20, respectively.

9. The use according to claim 8, characterized in that The monoclonal antibody 8F3 or an antigen-binding portion thereof comprises: A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID No. 12; and The light chain variable region, the amino acid sequence of which is shown in SEQ ID No.

17.

10. A kit, characterized in that: The invention comprises the anti-Aeromonas hydrophila monoclonal antibody 2D5 or an antigen-binding portion thereof according to any one of claims 1 to 3.

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