An indirect elisa kit for detecting flavidobacterium columnare antibody and application thereof

Abstract

The application discloses an indirect ELISA kit for detecting Flavobacterium psychrophilum antibody and application thereof, and belongs to the technical field of biology. The indirect ELISA kit comprises an enzyme-labeled plate coated with Flavobacterium psychrophilum OmpH recombinant protein, and the amino acid sequence of the Flavobacterium psychrophilum OmpH recombinant protein is shown as SEQ ID NO. 1. The indirect ELISA kit is established by using the purified recombinant protein OmpH as a coating antigen and HRP-labeled mouse anti-grass carp / Amur gudgeon IgM monoclonal antibody, and can be used not only for epidemiological investigation of the infection condition of the Flavobacterium psychrophilum of grass carp, but also for monitoring the antibody level of the immunized Flavobacterium psychrophilum of grass carp, and is beneficial to the health management of grass carp breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an indirect ELISA kit for detecting antibodies against *Flavobacterium psychrophilum* in grass carp and its application. Background Technology

[0002] Flavobacterium psychrophilum ( Flavobacterium psychrophilum Belonging to the genus *Xanthomonas* of the phylum Bacteroides, this bacterium is a major pathogen of bacterial cold-water diseases in salmonid fish and rainbow trout fry syndrome, causing significant economic losses to the global salmonid aquaculture industry. Although it primarily affects salmonid fish, it can also infect various commercially important fish species such as cyprinids, ayu, and anguilliformes. Infected fish often exhibit symptoms such as open ulcers on their body surface and splenomegaly.

[0003] Currently, common detection methods for *Flavobacterium psychrophilum* include bacterial isolation and culture, and PCR molecular identification. Bacterial isolation and culture have a relatively long cycle; PCR detection mainly targets pathogen nucleic acids, and although sensitive and efficient, it cannot distinguish between current and past infections, making it difficult to assess the immune status of fish populations. Serological antibody detection can serve as an effective supplementary method; however, there are currently no reports of indirect ELISA kits using recombinant *Flavobacterium psychrophilum* protein as the coating antigen for detecting specific antibodies in grass carp serum.

[0004] OmpH protein (also known as Skp or HlpA) is a member of the bacterial outer membrane protein family and is highly conserved in many Gram-negative bacteria. In *Flavobacterium psychrophilum*, OmpH has been shown to be a surface-exposed, immunodominant protein. Convalescent serum from naturally infected rainbow trout specifically recognizes this protein, indicating that OmpH protein effectively stimulates humoral immune responses in the host during natural infection. Furthermore, this protein is highly conserved among different strains of *Flavobacterium psychrophilum*. Given these characteristics, this protein has the potential to serve as a detection antigen; however, there are currently no reports on the use of OmpH in establishing serological detection methods for antibodies against *Flavobacterium psychrophilum* in grass carp. Summary of the Invention

[0005] The purpose of this invention is to provide an indirect ELISA kit for detecting antibodies against *Flavobacterium psychrophilum* in grass carp and its application, thereby addressing the problems existing in the prior art. Based on the immunogenicity and sequence conservation of the OmpH protein, this invention develops an indirect ELISA kit for detecting *Flavobacterium psychrophilum* antibodies in grass carp using recombinant OmpH protein as the coating antigen. This kit not only provides technical support for the epidemiological investigation of overwintering syndrome but also offers a convenient antibody detection method for evaluating the immunization efficacy of subsequent *Flavobacterium psychrophilum* vaccines.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an indirect ELISA kit for detecting antibodies against *Flavobacterium psychrophilum* in grass carp. The indirect ELISA kit includes an enzyme-labeled plate coated with recombinant *Flavobacterium psychrophilum* OmpH protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the coating concentration of OmpH recombinant protein in the ELISA plate is 2.0 μg / mL.

[0008] Preferably, the indirect ELISA kit further includes: a positive control, a negative control, an enzyme-labeled secondary antibody, a sample diluent, a stop solution, and a washing solution.

[0009] Preferably, the positive control is grass carp serum resistant to Flavobacterium psychrophilum; The negative control was grass carp serum that did not contain antibodies against Flavobacterium psychrophilum; The enzyme-labeled secondary antibody is an HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody.

[0010] Preferably, the sample diluent and the washing solution are both PBST buffer; the chromogenic solution is TMB chromogenic solution; and the stop solution is sulfuric acid.

[0011] The present invention also provides the use of the indirect ELISA kit in the following (1) or (2): (1) Application in the preparation of products for detecting the infection status of *Flavobacterium psychrophilum* in grass carp; (2) Application in the preparation of products for detecting antibodies against Flavobacterium psychrophilum in grass carp.

[0012] Preferably, the application method of the indirect ELISA kit includes the step of adding the grass carp serum sample to be tested into an enzyme-labeled plate coated with recombinant protein of Flavobacterium psychrophilum OmpH for detection.

[0013] Preferably, the detection method using the ELISA plate includes the following steps: 1) Dilute the grass carp serum sample to be tested 40 times with sample diluent, add it to an ELISA plate coated with recombinant protein of Flavobacterium psychrophilum OmpH, and incubate at 37°C for 1 hour; 2) Discard the reaction solution and wash with washing solution; 3) Add diluted HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody and incubate at 37°C for 1 hour; wherein the dilution ratio of the HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody is 1:4000. 4) Discard the reaction solution and wash with washing solution; 5) Add TMB colorimetric solution and react at 37°C in the dark for 3–5 minutes; 6) Add the stop solution and react at 37°C for 3–5 min; 7) OD of the final sample solution obtained by detection 450 Value, judgment result.

[0014] Preferably, the criterion for judgment is: simultaneously setting up positive and negative controls, when the average OD of the positive control is... 450 / Negative control mean OD 450 When the value is greater than 3, the experimental results are valid; the average OD of the negative control is used. 450 +3 standard deviations is used as the cut-off value, and the OD of the test sample is... 450 A value greater than the Cut-off value indicates a positive result, meaning the serum sample being tested contains antibodies against Flavobacterium psychrophilum.

[0015] When a serum sample tests positive for antibodies, it indicates that grass carp in their natural state (such as in aquaculture ponds, natural rivers, or reservoirs) are currently or have been infected with Flavobacterium psychrophilum; for grass carp that have been vaccinated, it indicates that a specific immune response against the bacterium has been generated.

[0016] The present invention also provides the use of the recombinant protein of Flavovirens psychrophilus OmpH in the following (1) or (2): (1) Application in the preparation of products for detecting the infection status of *Flavobacterium psychrophilum* in grass carp; (2) Application in the preparation of products for detecting antibodies against Flavobacterium psychrophilum in grass carp; The amino acid sequence of the recombinant protein of Flavovirens psychrophilus OmpH is shown in SEQ ID NO.1.

[0017] The present invention discloses the following technical effects: This invention constructs a psychrophilic flavipes. ompH A prokaryotic expression vector (pET-28b-OmpH) was used to transform the gene into *Escherichia coli* (BL21) for recombinant protein expression. Using the purified recombinant protein (rOmpH) as the coating antigen and HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody, an ELISA diagnostic kit for *Flavobacterium psychrophilum* antibody was developed to detect *Flavobacterium psychrophilum* antibody levels in grass carp serum. This kit can be used not only for epidemiological surveys of *Flavobacterium psychrophilum* infection status in grass carp but also for monitoring antibody levels in immunized grass carp, thus benefiting the health management of grass carp farming. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Construction of the pET-28b-OmpH recombinant expression vector; A: PCR amplification of the linearized pET-28b vector; B: PCR amplification of *Flavobacterium psychrophilum*. ompH Gene fragments; PCR identification of the C: pET-28b-OmpH recombinant expression vector; In each electrophoresis diagram, lane 1 is the DNA marker and lane 2 is the amplification product; Figure 2 Identification of purified OmpH recombinant protein; M is Protein Marker; 1 is purified OmpH recombinant protein. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Expression and purification of recombinant antigen protein (rOmpH) 1. PCR amplification of the linearized pET-28b vector and the outer membrane protein of Flavobacterium psychrophilum. ompH Gene fragments: Design and amplify the pET-28b linearized vector and Flavovirens outer membrane protein. ompH The PCR primers for the gene fragments (sequences are shown in Table 1) were synthesized by Zhejiang Shangya Biotechnology Co., Ltd.

[0026] Table 1. PCR amplification of linearized pET-28b and Flavobacterium psychrophilum ompH Primers for gene fragments Using the pET-28b vector plasmid (Invitrogen) and the psychrophilic flavipe genome as templates, and the corresponding primers listed in Table 1, linearized pET-28b vector and psychrophilic flavipepe were amplified by PCR. ompH Gene fragment. The PCR amplification reaction system was as follows: 1 μL template (10 ng / μL), 25 μL 2×HyPerFUsion High-Fidelity Master Mix (APE×BIO), 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, and sterile ultrapure water to a final volume of 50 μL. The PCR amplification reaction cycling parameters were: 98℃ pre-denaturation for 1 min, followed by 30 cycles (98℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s). ompH (3 min / 3 min (pET-28b vector)), extension at 72℃ for 5 min, storage at 4℃. After PCR, the PCR products were analyzed by electrophoresis on a 1% agarose gel (e.g., 3 min / 3 min (pET-28b vector)). Figure 1 (As shown in Figures A and B). The results show that the amplification was successful. ompH The gene fragment and pET-28b vector were consistent with the expected results.

[0027] 2. Construction of the pET-28b-OmpH recombinant expression vector: The purified linearized vector pET-28b and Flavobacterium psychrophilum were then used. ompHThe PCR product of the gene fragment was used for recombinant cloning using the recombinase Exnase MultiS (Vazyme). After sequence verification, the recombinant clone was named pET-28b-OmpH. The recombinant reaction system was as follows: purified Flavobacterium psychrophilum... ompH 10 ng of the gene fragment PCR product, 100 ng of purified pET-28b linearized vector, 2 μL of Exnase MultiS, 4 μL of 5× CE MultiS Buffer, and sterile ultrapure water were added to a final volume of 20 μL. The reaction mixture was incubated at 37°C for 30 min, then placed on ice for 5 min. Subsequently, 20 μL of the reaction mixture was transformed into *E. coli* competent cells DH5α and plated on LB agar plates supplemented with kanamycin. Positive clones were picked the following day for PCR identification. Figure 1 (C). The identified positive clones were transformed into BL21 cells for induced expression.

[0028] 3. Expression and identification of recombinant proteins: BL21 cells with positive clones were amplified, and the bacterial culture was inoculated at a volume ratio of 1:100 in LB medium supplemented with kanamycin. After shaking culture at 200 rpm and 37℃ for 3-4 h, IPTG was added and expression was induced at 37℃ for 4 h. The bacteria were then collected, resuspended in PBST, and sonicated (40 Hz, 4 s sonication with 8 s intervals). After disruption, the supernatant and precipitate were separated by centrifugation, and the supernatant and precipitate were detected by SDS-PAGE. The results showed that the expression product was mainly found in the supernatant.

[0029] 4. Preparation of antigen for detecting Flavobacterium psychrophilum antibodies: BL21 cells induced to express protein were lysed with an appropriate amount of lysis buffer (PBS pH 7.5, 10% Glyerol, 1 mM PMSF). After thorough mixing, the cells were sonicated for 20 min. The cell lysate was centrifuged at 12000 g for 10 min, and the supernatant (a mixture of supernatant and protein to be purified) was separated. A certain amount of pre-treated Ni resin was added to the supernatant mixture, and the mixture was incubated in a shaker at 4°C for 30 min. The resin was then collected and purified using PBS buffer (pH 7.5) containing 10 mM and 30 mM imidazole, respectively, to remove contaminating proteins. Finally, the target recombinant protein was eluted with PBS buffer (pH 7.5) containing 300 mM imidazole. The purified protein was analyzed by SDS-PAGE, indicating good purification results. Figure 2 ).

[0030] Flavovirens outer membrane protein gene ompHThe recombinant protein is a polypeptide having the amino acid sequence shown in SEQ ID NO.1 (where 1-13 are His tags and 14-19 are Thrombin sites): MGSSHHHHHHSSGLVPRGSHMAQTKVAHIDVSELLAKMPEMTAAKAQLDKLSKTFDTEYGTMVTEYQTKMKKYEAEATTATEAINETRAKEMQDMGQRIQQYRDSAQKQLQEKEQEIVKPIMDKAKTAIVKVGKSKGYQYVMDSASLILADGPNLFEDVKKELKF.

[0031] Example 2: Optimization of ELISA detection conditions 1. Determination of optimal antigen coating concentration and optimal serum dilution: The optimal concentrations of antigen and antibody were optimized using a checkerboard titration method. Positive and negative sera were diluted 1:10, 1:20, 1:40, 1:80, and 1:160 using antibody dilution buffer (PBST containing 0.05% Tween-20). The antigen protein was diluted to 8.0, 4.0, 2.0, and 1.0 μg / mL using coating buffer. Each concentration combination was performed in triplicate. After the ELISA reaction, OD was measured. 450 Absorbance value, calculate P / N value (OD of positive serum) 450 Mean / Negative Serum OD 450 (Average). The specific steps are as follows: (1) Solid-phase coating of recombinant protein of Flavobacterium psychrophilum (rOmpH): Different concentrations of antigen protein (8.0, 4.0, 2.0, 1.0 μg / mL) were coated onto the enzyme-labeled plate, 100 μL / well, and incubated overnight at 4℃. The next day, the reaction residue was discarded without washing. (2) Sealing: Add 250 μL of sealing solution per well and seal at 37°C for 1 h. Remove residual liquid, pat dry, and store at 4°C for later use; (3) Sample loading: Place the ELISA plate at room temperature for 30 min, and add positive and negative sera at different dilutions (1:10, 1:20, 1:40, 1:80, 1:160) at 50 μL / well, and incubate at 37℃ for 1 h. Add washing buffer (PBST containing 0.05% Tween-20) at 250 μL / well, wash 3 times, and pat dry. (4) Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody (Bogd Biotechnology Co., Ltd.) diluted 1:1000 to each well, and incubate at 37°C for 3 h. Add washing buffer at 250 μL per well, wash 5 times, and pat dry. (6) Add TMB colorimetric solution, 50 μL / well, and react at 37℃ for 3-5 min; (7) Add stop solution (2M sulfuric acid), 50 μL / well, and take readings within 5 min after termination. Measure OD using a microplate reader. 450 .

[0032] (8) Calculate the P / N ratio (OD of positive serum) 450 Mean / Negative Serum OD 450 The mean value was used to determine the optimal dilution ratio between the coated antigen and the serum sample. During the experiment, a blank control was set up without any sample added.

[0033] The results are shown in Tables 2 and 3. Based on the comparative analysis of P / N values: among the five serum dilutions, the 1:40 dilution showed the most outstanding P / N performance and good reproducibility at all antigen concentrations, and was therefore determined to be the optimal serum dilution. For the antigen coating concentration, the P / N values ​​for 2.0 μg / mL and 8.0 μg / mL at the optimal serum dilutions were 18.65 and 20.63, respectively, both far exceeding the generally accepted benchmark for excellent performance in methodological evaluation (generally, P / N > 10.0 is considered excellent), and there was no statistically significant difference in diagnostic efficacy. However, considering the high purification cost of recombinant antigens and the potential for increased non-specific adsorption due to high coating concentrations, the optimal working conditions were finally determined to be: antigen coating concentration of 2.0 μg / mL and a serum dilution of 1:40.

[0034] The positive serum was grass carp serum resistant to *Flavobacterium psychrophilum*. Its preparation method was as follows: 0.3% formalin-inactivated *Flavobacterium psychrophilum* (3.2 × 10⁻⁶) 8 A whole-cell vaccine was prepared by mixing CFU / mL with Freund's complete adjuvant at a volume ratio of 1:1. Healthy grass carp were immunized intraperitoneally with an immunization dose of 0.2 mL / fish, twice, with an interval of 14 days between immunizations. On day 28 after the second immunization, blood was collected from the tail vein, and serum was collected as positive serum for Flavobacterium psychrophilum antibodies and stored at -20℃ for later use.

[0035] The negative serum was grass carp serum that did not contain antibodies against Flavobacterium psychrophilum. It was collected from healthy grass carp free of specific pathogens.

[0036] Table 2. ELISA results of serum samples with different antigen concentrations and dilution factors. Table 3. ELISA readings for different concentrations of antigen and serum sample dilutions. 2. Determination of enzyme-labeled secondary antibody dilution: Coat an ELISA plate with 2 μg / mL OmpH protein, add 40-fold diluted positive and negative sera, and dilute the HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody to 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000 using antibody dilution buffer. Perform triplicate for each concentration. After the ELISA reaction, calculate the p / n ratio and analyze the results to determine the optimal dilution of the secondary antibody. The specific procedures are as follows: (1) Solid-phase coating of recombinant protein (rOmpH) of Flavovirens psychrophilus: 2.0 μg / mL of antigen protein rOmpH was added to the microplate at 100 μL / well and incubated overnight at 4℃. The next day, the reaction residue was discarded without washing. (2) Sealing: Add 250 μL of sealing solution per well, seal at 37℃ for 1 h, remove residual liquid and pat dry; (3) Sample loading: Add 50 μL of 1:40 diluted positive and negative serum samples to each well and incubate at 37°C for 1 h. Add 250 μL of washing buffer to each well, wash 3 times, and pat dry. (4) Add enzyme-labeled secondary antibody: Add HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody (Bogd Biotechnology Co., Ltd.) at different dilutions (1:1000, 1:2000, 1:4000, 1:8000, 1:16000), 100 μL / well, and incubate at 37℃ for 1 h. Add washing buffer at 250 μL / well, wash 5 times, and pat dry; (6) Add TMB colorimetric solution, 50 μL / well, and react at 37℃ for 3-5 min; (7) Add stop solution, 50 μL / well, and take readings within 5 min after termination. Measure OD using a microplate reader. 450 .

[0037] (8) Calculate the P / N ratio (OD of positive serum) 450 Mean / Negative Serum OD 450 The mean value was used to determine the optimal dilution ratio between the coated antigen and the serum sample. During the experiment, a blank control was set up without any sample added.

[0038] As shown in Table 4, the results indicate that at a dilution of 1:4000, positive OD 450 The value was 1.615, indicating a negative OD. 450 The P / N ratio was 19.23, and all three values ​​were within the ideal range, with the signal falling within the optimal linear detection range (0.2~2.5) of the microplate reader. At a 1:1000 dilution, the positive OD... 450A concentration of 3.536 indicates a risk of signal saturation; dilutions of 1:8000 and above result in low positive signals (<1.0), potentially affecting the detection of low-titer samples. Considering both detection performance and antibody cost, 1:4000 was ultimately chosen as the optimal working concentration for the HRP-labeled secondary antibody.

[0039] Table 4. ELISA readings at different secondary antibody dilutions Example 3: Indirect ELISA kit for detecting antibodies against Flavobacterium psychrophilum and its usage method The kit consists of an ELISA plate coated with Flavobacterium psychrophilum OmpH protein, a positive control (grass carp serum against Flavobacterium psychrophilum), a negative control (grass carp serum without Flavobacterium psychrophilum antibody), an HRP-labeled mouse anti-grass carp / black carp monoclonal antibody (BorgD Biotech), sample diluent (PBST containing 0.05% Tween-20), chromogenic reagent TMB, stop solution (2M sulfuric acid), and wash buffer (PBST containing 0.05% Tween-20).

[0040] The steps and positive criteria for detecting antibodies against Flavobacterium psychrophilum using this kit are as follows: 1) Sample addition and incubation: Add 50 μL of the serum sample to be tested diluted 1:40 to the microplate and incubate at 37°C for 1 h. 2) Washing: Discard the reaction solution and wash the plate three times with PBST containing 0.05% Tween-20; 3) Antibody incubation: Add HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody diluted 1:4000 and incubate at 37℃ for 1 h; 4) Washing: Discard the reaction solution and wash 5 times with PBST containing 0.05% Tween-20; 5) Color development: Add 100 μL of TMB color development solution to each well and react at 37℃ for 3–5 min. 6) Termination: Add stop solution, 50 μL / well, and react at 37℃ for 3–5 min; 7) Detection and judgment: Measure the OD of the final sample solution obtained in step 6) using an ELISA reader. 450 , the judgment result.

[0041] The method further includes a positive control test and a negative control test; when conducting a positive control test, the test sample in step 1) is replaced with a positive control; when conducting a negative control test, the test sample in step 1) is replaced with a negative control.

[0042] Criteria for interpreting test results: Average OD of positive control 450 / Negative control mean OD 450 When the value is >3, the experimental results are valid; the average OD value of the negative control is >3. 450 + 3 standard deviations (SD) as cut-off, sample OD 450 >Cut-off means the result is positive.

[0043] Example 5: Clinical Sample Testing Results Serum samples from grass carp vaccinated with inactivated *Flavobacterium psychrophilum* (collected 21-90 days post-immunization, 6 samples), grass carp infected with *Flavobacterium psychrophilum* (collected 28-60 days post-infection, 6 samples), and healthy grass carp (6 samples) were tested. As shown in Table 5, serum samples from grass carp vaccinated with inactivated *Flavobacterium psychrophilum* and grass carp infected with *Flavobacterium psychrophilum* were all ELISA positive. Serum samples from healthy grass carp were all ELISA negative. These results indicate that the ELISA kit for detecting *Flavobacterium psychrophilum* antibodies based on OmpH protein of this invention can not only be used for epidemiological surveys of *Flavobacterium psychrophilum* infection, but also for the detection of specific antibodies against immunized grass carp, showing promising application prospects.

[0044] Table 5 Results of antibody detection for Flavobacterium psychrophilum in serum samples The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An indirect ELISA kit for detecting antibodies against *Flavobacterium psychrophilum* in grass carp, characterized in that... The indirect ELISA kit includes an enzyme-labeled plate coated with recombinant Flavobacterium psychrophilum OmpH protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The indirect ELISA kit as described in claim 1, characterized in that, The coating concentration of OmpH recombinant protein in the ELISA plate was 2.0 μg / mL.

3. The indirect ELISA kit as described in claim 1, characterized in that, The indirect ELISA kit also includes: a positive control, a negative control, an enzyme-labeled secondary antibody, a sample diluent, a stop solution, and a washing solution.

4. The indirect ELISA kit as described in claim 3, characterized in that, The positive control was grass carp serum resistant to Flavobacterium psychrophilum; The negative control was grass carp serum that did not contain antibodies against Flavobacterium psychrophilum; The enzyme-labeled secondary antibody is an HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody.

5. The indirect ELISA kit as described in claim 3, characterized in that, The sample diluent and the washing solution are both PBST buffer; the chromogenic solution is TMB chromogenic solution; and the stop solution is sulfuric acid.

6. The use of the indirect ELISA kit according to any one of claims 1-5 in the following (1) or (2): (1) Application in the preparation of products for detecting the infection status of *Flavobacterium psychrophilum* in grass carp; (2) Application in the preparation of products for detecting antibodies against Flavobacterium psychrophilum in grass carp.

7. The application as described in claim 6, characterized in that, The application method of the indirect ELISA kit includes the step of adding the grass carp serum sample to be tested into an enzyme-labeled plate coated with recombinant protein of Flavobacterium psychrophilum OmpH for detection.

8. The application as described in claim 7, characterized in that, The method for detection using the aforementioned ELISA plate includes the following steps: 1) Dilute the grass carp serum sample to be tested 40 times with sample diluent, add it to the ELISA plate coated with recombinant protein of Flavobacterium psychrophilum OmpH, and incubate at 37°C for 1-3 hours; 2) Discard the reaction solution and wash with washing solution; 3) Add diluted HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody and incubate at 37°C for 1–3 h; wherein the dilution ratio of the HRP-labeled mouse anti-grass carp / black carp IgM monoclonal antibody is 1:4000. 4) Discard the reaction solution and wash with washing solution; 5) Add TMB colorimetric solution and react at 37°C in the dark for 3–5 minutes; 6) Add the stop solution and react at 37°C for 3–5 min; 7) OD of the final sample solution obtained by detection 450 Value, the result of the judgment.

9. The application as described in claim 8, characterized in that, The criterion for judgment is as follows: Positive and negative controls are set up simultaneously; when the average OD of the positive control is... 450 / Negative control mean OD 450 When the value is greater than 3, the experimental results are valid; the average OD of the negative control is used. 450 +3 standard deviations is used as the cut-off value, and the OD of the test sample is... 450 A value greater than the Cut-off value indicates a positive result, meaning the serum sample being tested contains antibodies against Flavobacterium psychrophilum.

10. Application of the recombinant protein OmpH from Flavovirens psychrophilus in the following (1) or (2): (1) Application in the preparation of products for detecting the infection status of *Flavobacterium psychrophilum* in grass carp; (2) Application in the preparation of products for detecting antibodies against Flavobacterium psychrophilum in grass carp; in, The amino acid sequence of the recombinant protein of Flavobacterium psychrophilum OmpH is shown in SEQ ID NO.1.

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