ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting bacteroides nodosus Fimb A protein antibody and application of ELISA kit

Abstract

The invention relates to the technical field of bacterial detection, in particular to an ELISA (enzyme-linked immuno sorbent assay) kit for detecting a bacteroides nodosus FimbA protein antibody and application of the ELISA kit. The ELISA kit provided by the invention comprises the following components which are independently packaged: a solid-phase carrier coated with bacteroides nodulosus FimbA protein, an enzyme-labeled secondary antibody, a diluent, a washing solution, a developing solution and a stop solution. The ELISA detection kit provided by the invention adopts the bacteroides nodules Fimb A protein as a detection antigen, can detect whether a to-be-detected sample contains bacteroides nodules or not, can be used for realizing early screening of bacteroides nodules infection and foot and limb diseases, and has the characteristics of simplicity in operation, good specificity, high sensitivity, strong repeatability and accurate result. Experimental results show that the P / N value of bovine clinical positive serum of bacteroides nodosus is greater than or equal to 0.212, and the kit has good accuracy.

Description

Technical Field

[0001] The invention relates to the technical field of bacteria detection, in particular to an ELISA antibody kit for detecting Fimb A protein of Bacteroides nodosa. Background Art

[0002] Ruminant hoof disease is a general term for ruminant limb and hoof diseases. It is caused by mixed infection of multiple bacteria and causes limb inflammation, including deformed hoof, hoof rot, hoof dermatitis, interdigital dermatitis, etc. The main clinical manifestations are lameness, difficulty in getting up and lying down, weight loss, decreased fertility, etc. Lameness of dairy cows caused by hoof disease is one of the important reasons for early elimination of high-yielding dairy cows. Most of the dairy cows eliminated due to lameness caused by hoof disease are high-yielding dairy cows, causing huge economic losses to the dairy farming industry.

[0003] Nodular Bacteroides (D.nodosus) is a strictly anaerobic Gram-negative bacillus belonging to the Cardiobacteriaceae family. It is non-motile, has no spores and capsules, and can survive for up to 40 days in 5°C sticky soil. It has multiple serotypes, among which type A Nodular Bacteroides was first discovered in the lesions of sheep hoof disease in 1938. It is one of the main bacterial types that cause hoof disease in ruminants such as deer, cattle, and sheep. Nodular Bacteroides can secrete a large amount of highly active proteases to soften and denature the tissue proteins of the animal's hooves, resulting in a decrease in the hardness of the tissue on the surface and base of the animal's hoof, thereby causing interdigital dermatitis. At the same time, studies have also found that Nodular Bacteroides is the primary pathogen causing cow hoof rot, and plays an important role in the occurrence of cow hoof dermatitis and interdigital dermatitis. Therefore, it is necessary to continuously monitor the infection of Nodular Bacteroides in dairy cows and establish relevant diagnostic detection methods.

[0004] Currently in veterinary clinics, screening for Bacteroides nodosa infection and limb and hoof disease in dairy cows mainly relies on PCR diagnosis and bacterial isolation and identification. The detection results of early Bacteroides nodosa infection in cattle are not accurate and have poor repeatability. Currently, there is no effective antigen for indirect ELISA detection of Bacteroides nodosa in cattle. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an ELISA kit for detecting antibodies to Fimb A protein of Bacteroides nodules and its application. The ELISA kit provided by the present invention is an indirect ELISA kit, which can determine whether the sample to be tested contains antibodies to Fimb A protein of Bacteroides nodules or whether the sample to be tested is infected with Bacteroides nodules by detecting whether the sample to be tested contains antibodies to Fimb A protein of Bacteroides nodules. It has the characteristics of good specificity and high accuracy, and can be used to achieve early screening of Bacteroides nodules infection and limb and hoof diseases.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an ELISA kit for detecting antibodies to Bacteroides nodosa Fimb A protein, comprising the following independently packaged components: a solid phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing solution, a color developing solution and a stop solution, wherein the solid phase carrier is a solid phase carrier coated with Bacteroides nodosa Fimb A protein, and the amino acid sequence of the Bacteroides nodosa Fimb A protein is shown in SEQ ID NO.1.

[0008] Preferably, the preparation method of the Bacteroides nodosa Fimb A protein comprises: transferring a recombinant vector inserted with a Fimb A protein encoding gene into Escherichia coli, inducing expression with isopropylthiogalactoside to obtain a supernatant containing the Fimb A protein; purifying the supernatant containing the Fimb A protein to obtain the Bacteroides nodosa Fimb A protein.

[0009] Preferably, the primer pair for amplifying the Fimb A protein encoding gene includes primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4; and the initial vector of the recombinant vector includes a pET-32a vector.

[0010] Preferably, the preparation method of the solid phase carrier coated with Bacteroides nodosa Fimb A protein comprises: mixing 0.01M phosphate buffer and the Bacteroides nodosa Fimb A protein to obtain a 0.25μg / mL Bacteroides nodosa Fimb A protein solution; adding the Bacteroides nodosa Fimb A protein solution to the solid phase carrier, coating at 4°C for 12h, discarding the Bacteroides nodosa Fimb A protein solution, adding a skim milk solution with a mass concentration of 5% to the solid phase carrier, and sealing at 37°C for 2h to obtain the solid phase carrier coated with Bacteroides nodosa Fimb A protein.

[0011] Preferably, the enzyme-labeled secondary antibody includes HRP-labeled IgG enzyme-labeled secondary antibody; the sample diluent includes PBST buffer; the washing solution includes PBST buffer; the color developing solution includes TMB substrate color developing solution; and the stop solution includes 2 mol / L sulfuric acid solution.

[0012] Preferably, the ELISA kit further comprises independently packaged standard positive control serum and independently packaged standard negative control serum; the standard positive control serum is serum from animals infected with Bacteroides nodules; the standard negative control serum is serum from animals not infected with Bacteroides nodules.

[0013] The present invention provides the use of the ELISA kit described in the above technical solution in detecting antibodies against Fimb A protein of Bacteroides nodosa and / or Bacteroides nodosa, and the direct purpose of the use is non-diagnosis and non-treatment.

[0014] Preferably, the application includes:

[0015] Mixing the sample to be tested and the sample diluent to obtain a diluted sample to be tested; the volume ratio of the sample to be tested and the sample diluent is 1:819200;

[0016] The diluted sample to be tested and the sample diluent are added to different positions of the solid phase carrier respectively, and incubated at 37° C. for 1 hour to obtain a first solid phase carrier;

[0017] Mixing the enzyme-labeled secondary antibody and the sample diluent to obtain a diluted enzyme-labeled secondary antibody; the volume ratio of the enzyme-labeled secondary antibody to the sample diluent is 1:30000;

[0018] Adding the diluted enzyme-labeled secondary antibody to the first solid phase carrier, incubating at 37° C. for 1 h to obtain a second solid phase carrier;

[0019] Add a color developing solution to the second solid phase carrier, and color develop at 37° C. in the dark for 3 to 10 minutes to obtain a third solid phase carrier;

[0020] Add the stop solution to the third solid phase carrier, and read the OD at the position where the diluted sample to be tested and the sample dilution solution are added. 450nm The value of the diluted sample to be tested is 450nm The value is recorded as P, and the OD at the position where the sample diluent is added 450nm The value is recorded as N, and the test results of the sample to be tested are determined according to the P / N value, including:

[0021] If the P / N value is ≥ 0.212, the sample to be tested is determined to be positive, that is, the sample to be tested contains Bacteroides nodosa Fimb A protein antibodies and / or Bacteroides nodosa;

[0022] If the P / N value is ≤0.187, the sample to be tested is judged to be negative, that is, the sample to be tested does not contain Bacteroides nodosa FimbA protein antibodies and Bacteroides nodosa;

[0023] If 0.187<P / N value<0.212, the sample to be tested is a suspicious sample and retest is performed.

[0024] Preferably, after each incubation or color development, the solid phase carrier is washed with the washing solution; the number of washings is 3 times; and the time for each washing is 3 minutes.

[0025] Preferably, the sample to be tested includes serum.

[0026] Beneficial effects:

[0027] The present invention provides an ELISA kit for detecting antibodies to Fimb A protein of Bacteroides nodules, comprising the following independently packaged components: a solid phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing solution, a color developing solution and a stop solution, wherein the solid phase carrier is a solid phase carrier coated with Fimb A protein of Bacteroides nodules, and the amino acid sequence of the Fimb A protein of Bacteroides nodules is shown in SEQ ID NO.1. The ELISA detection kit provided by the present invention adopts Fimb A protein of Bacteroides nodules as a detection antigen, and can detect whether the sample to be tested contains antibodies to Fimb A protein of Bacteroides nodules, thereby determining whether the sample to be tested contains Bacteroides nodules, and can be used to achieve early screening of Bacteroides nodules infection and limb and hoof disease, and has the characteristics of simple operation, good specificity, high sensitivity, strong repeatability and accurate results. The experimental results show that the P / N value of clinical positive serum of Bacteroides nodules cattle is ≥0.212, indicating that the kit has good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 This is the SDS-PAGE diagram of the prokaryotic expression of the recombinant protein Fimb A of Bacteroides nodosa; wherein, M represents the pre-stained rainbow protein Marker, 1 represents the whole bacteria of the uninduced pET-32a empty vector ultrasonically broken, 2 represents the supernatant of the pET-32a empty vector ultrasonically broken after induction, 3 represents the precipitate of the pET-32a empty vector ultrasonically broken after induction, 4 represents the whole bacteria of the uninduced pET-32a-Fimb A recombinant protein ultrasonically broken, 5 represents the whole bacteria of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction, 6 represents the supernatant of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction, and 7 represents the precipitate of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction;

[0030] Figure 2 This is the SDS-PAGE image of the purified recombinant protein of Bacillus nodosa Fimb A; where M represents the pre-stained rainbow protein marker, and 1 represents the purified recombinant protein of pET-32a-Fimb A;

[0031] Figure 3This is a Western blot identification diagram of the prokaryotic expression of the recombinant protein Fimb A of Bacteroides nodosa; wherein, M represents the pre-stained rainbow protein Marker, 1 represents the whole bacteria of the uninduced pET-32a empty vector ultrasonically broken, 2 represents the supernatant of the pET-32a empty vector ultrasonically broken after induction, 3 represents the precipitate of the pET-32a empty vector ultrasonically broken after induction, 4 represents the whole bacteria of the uninduced pET-32a-Fimb A recombinant protein ultrasonically broken, 5 represents the whole bacteria of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction, 6 represents the supernatant of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction, and 7 represents the precipitate of the pET-32a-Fimb A recombinant protein ultrasonically broken after induction;

[0032] Figure 4 This is a Western blot identification image of the purified Bacteroides nodosa Fimb A recombinant protein; where M represents the pre-stained rainbow protein Marker, and 1 represents the purified pET-32a-Fimb A recombinant protein. DETAILED DESCRIPTION

[0033] The present invention provides an ELISA kit for detecting antibodies to Bacteroides nodosa Fimb A protein, comprising the following independently packaged components: a solid phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing solution, a color developing solution, and a stop solution, wherein the solid phase carrier is a solid phase carrier coated with Bacteroides nodosa Fimb A protein, and the amino acid sequence of the Bacteroides nodosa Fimb A protein is shown in SEQ ID NO.1, and is specifically as follows:

[0034] IPAYNDYIARSQAAEGLTLADGLKVRISDHLESGECKGDANPASGSLGNDDKGKYALATIDGDYNKDAKTADEKNGCKVVITYGQGTAGEKISKLIVGKKLVLDQFVNGSYKYNEGETDLELKFIPNAVKN.

[0035] As an embodiment, the preparation method of the nodular Bacteroides Fimb A protein comprises: transferring a recombinant vector inserted with a Fimb A protein encoding gene into Escherichia coli, inducing expression with isopropylthiogalactoside to obtain a supernatant containing the Fimb A protein; purifying the supernatant containing the Fimb A protein to obtain the nodular Bacteroides Fimb A protein; the nucleotide sequence of the Fimb A protein encoding gene is shown in SEQ ID NO.2, and is specifically as follows:

[0036] 5'-atccctgcatacaacgactacatcgctcgttcacaagcagctgaaggcttaacattggctgatggtttgaaggttcgcatttctgatcacttagaaa gcggtgaatgtaagggagatgcgaacccagcttcaggatctttaggtaatgatgataaaggtaaatacgctcttgctacaattgatggtgattataataaa gacgcgaaaactgctgatgagaagaatggttgtaaagttgtaatcacttatggtcaaggtactgcaggcgagaaaatttctaagttaatcgttggtaagaa attggttttagatcaatttgttaatggttcatacaaatataatgaaggcgaaactgatttggaacttaaatttattccgaatgctgttaaaaactaa-3'.

[0037] As an embodiment, the primer pair for amplifying the Fimb A protein encoding gene includes primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4; the initial vector of the recombinant vector includes a pET-32a vector.

[0038] The present invention adopts Bacteroides nodosa Fimb A protein as a detection antigen, and can detect whether a sample to be tested contains Bacteroides nodosa Fimb A protein antibodies, thereby determining whether the sample to be tested contains Bacteroides nodosa Fimb A protein antibodies.

[0039] As an embodiment, the preparation method of the solid phase carrier coated with the nodular Bacteroides Fimb A protein comprises: mixing 0.01M phosphate buffer and the nodular Bacteroides Fimb A protein to obtain a 0.25μg / mL nodular Bacteroides Fimb A protein solution; adding the nodular Bacteroides Fimb A protein solution to the solid phase carrier, coating at 4°C for 12h, discarding the nodular Bacteroides Fimb A protein solution, adding a skim milk solution with a mass concentration of 5% to the solid phase carrier, and sealing at 37°C for 2h to obtain the solid phase carrier coated with the nodular Bacteroides Fimb A protein. As another embodiment, the pH value of the phosphate buffer is 7.4. The solid phase carrier coated with the nodular Bacteroides Fimb A protein prepared by the present invention under suitable conditions can effectively detect whether the sample to be tested contains nodular Bacteroides Fimb A protein antibodies, thereby determining whether the sample to be tested contains nodular Bacteroides, with the advantages of good specificity, high sensitivity and accurate results.

[0040] As an embodiment, the solid phase carrier can be a 96-well enzyme labeling strip; the specification of the 96-well enzyme labeling strip is 8 holes×12 strips, and it can be disassembled by itself; as another embodiment, the 96-well enzyme labeling strip is purchased from Costar.

[0041] As one embodiment, the enzyme-labeled secondary antibody includes HRP-labeled IgG enzyme-labeled secondary antibody; as another embodiment, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG.

[0042] As an embodiment, the sample diluent can be a PBST buffer solution; as another embodiment, the preparation method of the PBST solution is to mix a PBS solution and Tween-20 to obtain a PBST solution; the volume ratio of Tween-20 to PBS is 1:2000.

[0043] As an embodiment, the washing solution may be PBST washing solution.

[0044] As an embodiment, the color developing solution can be a TMB substrate color developing solution.

[0045] As an embodiment, the stop solution may be a 2 mol / L sulfuric acid solution.

[0046] The ELISA kit of the present invention adopts an indirect method, wherein the Fimb A recombinant protein of Bacteroides nodosa is coated in an ELISA plate, the ELISA plate is blocked with 5% skim milk, and a serum sample to be tested is added to a solid phase carrier coated with the Fimb A protein of Bacteroides nodosa. If the serum contains a specific antibody of Bacteroides nodosa, the antibody will bind to the Fimb A protein antigen on the solid phase carrier, and then an enzyme-labeled secondary antibody capable of bonding with the specific antibody is added, and then a color developing solution is added for color development, and a stop solution is used to stop the color development, and the OD value is measured by an ELISA instrument. 450nm The absorbance value of each well is measured at the wavelength. The size of the OD value (the depth of the color after the color development reaction is terminated) is positively correlated with the content of Bacteroides nodosa antibodies in the sample to be tested. The protective effect of the antibody is judged according to the antibody content level in the sample to be tested. In addition, the coating antigen (Bacteroides nodosa Fimb A recombinant protein) used in the kit is a prokaryotically expressed recombinant protein with mass production and stability, providing a set of practical and easy-to-operate diagnostic kit products for large-scale farms.

[0047] As an embodiment, the ELISA kit also includes independently packaged standard positive control serum and independently packaged standard negative control serum; the standard positive control serum is animal serum infected with Bacteroides nodules; the standard negative control serum is animal serum not infected with Bacteroides nodules.

[0048] The standard positive control serum and the standard negative control serum provided by the present invention can determine the positive critical value and the negative critical value. The present invention determines the results as follows: OD of the sample to be tested 450nm ≥ positive critical value, judged as positive, the OD of the sample to be tested 450nm ≤ negative critical value, judged as negative, the OD of the sample to be tested 450nm If the sample is between the positive critical value and the negative critical value, it is a suspicious sample and needs to be retested.

[0049] The present invention provides the use of the ELISA kit described in the above technical solution in detecting Bacteroides nodosa Fimb A protein antibodies and / or Bacteroides nodosa, and the direct purpose of the application is non-diagnosis and non-treatment. When the direct purpose of the application of the present invention is non-diagnosis and non-treatment, it is only to obtain an intermediate value of whether the sample to be tested contains Bacteroides nodosa Fimb A protein antibodies and / or Bacteroides nodosa.

[0050] The present invention uses an ELISA kit to detect cattle positive serum for Bacteroides nodosa, foot-and-mouth disease, Brucella, and Staphylococcus aureus. The results show that the P / N values ​​of cattle clinical positive serum for foot-and-mouth disease, Brucella, and Staphylococcus are less than 0.187, and the P / N value of cattle clinical positive serum for Bacteroides nodosa is greater than 0.212. This shows that the indirect ELISA detection method for Bacteroides nodosa Fimb A protein has good specificity.

[0051] As an implementation method, the application includes:

[0052] Mixing the sample to be tested and the sample diluent to obtain a diluted sample to be tested; the volume ratio of the sample to be tested and the sample diluent is 1:819200;

[0053] The diluted sample to be tested and the sample diluent are added to different positions of the solid phase carrier respectively, and incubated at 37° C. for 1 hour to obtain a first solid phase carrier;

[0054] Mixing the enzyme-labeled secondary antibody and the sample diluent to obtain a diluted enzyme-labeled secondary antibody; the volume ratio of the enzyme-labeled secondary antibody to the sample diluent is 1:30000;

[0055] Adding the diluted enzyme-labeled secondary antibody to the first solid phase carrier, incubating at 37° C. for 1 h to obtain a second solid phase carrier;

[0056] Add a color developing solution to the second solid phase carrier, and color develop at 37° C. in the dark for 5 to 10 minutes to obtain a third solid phase carrier;

[0057] Add the stop solution to the third solid phase carrier, and read the OD at the position where the diluted sample to be tested and the sample dilution solution are added. 450nmThe value of the diluted sample to be tested is 450nm The value is recorded as P, and the OD at the position where the sample diluent is added 450nm The value is recorded as N, and the test results of the sample to be tested are determined according to the P / N value, including:

[0058] If the P / N value is ≥ 0.212, the sample to be tested is determined to be positive, that is, the sample to be tested contains Bacteroides nodosa Fimb A protein antibodies and / or Bacteroides nodosa;

[0059] If the P / N value is ≤0.187, the sample to be tested is judged to be negative, that is, the sample to be tested does not contain Bacteroides nodosa FimbA protein antibodies and Bacteroides nodosa;

[0060] If 0.187<P / N value<0.212, the sample to be tested is a suspicious sample and retest is performed.

[0061] As an embodiment, after each incubation or color development, the solid phase carrier is washed with the washing solution; the number of washings is 3 times; and the time for each washing is 3 minutes.

[0062] As one embodiment, the color development time is 4 to 9 minutes; as another embodiment, the color development time is 4 to 8 minutes.

[0063] As an embodiment, if the color-developing solution is not used immediately, the color-developing solution is stored at 4° C. When used, the color-developing solution stored at 4° C. is warmed up to room temperature before use.

[0064] As an embodiment, the sample to be tested may be serum.

[0065] To further illustrate the present invention, an ELISA kit for detecting Fimb A protein of Bacteroides nodosa provided by the present invention and its application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] 1. Antigen Preparation

[0068] The sequencing and identification were performed by Harbin Boshi Biotechnology Co., Ltd. The specific steps are as follows:

[0069] The Bacteroides nodosa Fimb A recombinant protein (purified Bacteroides nodosa Fimb A recombinant protein) was obtained by the following method:

[0070] 1) The whole genome DNA of Bacteroides nodosa A198 strain (purchased from DSMZ, Germany, DSM20708) was extracted using the bacterial genome extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0071] 2) Using the whole genome DNA extracted in step 1) as a template, PCR amplified the Fimb A fragment to obtain an amplified product, and purified the amplified product using the Tiangen Genome Purification Kit to obtain a purified product of the target gene; the amplification primer sequences are as follows:

[0072] Fimb AF: 5'-CGCggatccATGATCCCTGCATACAACGAC-3', SEQ ID No. 3;

[0073] Fimb AR: 5'-CCGctcgagTTAGTTTTTAACAGCATTCGG-3', SEQ ID No. 4;

[0074] The PCR amplification system was: Fimb AF 0.5 μL, Fimb AR 0.5 μL, 2× Taq 6.75 μL, ddH2O 14.75 μL, and whole genomic DNA 2.5 μL;

[0075] The reaction program of PCR amplification was 95°C for 5 min; 94°C for 1 min, 58°C for 50 s, 72°C for 1 min, 30 cycles; 72°C for 10 min.

[0076] 3) The pET-32a vector and the purified product of the target gene were double-digested with BamHI and XhoI to obtain a linearized pET-32a vector (5860 bp) and a linearized purified product of the target gene (399 bp);

[0077] 4) Using T4 DNA ligase, the linearized pET-32a vector obtained in step 3) and the purified product of the linearized target gene were connected to obtain a pET-32a-Fimb A recombinant vector;

[0078] 5) The pET-32a-Fimb A recombinant vector was transformed into E. coli BL21 (DE3) competent cells, and after shaking culture at 220 r / min and 37°C, a bacterial solution containing the recombinant vector was obtained; at the same time, a bacterial solution transformed with the pET-32a empty vector was set as a blank control group;

[0079] 6) OD using a spectrophotometer 600nmWhen the concentrations of the two bacterial solutions in step 5) are measured to be 0.6-0.8, IPTG with a final concentration of 1 mmol / L is added respectively, and the mixture is induced at 220 r / min at 37°C for 6 h. The two bacterial solutions are centrifuged and the precipitates are ultrasonically broken to separate the supernatant and the precipitate, as follows: 100 mL of the induced bacterial solution is added to a centrifuge tube, centrifuged at 8000 r / min for 10 min at room temperature, and the supernatant is discarded; 20 mL of pre-cooled PBS is added to the precipitate after centrifugation and mixed, and ultrasonically broken under ice-water mixture conditions (0°C) until it is clarified, and the ultrasonic program is set to 42 Hz, ultrasonic for 5 s, and intermittent for 10 s. The product obtained after ultrasonication is the whole bacterial body; the ultrasonic product is centrifuged at 8000 r / min for 20 min at 4°C to separate the supernatant and the precipitate, and the precipitate is resuspended with 200 μL PBS;

[0080] At the same time, two bacterial liquids were set up without IPTG induction as the control group, and 7 substances were collected, namely: 1. ultrasonic crushing of whole bacteria by uninduced pET-32a empty vector, 2. ultrasonic crushing supernatant of induced pET-32a empty vector, 3. ultrasonic crushing precipitate of induced pET-32a empty vector, 4. ultrasonic crushing of whole bacteria by uninduced pET-32a-Fimb A recombinant protein, 5. ultrasonic crushing of whole bacteria by induced pET-32a-Fimb A recombinant protein, 6. ultrasonic crushing supernatant of induced pET-32a-Fimb A recombinant protein, 7. ultrasonic crushing precipitate of induced pET-32a-Fimb A recombinant protein;

[0081] 7) The 7 substances obtained in step 6) were subjected to SDS-PAGE analysis, and the results were as follows: Figure 1 The results showed that the Fimb A recombinant protein was successfully expressed at 32 kDa in the form of supernatant after induction, the pET-32a empty vector was expressed at 18 kDa after induction, and the uninduced pET-32a-Fimb A recombinant protein was not expressed, which was consistent with the expected results.

[0082] 8) The Fimb A recombinant protein expressed in the supernatant was purified by nickel column (HIS tag) affinity chromatography to obtain the purified Bacteroides nodosa Fimb A recombinant protein. The purified Bacteroides nodosa Fimb A recombinant protein was subjected to polyacrylamide gel electrophoresis. The results were as follows: Figure 2 As shown in the results, the recombinant protein of Bacteroides nodosa Fimb A purified by nickel column affinity chromatography had a single band at 32 kDa, and the protein concentration was 2548 mg / mL, indicating that the recombinant protein of Bacteroides nodosa Fimb A was successfully purified.

[0083] 9) Using HIS tag antibody as primary antibody, the 7 substances obtained in step 6) were identified by Western blot. The results are as follows: Figure 3As shown in the results, the Fimb A recombinant protein expressed in the supernatant obtained after IPTG induction had a reaction band at about 32kDa in the supernatant of ultrasonic fragmentation, and the pET-32a empty vector ultrasonic fragmentation supernatant and precipitate after IPTG induction had a reaction band at about 18kDa, and the uninduced Bacteroides nodosa Fimb A recombinant protein had no reaction band. The results proved that the Bacteroides nodosa Fimb A recombinant protein was successfully expressed and had reactogenicity.

[0084] 10) The purified Bacteroides nodosa Fimb A recombinant protein obtained in step 8) was identified by Western blot. The results were as follows: Figure 4 As shown in the results, the antigen-antibody reaction result was a single band without other mixed bands, proving that the recombinant protein of Bacteroides nodosa Fimb A was successfully purified.

[0085] 2. Antigen emulsification

[0086] The most important thing in antigen-antibody immunoassay is the emulsification of antigen. The quality of antigen emulsification directly affects the final result of the immunoassay. The specific emulsification experiment steps are as follows:

[0087] 1) Adjust the concentration of purified antigen (i.e., Fimb A recombinant protein) to 1 mg / mL.

[0088] 2) Prepare a 5mL centrifuge tube, add equal proportions of antigen and adjuvant into it, and then vortex on a vortexer until a viscous emulsion is formed. In order to avoid the waste of antigen and adjuvant caused by the process of evacuating bubbles, it is usually necessary to absorb more antigen and adjuvant than the normal amount. First, use drops of emulsified antigen sample on the surface of distilled water. The completely emulsified sample will appear as oil droplets on the water surface and will not disperse. This indicates that the emulsification is complete and can be used for immunoassays. If the sample is dispersed on the water surface, the emulsification is incomplete and it is necessary to continue emulsification according to the above steps until the antigen is completely emulsified.

[0089] 3. Immunization of Animals

[0090] The Bacillus nodosa Fimb A recombinant protein obtained above was used to immunize 6-8 week old female rabbits (weight 2 kg), and the specific operation steps were as follows:

[0091] (1) After the rabbits have adapted to the new environment for one week, blood is collected from the ear vein. The blood volume is about 2 mL as a negative control. After blood collection, the wound is pressed and disinfected with alcohol cotton swabs 2 minutes later. The collected blood should first be placed in a 37°C incubator for 30 minutes to prevent activation of the complement system in the blood. It is then transferred to a 4°C environment and left overnight to allow the serum to precipitate. The next day, the completely stratified blood sample is transferred to a centrifuge tube and centrifuged for 10 minutes at 4°C and 3000 r / min. The supernatant is stored in a -80°C refrigerator.

[0092] (2) Basic immunization: Freund's adjuvant (Sigma, SA-F5881) and the above-obtained Bacillus nodosa Fimb A recombinant protein were emulsified according to the method in step 2, and 1 mL of the fully emulsified antigen sample with a concentration of 0.5 mg / ml was drawn into a 2 mL disposable syringe and injected subcutaneously in the back. The total amount of antigen protein used for each rabbit during the first immunization was 500 μg.

[0093] (3) Booster immunization: 14 days after the first immunization, emulsify the antigen and Freund's incomplete adjuvant (Sigma, F5506) according to step 2. After emulsification, use a 2mL disposable syringe to draw 1mL of the antigen sample with a concentration of 0.5mg / ml, and use the back subcutaneous injection method. When boosting immunization, avoid the site of previous injection. Each injection is 0.2mL, and the protein amount of the antigen for boosting immunization is 200μg. Perform another booster immunization on the 21st day and the 28th day. Usually, blood samples need to be collected after the third immunization. The blood sampling time is 7 days after each booster immunization and before the next immunization. Blood is collected from the ear vein. The antibody titer is determined by indirect ELISA method to determine the number of booster immunizations. Seven days after the last booster immunization, whole blood is collected from the animal by heart blood sampling, and serum is collected according to the method of the above step (1).

[0094] 4. The antibody titer was determined by indirect ELISA method. The specific steps are as follows:

[0095] (1) Antigen coating: The purified Bacteroides nodosa Fimb A recombinant protein was diluted to 1 μg / mL with pH=7.4, 0.01M phosphate coating solution, mixed evenly, and added to a 96-well ELISA plate at 100 μL / well. The plate was coated with Bacteroides nodosa Fimb A recombinant protein overnight at 4°C. The wells without sample were used as blank control groups, the wells with pre-immune serum were used as negative control groups, and the wells with post-immune antiserum were used as experimental groups.

[0096] (2) Washing: The next day, discard the coating solution and wash with PBST solution three times with shaking, each time for 3 min.

[0097] (3) Blocking: Add 200 μL of 5% skim milk to each well as blocking solution and block at 37°C for 2 h. Discard the blocking solution and wash with PBST for 3 times with shaking, 3 min each time;

[0098] (4) Primary antibody incubation: The pre-immune serum and the antiserum to be tested (post-immune serum) of the rabbit were diluted with PBST solution at a dilution gradient of 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, etc., and 100 μL of the diluted serum was added to each well. The cells were incubated at 37°C for 1 h. A blank control was set up at the same time. The cells were washed with PBST for 3 times with shaking for 3 min each time.

[0099] (5) Secondary antibody incubation: Dilute commercial HRP-labeled goat anti-rabbit IgG with PBST solution at a volume ratio of 1:50,000, 100 μL / well, incubate at 37°C for 1 h;

[0100] (6) Color development: Add 100 μL of TMB substrate color development solution to each well and develop the color for 15 min at room temperature in the dark.

[0101] (7) Stop color development: Add 100 μL 2 mol / L H2SO4 to each well to stop color development and set the microplate reader to read the OD 450nm The optimal antigen coating concentration and serum dilution criteria: OD of positive serum 450nm The value is above 1.5, and the OD of negative serum 450nm The best value is below 0.2 and the P / N (positive / negative) value is the largest.

[0102] Before each step, wash the plate and pat dry the liquid in the plate.

[0103] Example 2

[0104] An indirect ELISA kit for detecting Fimb A protein of Bacteroides nodosa, comprising the following components: positive control serum, negative control serum, 96-well enzyme-labeled plates, HRP-labeled goat anti-rabbit IgG enzyme-labeled secondary antibody, coating solution, sample diluent, washing solution, blocking solution, TMB substrate colorimetric solution (Solebol colorimetric solution, PR1200), and stop solution; the positive control serum is serum from rabbits infected with Bacteroides nodosa, and the negative control serum is serum from rabbits that are not immunized or infected with Bacteroides nodosa.

[0105] Coating solution: 1 pack of commercial PBS powder (White Shark, BL601A) was mixed with 2000 mL of deionized water to obtain a 0.01 M PBS solution.

[0106] Sample diluent or washing solution: PBS solution and Tween-20 are mixed, and the volume ratio of Tween-20 to PBS is 1:2000.

[0107] Blocking solution: 1 g skim milk powder (Biofroxx, 68514-61-4) is prepared into 20 mL sample diluent to obtain a mass concentration of 5% skim milk.

[0108] TMB substrate colorimetric solution (commercial Solebol colorimetric solution PR1200).

[0109] Stop solution (2 mol / L H2SO4): distilled water 178.3 mL, add concentrated sulfuric acid (98%) 21.7 mL dropwise.

[0110] Preparation method of ELISA plate: Use 0.01M phosphate buffer (pH=7.4) as coating solution, dilute the purified Bacillus nodosa Fimb A recombinant protein prepared in Example 1, and add 100μl / well to 96-well ELISA plate to ensure that the concentration of Bacillus nodosa Fimb A recombinant protein in each well is 0.25μg / mL. Coat for 12h at 4℃, and the next day, discard the coating solution, wash (wash 3 times; each time, the amount of washing solution is 300μL / well, and the washing time is 3min), pat dry, add 5% skim milk blocking solution at 200μL / well, stand at 37℃ for 2h, wash 3 times, the washing method is the same as before, and pat dry.

[0111] Example 3

[0112] The basic operation steps of the indirect ELISA kit described in Example 2 are as follows:

[0113] 1) The purified Bacteroides nodosa Fimb A recombinant protein prepared in Example 1 was diluted to 2 μg / mL with 0.01 M phosphate buffer (pH 7.4) and added to a 96-well ELISA plate, 100 μL per well, to ensure that the concentration of the Bacteroides nodosa Fimb A recombinant protein in each well was 0.25 μg / mL, and coated at 4° C. for 12 h;

[0114] 2) Block the 96-well ELISA plate coated overnight by adding 5% skim milk at 200 μL / well and blocking at 37°C for 2 h, followed by conventional washing with PBST;

[0115] 3) According to the optimal dilution multiple of 1:819200 (volume ratio), the serum to be tested, the negative serum control and the positive serum control were diluted with PBST buffer, and the diluted serum and blank control (PBST buffer) were added to the 96-well ELISA plate coated with Bacteroides nodosa Fimb A protein at 100 μL / well, incubated at 37°C for 1 hour, and then washed with PBST;

[0116] 4) HRP-labeled goat anti-rabbit IgG was diluted at a ratio of 1:30,000 (i.e., 1 μL HRP-labeled goat anti-rabbit IgG was mixed with 30,000 μL PBST solution), and then added to a 96-well ELISA plate at a volume of 100 μL / well. After incubation at 37°C for 30 min, conventional washing with PBST was performed;

[0117] 5) Take out the TMB substrate colorimetric solution from the 4°C refrigerator and warm it to room temperature, add 100 μL / well of the TMB substrate colorimetric solution, and develop the color at 37°C in the dark for 5-10 min;

[0118] 6) Add stop solution at 100 μL / well to stop color development and set the microplate reader to read OD 450nm The value of positive serum OD 450nm The value is recorded as P1, the OD of negative serum 450nm The value is recorded as N1, and the OD of the serum to be tested 450nm The value is recorded as P, and the OD of the blank control 450nm The value is recorded as N. Before entering the next step each time, the plate must be washed and the liquid in the plate must be patted dry.

[0119] Calculation method of judgment criteria:

[0120]

[0121] in is the mean value of positive serum / negative serum, and SD is the standard deviation of positive serum / negative serum.

[0122] The P / N value is ≥0.212, and the serum to be tested is judged as positive;

[0123] P / N value ≤ 0.187, the serum to be tested is judged as negative;

[0124] When the P / N value is 0.187<0.212, the serum sample to be tested is suspicious and needs to be retested.

[0125] Example 4

[0126] Screening of optimal antigen coating concentration and negative and positive serum working concentrations

[0127] The Fimb A recombinant protein of Bacteroides nodosa prepared in Example 1 was diluted and coated at 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. At the same time, the negative and positive sera of Bacteroides nodosa were diluted at a gradient of 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200. The goat anti-rabbit enzyme-labeled secondary antibody was diluted at 1:50000. The establishment of the indirect ELISA kit was the same as in Example 2, and the basic operating steps were the same as in Example 3. The P / N value was calculated using the square matrix method. The P / N value calculation formula is: P / N value=P / N×100%, N is the negative serum OD 450nm , P is positive serum OD 450nm , calculate the optimal concentration of antigen coating and the optimal working concentration of serum.

[0128] When the P / N value was the highest, the optimal antigen coating concentration was 0.25 μg / mL and the optimal serum working concentration was 1:819200.

[0129] The results are shown in Table 1. The best results are highlighted in bold in the table below.

[0130] Table 1 Optimal antigen coating amount and optimal serum dilution

[0131]

[0132]

[0133] Example 5

[0134] Screening of the Optimal Enzyme-labeled Antibody Working Concentration

[0135] The purified Fimb A protein was coated at an optimal antigen coating concentration of 0.25 μg / mL. At the same time, the standard Bacteroides nodosa negative and positive sera were diluted at an optimal serum dilution multiple of 1:819200. The enzyme-labeled secondary antibody was diluted at 1:5000, 1:10000, 1:20000, 1:30000, 1:40000, etc. according to the working range of the instruction manual. The establishment of the indirect ELISA kit was the same as in Example 2, and the basic operating steps were the same as in Example 3. The P / N value was calculated using the square matrix method, and the optimal enzyme-labeled antibody working concentration was calculated. The results are shown in Table 2.

[0136] Table 2 Optimal enzyme-labeled antibody working concentration

[0137]

[0138] The results show that when the P / N value is the highest, the optimal enzyme-labeled antibody working concentration is 1:30000.

[0139] Example 6

[0140] Screening of antigen coating solution and coating conditions

[0141] According to the optimal experimental conditions of Example 4 and Example 5, different coating temperature conditions and coating times were set (see Table 3 for details), and each treatment was repeated 3 times. The establishment of the indirect ELISA kit was the same as in Example 2, and the basic operating steps were the same as in Example 3. The square matrix method was used to calculate the P / N value, the optimal antigen coating solution and coating conditions, and the results are shown in Table 3.

[0142] Table 3 Antigen coating solution and coating conditions

[0143]

[0144]

[0145] The results showed that under 4°C conditions, using 0.01 mol / L phosphate buffer as the coating solution to dilute the antigen and coating for 12 hours, the obtained P / N value was the highest.

[0146] Example 7

[0147] Screening of blocking solution and blocking conditions

[0148] According to the optimal experimental conditions of Examples 4 to 6, different blocking solutions and blocking times were set (see Table 4 for details), and each treatment was repeated 3 times. The indirect ELISA kit was established in the same way as in Example 2, and the basic operating steps were the same as in Example 3. The square matrix method was used to calculate the P / N value, the optimal blocking solution and blocking time, and the results are shown in Table 4.

[0149] Table 4 Optimal blocking solution and blocking conditions

[0150]

[0151] The results showed that the best blocking conditions were as follows: 5% skim milk, 37°C for 2h, and the highest P / N value.

[0152] Example 8

[0153] Screening of the reaction conditions of TMB substrate colorimetric solution

[0154] TMB substrate colorimetric solution was added at 100 μL / well, the temperature was room temperature and 37°C, the color development time was 5 min, 10 min, 15 min, and 20 min, respectively (see Table 5 for details), each treatment was repeated 3 times, and a blank control was set up. The establishment of the indirect ELISA kit was the same as in Example 2, and the basic operating steps were the same as in Example 3. The square matrix method was used to calculate the P / N value and the action conditions of the TMB substrate colorimetric solution. The results are shown in Table 5.

[0155] Table 5 Results of the color development solution conditions

[0156]

[0157]

[0158] The results show that the best condition for the color development solution is 5 minutes at 37°C.

[0159] Example 9

[0160] Tests on positive / negative cutoff value, repeatability, sensitivity and specificity of indirect ELISA kits

[0161] 1. Determined by indirect ELISA test, 30 negative bovine serum samples with known background were tested to determine the negative and positive critical values, and blank serum was used as a control to obtain The negative and positive results are as follows: When the negative and positive judgment criteria are obtained: the sample P / N value ≥ 0.212 is judged as positive, and the sample P / N value ≤ 0.187 is judged as negative. If the sample P / N is between 0.187 and 0.212, it is a suspicious sample and needs to be retested. If it is still between the two, the result is judged as positive.

[0162] 2. The indirect ELISA detection method for detecting Fimb A protein of Bacteroides nodosa and the negative and positive judgment criteria provided by the present invention were used to detect bovine Bacteroides nodosa positive serum, bovine foot-and-mouth disease virus positive serum, brucellosis positive serum and bovine Staphylococcus aureus positive serum respectively (wherein the bovine Bacteroides nodosa strain model is A198 strain, purchased from DSMZ, Germany, DSM20708; the bovine foot-and-mouth disease virus positive serum was purchased from the Central Prison and numbered VIP(P)10121; the brucellosis positive serum was purchased from the Central Prison and numbered VIP(SY)1068; the bovine Staphylococcus aureus positive serum was donated by Professor Wang Jianfa of Heilongjiang Bayi Agricultural University). The basic operating steps of the indirect ELISA kit are the same as those in Example 3, and the positive / negative P / N values ​​are determined. The results are shown in Table 6.

[0163] Table 6 Specificity test results

[0164] serum Bacteroides nodosa Foot-and-mouth disease virus Brucella staphylococcus P / N Value 3.846 0.179 0.148 0.126

[0165] The results show that the indirect ELISA kit for detecting antibodies to the Fimb A protein of Bacteroides nodosa provided by the present invention has specificity.

[0166] 3. The indirect ELISA detection method for detecting Fimb A protein of Bacteroides nodosa provided by the present invention was used to dilute the positive serum of Bacteroides nodosa from 100 times and then to 1:104857600 by double dilution method to determine the sensitivity of this method. The basic operation steps of the indirect ELISA kit were the same as those in Example 3, and the P / N value was calculated. The results are shown in Table 7.

[0167] Table 7 Kit sensitivity test results

[0168]

[0169] The results show that when the serum is diluted to 1:26214400, the OD 450nm The value is 0.269, and the result is judged to be positive. Therefore, the sensitivity of the indirect ELISA detection method established in the present invention is 1:26214400.

[0170] 4. The indirect ELISA detection kit for detecting Fimb A protein of Bacteroides nodosa provided by the present invention was used to perform intra-batch and inter-batch repeatability tests. Intra-batch repeatability test: 96-well ELISA plates coated with the same batch were used to test three known negative sera and three known positive sera. Inter-batch repeatability test: 96-well ELISA plates coated with different batches were used to test three known negative sera and three known positive sera.

[0171] The basic operation steps of the indirect ELISA kit are the same as those in Example 3. The average P / N value of each sample in the intra-batch repeatability test and the inter-batch repeatability test is calculated. Standard deviation (SD) and coefficient of variation CV, Samples 1 to 3 are known positive samples, and samples 4 to 6 are known negative samples. The test results are shown in Tables 8 and 9.

[0172] Table 8 Results of intra-batch repeatability test of the kit

[0173]

[0174] Table 9 Results of the kit batch reproducibility test

[0175]

[0176] The results show that the CV% values ​​of the kits of the present invention are all less than 10%, which proves that the established indirect ELISA antibody detection kit for Bacteroides nodosa has good repeatability.

[0177] 5. The indirect ELISA kit and PCR identification method for detecting Fimb A protein of Bacteroides nodosa provided by the present invention were used, wherein the PCR identification method was performed with reference to the method in the following literature (Li Lin, Dong Jing, Cui Jixian, et al. Study on PCR detection method of Bacteroides nodosa for foot rot in dairy cows [J]. Modern Animal Husbandry and Veterinary Medicine, 2014, (06): 37-40.). 92 clinical bovine serum samples with known identification results (3 positive samples and 89 negative samples) were tested. The basic operating steps of the indirect ELISA kit were the same as those in Example 3. The P / N value was calculated. The results are shown in Table 10.

[0178] Table 10 Test results of kit compliance rate

[0179] Detection Methods Positive Negative total Compliance rate PCR 3 89 92 100％ ELISA 4 88 92 96.3％

[0180] As shown in Table 10, the PCR identification results included 3 positive sera and 89 negative sera, and the indirect ELISA results included 4 positive sera and 88 negative sera. Finally, the consistency rate of the indirect ELISA and PCR identification of Necrobacterium necrotum of the present invention was 96.3%.

[0181] Example 10

[0182] Indirect ELISA kits for testing clinical samples

[0183] The indirect ELISA kit for detecting antibodies to Fimb A protein of Bacteroides nodosa provided by the present invention was used to detect 100 clinical serum samples from a cattle farm in Heilongjiang Province. The basic operation steps of the indirect ELISA kit were the same as those in Example 3, and the test results are shown in Table 11.

[0184] Table 11 Clinical sample testing results

[0185] Negative Positive total Positive rate Clinical bovine serum 100 3 103 3％

[0186] The results show that the kit of the present invention can specifically detect Bacteroides nodosa, and the positive rate is 3%.

[0187] In summary, the application of the Bacteroides nodosa Fimb A recombinant protein of the present invention in the preparation of polyclonal antibodies for detecting the Bacteroides nodosa Fimb A protein, and the use of the recombinant protein of the present invention can prepare polyclonal antibodies for accurately detecting Bacteroides nodosa, and the obtained polyclonal antibodies have good repeatability. At the same time, the present invention provides a polyclonal antibody prepared by the recombinant protein of the present invention, which can realize the screening of Bacteroides nodosa infection and limb and hoof disease, and has the characteristics of simple operation, good specificity, high sensitivity, strong repeatability and accurate results. The experimental results show that the P / N value of the clinical positive serum of Bacteroides nodosa cattle is greater than 0.212, indicating that the polyclonal antibody has good specificity.

[0188] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An ELISA kit for detecting antibodies to the FimbA protein of Bacteroides nodosa, comprising the following independently packaged components: a solid phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing solution, a color developing solution, and a stop solution, characterized in that: The solid phase carrier is a solid phase carrier coated with Bacteroides nodosa FimbA protein, and the amino acid sequence of the Bacteroides nodosa FimbA protein is shown in SEQ ID NO.

1.

2. The ELISA kit according to claim 1, characterized in that The preparation method of the Bacteroides nodosa Fimb A protein comprises: transferring a recombinant vector inserted with a FimbA protein encoding gene into Escherichia coli, inducing expression with isopropylthiogalactoside to obtain a supernatant containing the FimbA protein; and purifying the supernatant containing the FimbA protein to obtain the Bacteroides nodosa FimbA protein.

3. The ELISA kit according to claim 2, characterized in that The primer pair for amplifying the FimbA protein encoding gene includes primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4; the initial vector of the recombinant vector includes the pET-32a vector.

4. The ELISA kit according to claim 1, characterized in that The preparation method of the solid phase carrier coated with the nodule Bacteroides FimbA protein comprises: mixing 0.01M phosphate buffer and the nodule Bacteroides FimbA protein to obtain a 0.25μg / mL nodule Bacteroides FimbA protein solution; adding the nodule Bacteroides FimbA protein solution to the solid phase carrier, coating at 4°C for 12h, discarding the nodule Bacteroides FimbA protein solution, adding a skim milk solution with a mass concentration of 5% to the solid phase carrier, sealing at 37°C for 2h, and obtaining the solid phase carrier coated with the nodule Bacteroides FimbA protein.

5. The ELISA kit according to claim 1, characterized in that The enzyme-labeled secondary antibody includes an HRP-labeled IgG enzyme-labeled secondary antibody; the sample diluent includes a PBST buffer; the washing solution includes a PBST buffer; the color developing solution includes a TMB substrate color developing solution; and the stop solution includes a 2 mol / L sulfuric acid solution.

6. The ELISA kit according to any one of claims 1 to 5, characterized in that The ELISA kit also includes independently packaged standard positive control serum and independently packaged standard negative control serum; the standard positive control serum is animal serum infected with Bacteroides nodules; the standard negative control serum is animal serum not infected with Bacteroides nodules.

7. Use of the ELISA kit according to any one of claims 1 to 6 in detecting antibodies against FimbA protein of Bacteroides nodosa and / or Bacteroides nodosa, wherein the direct purpose of the use is non-diagnosis and non-treatment.

8. The use according to claim 7, characterized in that: The applications include: Mixing the sample to be tested and the sample diluent to obtain a diluted sample to be tested; the volume ratio of the sample to be tested and the sample diluent is 1:819200; The diluted sample to be tested and the sample diluent are added to different positions of the solid phase carrier respectively, and incubated at 37° C. for 1 hour to obtain a first solid phase carrier; Mixing the enzyme-labeled secondary antibody and the sample diluent to obtain a diluted enzyme-labeled secondary antibody; the volume ratio of the enzyme-labeled secondary antibody to the sample diluent is 1:30000; Adding the diluted enzyme-labeled secondary antibody to the first solid phase carrier, incubating at 37° C. for 1 h to obtain a second solid phase carrier; Add a color developing solution to the second solid phase carrier, and color develop at 37° C. in the dark for 3 to 10 minutes to obtain a third solid phase carrier; Add the stop solution to the third solid phase carrier, and read the OD at the position where the diluted sample to be tested and the sample dilution solution are added. 450nm The value of OD at the position of the diluted sample to be tested is 450nm The value is recorded as P, and the OD at the position where the sample diluent is added 450nm The value is recorded as N, and the test results of the sample to be tested are determined according to the P / N value, including: If the P / N value is ≥ 0.212, the sample to be tested is determined to be positive, that is, the sample to be tested contains Bacteroides nodosa Fimb A protein antibodies and / or Bacteroides nodosa; If the P / N value is ≤0.187, the sample to be tested is judged to be negative, that is, the sample to be tested does not contain Bacteroides nodosa Fimb A protein antibodies and Bacteroides nodosa; If 0.187<P / N value<0.212, the sample to be tested is a suspicious sample and retest is performed.

9. The use according to claim 8, characterized in that: After each incubation or color development, the solid phase carrier is washed with the washing solution; the number of washings is 3 times; and the time for each washing is 3 minutes.

10. The use according to claim 8, characterized in that: The sample to be tested includes serum.

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