An elisa kit for detecting fimb a protein antibody of bacteroides pleomorphica and application thereof

Abstract

The present application relates to the technical field of bacterial detection, in particular to an ELISA kit for detecting FimbA protein antibody of Bacteroides nodosus and application thereof.The ELISA kit provided by the present application comprises the following components packaged independently: solid phase carrier coated with FimbA protein of Bacteroides nodosus, enzyme-labeled secondary antibody, diluent, washing solution, color developing solution and termination solution.The ELISA kit provided by the present application uses FimbA protein of Bacteroides nodosus as detection antigen, and can detect whether Bacteroides nodosus is contained in the sample to be detected, and can be used for early screening of Bacteroides nodosus infection and laminitis, and has the characteristics of simple operation, good specificity, high sensitivity, strong repeatability and accurate result.The experimental results show that the P / N value of Bacteroides nodosus bovine clinical positive serum is greater than or equal to 0.212, indicating that the kit has good accuracy.

Description

Technical Field

[0001] This invention relates to the field of bacterial detection technology, and in particular to an ELISA antibody kit for detecting Fimb A protein in Bacteroides arthriticum and its application. Background Technology

[0002] Ruminant hoof diseases are a general term for diseases of the limbs and hooves of ruminants. They are inflammatory conditions of the limbs caused by mixed bacterial infections, including deformed hooves, foot rot, hoof dermatitis, and interdigital dermatitis. The main clinical manifestations are lameness, difficulty getting up and down, emaciation, and decreased fertility. Lameness in dairy cows caused by hoof diseases is one of the major reasons for the premature culling of high-producing dairy cows. Most of the dairy cows culled due to lameness caused by hoof diseases are high-producing dairy cows, resulting in huge economic losses to the dairy farming industry.

[0003] *Dendrobacter nodosus* is a strictly anaerobic, Gram-negative bacillus belonging to the Cardiobacteriaceae family. It is non-motile, non-spore-forming, and non-capsulated. It can survive for up to 40 days in clayey soil at 5°C. It has multiple serotypes, with serotype A first discovered in sheep limb and hoof lesions in 1938. It is one of the main bacterial serotypes causing limb and hoof diseases in ruminants such as deer, cattle, and sheep. *Dendrobacter nodosus* secretes large amounts of highly active proteases that soften and denature the tissue proteins in animal hooves, leading to a decrease in the hardness of the hoof surface and basal layer, thus causing interdigital dermatitis. Studies have also found that *Dendrobacter nodosus* is the primary pathogen causing foot rot in dairy cows and plays an important role in the development of hoof dermatitis and interdigital dermatitis in dairy cows. Therefore, it is necessary to continuously monitor *Dendrobacter nodosus* infection in dairy cows and establish relevant diagnostic methods.

[0004] Currently in veterinary clinical practice, screening for Arthrozobacter infection and hoof diseases in dairy cattle mainly relies on PCR diagnosis and bacterial isolation and identification. The accuracy and reproducibility of detection results for early Arthrozobacter infection in cattle are not high, and there is currently no effective antigen for indirect ELISA detection of Arthrozobacter in cattle. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an ELISA kit for detecting antibodies against Bacteroides Fimb A protein and its application. The ELISA kit provided by this invention is an indirect ELISA kit. By detecting whether the sample contains antibodies against Bacteroides Fimb A protein, it determines whether the sample contains Bacteroides Fimb A or whether the sample is infected with Bacteroides Fimb A. It features high specificity and accuracy and can be used for early screening of Bacteroides Fimb A infection and hoof diseases.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

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

[0008] Preferably, the method for preparing the Bacteroides Fimb A protein includes: transforming a recombinant vector containing the Fimb A protein encoding gene into Escherichia coli, inducing expression with isopropyl thiogalactoside to obtain a supernatant containing the Fimb A protein; and purifying the supernatant containing the Fimb A protein to obtain the Bacteroides 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; the initial vector of the recombinant vector includes the pET-32a vector.

[0010] Preferably, the method for preparing the solid-phase carrier coated with Bacteroides Fimb A protein includes: mixing 0.01M phosphate buffer and Bacteroides Fimb A protein to obtain a 0.25 μg / mL Bacteroides Fimb A protein solution; adding the Bacteroides Fimb A protein solution to the solid-phase carrier, coating at 4°C for 12 h, discarding the Bacteroides Fimb A protein solution, adding a 5% (w / w) skim milk solution to the solid-phase carrier, and blocking at 37°C for 2 h to obtain the solid-phase carrier coated with Bacteroides Fimb A protein.

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

[0012] Preferably, the ELISA kit further includes separately packaged standard positive control serum and separately packaged standard negative control serum; the standard positive control serum is animal serum infected with Bacteroides argentis; the standard negative control serum is animal serum not infected with Bacteroides argentis.

[0013] The present invention provides the application of the ELISA kit described above in the detection of Bacteroides Fimb A protein antibody and / or Bacteroides argentis, the direct purpose of which is non-diagnostic and non-therapeutic.

[0014] Preferably, the application includes:

[0015] The sample to be tested and the sample diluent are mixed to obtain the diluted sample to be tested; the volume ratio of the sample to the sample diluent is 1:819200.

[0016] The diluted sample to be tested and the sample dilution solution were added to different positions of the solid support and incubated at 37°C for 1 hour to obtain the first solid support.

[0017] The enzyme-labeled secondary antibody and the sample dilution solution are mixed to obtain the diluted enzyme-labeled secondary antibody; the volume ratio of the enzyme-labeled secondary antibody to the sample dilution solution is 1:30000.

[0018] The diluted enzyme-labeled secondary antibody was added to the first solid-phase carrier and incubated at 37°C for 1 hour to obtain the second solid-phase carrier.

[0019] Add a colorimetric solution to the second solid support and develop the color at 37°C in the dark for 3-10 minutes to obtain the third solid support.

[0020] A stop solution is added to the third solid support, and the OD values ​​at the locations where the diluted test sample and the sample dilution solution are added are read. 450nm The value of OD at the location where the diluted sample to be tested is added. 450nm The value is denoted as P, representing the OD at the location where the sample diluent was added. 450nm The value is denoted as N. The test result of the sample is determined based on the P / N value, including:

[0021] If the P / N value is ≥0.212, the sample is considered positive, meaning that the sample contains Bacteroides Fimb A protein antibody and / or Bacteroides Fimb A.

[0022] If the P / N value is ≤0.187, the sample is considered negative, meaning that the sample does not contain Bacteroides fimbA protein antibody or Bacteroides.

[0023] If 0.187 < P / N value < 0.212, the sample to be tested is a suspected sample and should be retested.

[0024] Preferably, after each incubation or color development, the solid support is washed with the washing solution; the washing is performed 3 times; and each washing takes 3 minutes.

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

[0026] Beneficial effects:

[0027] This invention provides an ELISA kit for detecting antibodies against Bacteroides arbuscularense Fimb A protein, comprising the following individually packaged components: a solid-phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing buffer, a chromogenic solution, and a stop solution. The solid-phase carrier is coated with Bacteroides arbuscularense Fimb A protein, the amino acid sequence of which is shown in SEQ ID NO.1. The ELISA kit provided by this invention uses Bacteroides arbuscularense Fimb A protein as the detection antigen, enabling the detection of whether a sample contains antibodies against Bacteroides arbuscularense Fimb A protein, thereby determining whether the sample contains Bacteroides arbuscularense. It can be used for early screening of Bacteroides arbuscularense infection and hoof diseases, and features simple operation, high specificity, high sensitivity, strong reproducibility, and accurate results. Experimental results show that the P / N value of clinically positive bovine serum for Bacteroides arbuscularense is ≥0.212, indicating that the kit has good accuracy. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 SDS-PAGE image of prokaryotic expression of Bacteroides Fimb A recombinant protein; where M represents pre-stained rainbow protein marker, 1 represents whole cells of uninduced pET-32a empty vector sonicated, 2 represents supernatant of pET-32a empty vector sonicated after induction, 3 represents precipitate of pET-32a empty vector sonicated after induction, 4 represents whole cells of uninduced pET-32a-Fimb A recombinant protein sonicated, 5 represents whole cells of pET-32a-Fimb A recombinant protein sonicated after induction, 6 represents supernatant of pET-32a-Fimb A recombinant protein sonicated after induction, and 7 represents precipitate of pET-32a-Fimb A recombinant protein sonicated after induction.

[0030] Figure 2 SDS-PAGE image of purified Bacteroides Fimb A recombinant protein; where M represents the prestained rainbow protein marker and 1 represents the purified pET-32a-Fimb A recombinant protein;

[0031] Figure 3This is a Western blot diagram identifying the prokaryotic expression of Bacteroides Fimb A recombinant protein; where M represents the pre-stained rainbow protein marker, 1 represents whole cells of uninduced pET-32a empty vector sonicated, 2 represents the supernatant of pET-32a empty vector sonicated after induction, 3 represents the precipitate of pET-32a empty vector sonicated after induction, 4 represents whole cells of uninduced pET-32a-Fimb A recombinant protein sonicated, 5 represents whole cells of pET-32a-Fimb A recombinant protein sonicated after induction, 6 represents the supernatant of pET-32a-Fimb A recombinant protein sonicated after induction, and 7 represents the precipitate of pET-32a-Fimb A recombinant protein sonicated after induction.

[0032] Figure 4 The image shows the Western blot identification of purified Bacteroides Fimb A recombinant protein; where M represents the prestained rainbow protein marker and 1 represents the purified pET-32a-Fimb A recombinant protein. Detailed Implementation

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

[0034] IPAYNDYIARSQAAEGLTLADGLKVRISDHLESGECKGDANPASGSLGNDDKGKYALATIDGDYNKDAKTADEKNGCKVVITYGQGTAGEKISKLIVGKKLVLDQFVNGSYKYNEGETDLELKFIPNAVKN.

[0035] As one embodiment, the method for preparing the *Bacteroides argentifera* Fimb A protein includes: transforming a recombinant vector containing the Fimb A protein encoding gene into *Escherichia coli*, inducing expression with isopropyl thiogalactoside, and obtaining a supernatant containing the Fimb A protein; purifying the supernatant containing the Fimb A protein to obtain the *Bacteroides argentifera* Fimb A protein; the nucleotide sequence of the Fimb A protein encoding gene is shown in SEQ ID NO.2, and is as follows:

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

[0037] In one 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 the pET-32a vector.

[0038] This invention uses Bacteroides Fimb A protein as a detection antigen to detect whether the sample contains Bacteroides Fimb A protein antibody, thereby determining whether the sample contains Bacteroides Fimb A.

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

[0040] In one embodiment, the solid-phase carrier can be a 96-well ELISA strip; the 96-well ELISA strip has a specification of 8 wells × 12 strips and can be disassembled; in another embodiment, the 96-well ELISA strip is purchased from Costar.

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

[0042] In one embodiment, the sample diluent can be PBST buffer; in another embodiment, the PBST solution is prepared by mixing PBS solution and Tween-20 to obtain PBST solution; the volume ratio of Tween-20 to PBS is 1:2000.

[0043] In one embodiment, the washing solution can be PBST washing solution.

[0044] As one implementation method, the colorimetric solution can be a TMB substrate colorimetric solution.

[0045] As one implementation method, the terminating solution can be a 2 mol / L sulfuric acid solution.

[0046] The ELISA kit of this invention uses an indirect method. The recombinant Bacteroides Fimb A protein is coated onto an ELISA plate, which is then blocked with 5% skim milk. Serum samples to be tested are added to the solid-phase carrier coated with Bacteroides Fimb A protein. If the serum contains Bacteroides-specific antibodies, the antibodies will bind to the Fimb A protein antigen on the solid-phase carrier. Then, an enzyme-labeled secondary antibody that binds to the specific antibody is added, followed by a colorimetric reaction solution. The colorimetric reaction is terminated with a stop solution. The sample is then measured using an ELISA reader at OD500. 450nm At the specified wavelength, the absorbance values ​​of each well were measured. The OD value (the intensity of the color after the colorimetric reaction is terminated) is positively correlated with the amount of *Bacteroides argentis* antibody in the sample. The protective effect of the antibody is determined based on the antibody content level in the sample. Furthermore, the coating antigen (*Bacteroides argentis* Fimb A recombinant protein) used in this kit is a prokaryotically expressed recombinant protein, possessing characteristics of mass production and stability. This provides a highly practical and easy-to-use diagnostic kit for large-scale farms.

[0047] In one embodiment, the ELISA kit further includes individually packaged standard positive control serum and individually packaged standard negative control serum; the standard positive control serum is animal serum infected with Bacteroides argentis; the standard negative control serum is animal serum not infected with Bacteroides argentis.

[0048] The standard positive control serum and standard negative control serum provided by this invention can determine the positive and negative cutoff values. This invention interprets the results as follows: OD of the test sample... 450nm If the value is greater than or equal to the positive threshold, the sample is considered positive. 450nm If the sample's OD value is less than or equal to the negative threshold, it is considered negative. 450nm Samples between the positive and negative thresholds are considered suspicious and require retesting.

[0049] This invention provides the application of the ELISA kit described above in the detection of *Bacteroides argentis* Fimb A protein antibody and / or *Bacteroides argentis*, wherein the direct purpose of the application is non-diagnostic and non-therapeutic. When the direct purpose of the application of this invention is non-diagnostic and non-therapeutic, it is merely to obtain an intermediate value as to whether the sample to be tested contains *Bacteroides argentis* Fimb A protein antibody and / or *Bacteroides argentis*.

[0050] This invention utilizes an ELISA kit to detect positive bovine serum for Bacteroides arthriticum, foot-and-mouth disease, Brucella, and Staphylococcus aureus. The results show that the P / N value of clinically positive bovine serum for foot-and-mouth disease, Brucella, and Staphylococcus is <0.187, while the P / N value of clinically positive bovine serum for Bacteroides arthriticum is >0.212. This demonstrates that the indirect ELISA detection method for Bacteroides arthriticum Fimb A protein has good specificity.

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

[0052] The sample to be tested and the sample diluent are mixed to obtain the diluted sample to be tested; the volume ratio of the sample to the sample diluent is 1:819200.

[0053] The diluted sample to be tested and the sample dilution solution were added to different positions of the solid support and incubated at 37°C for 1 hour to obtain the first solid support.

[0054] The enzyme-labeled secondary antibody and the sample dilution solution are mixed to obtain the diluted enzyme-labeled secondary antibody; the volume ratio of the enzyme-labeled secondary antibody to the sample dilution solution is 1:30000.

[0055] The diluted enzyme-labeled secondary antibody was added to the first solid-phase carrier and incubated at 37°C for 1 hour to obtain the second solid-phase carrier.

[0056] Add a colorimetric solution to the second solid support and develop the color at 37°C in the dark for 5-10 minutes to obtain the third solid support.

[0057] A stop solution is added to the third solid support, and the OD values ​​at the locations where the diluted test sample and the sample dilution solution are added are read. 450nmThe value of OD at the location where the diluted sample to be tested is added. 450nm The value is denoted as P, representing the OD at the location where the sample diluent was added. 450nm The value is denoted as N. The test result of the sample is determined based on the P / N value, including:

[0058] If the P / N value is ≥0.212, the sample is considered positive, meaning that the sample contains Bacteroides Fimb A protein antibody and / or Bacteroides Fimb A.

[0059] If the P / N value is ≤0.187, the sample is considered negative, meaning that the sample does not contain Bacteroides fimbA protein antibody or Bacteroides.

[0060] If 0.187 < P / N value < 0.212, the sample to be tested is a suspected sample and should be retested.

[0061] In one implementation, after each incubation or color development, the solid support is washed with the washing solution; the washing is performed 3 times; and each washing takes 3 minutes.

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

[0063] As one implementation method, if the colorimetric solution is not used immediately, it can be stored at 4°C. When using it, the colorimetric solution stored at 4°C can be allowed to warm to room temperature before use.

[0064] As one implementation method, the sample to be tested can be serum.

[0065] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an ELISA kit for detecting Bacteroides Fimb A protein and its application, but these descriptions 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 carried out by Harbin Bosheng Biotechnology Co., Ltd., and the specific steps are as follows:

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

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

[0071] 2) Using the whole-genome DNA extracted in step 1) as a template, the Fimb A fragment was amplified by PCR to obtain the amplification product. The amplification product was purified using the Tiangen Genomics Purification Kit to obtain the purified target gene product. 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 consisted of: 0.5 μL Fimb AF, 0.5 μL Fimb AR, 6.75 μL 2×Taq, 14.75 μL ddH2O, and 2.5 μL whole genomic DNA.

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

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

[0077] 4) The linearized pET-32a vector obtained in step 3) and the purified linearized target gene product were ligated using T4 DNA ligase to obtain the 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 cultured with shaking at 220 r / min and 37 ℃ to obtain a bacterial culture containing the recombinant vector; at the same time, a bacterial culture transformed with the pET-32a empty vector was set up as a blank control group.

[0079] 6) Use a spectrophotometer OD 600nmWhen the concentrations of the two bacterial cultures in step 5) were measured to be 0.6–0.8, IPTG with a final concentration of 1 mmol / L was added, and the cultures were induced at 220 r / min and 37℃ for 6 h. After centrifugation, the precipitates of the two bacterial cultures were ultrasonically disrupted to separate the supernatant and precipitate. Specifically, 100 mL of the induced bacterial culture was added to a centrifuge tube and centrifuged at 8000 r / min for 10 min at room temperature. The supernatant was discarded. 20 mL of pre-cooled PBS was added to the precipitate after centrifugation and mixed well. The culture was ultrasonically disrupted under ice-water mixture conditions (0℃) until clear. The ultrasonic program was set to 42 Hz, with 5 s of sonication followed by a 10 s interval. The product obtained after sonication was whole cells. The product after sonication was centrifuged at 8000 r / min for 20 min at 4℃ to separate the supernatant and precipitate. The precipitate was resuspended in 200 μL of PBS.

[0080] Meanwhile, two bacterial cultures without IPTG induction were set up as a control group. Seven substances were collected: 1. Uninduced pET-32a empty vector whole cells were sonicated and disrupted; 2. Induced pET-32a empty vector supernatant was sonicated and disrupted; 3. Induced pET-32a empty vector precipitate was sonicated and disrupted; 4. Uninduced pET-32a-Fimb A recombinant protein whole cells were sonicated and disrupted; 5. Induced pET-32a-Fimb A recombinant protein whole cells were sonicated and disrupted; 6. Induced pET-32a-Fimb A recombinant protein supernatant was sonicated and disrupted; 7. Induced pET-32a-Fimb A recombinant protein precipitate was sonicated and disrupted.

[0081] 7) The seven substances obtained in step 6) were analyzed by SDS-PAGE, and the results are as follows: Figure 1 As shown in the figure. The results indicate that the recombinant Fimb A protein was successfully expressed at 32 kDa in supernatant form after induction, and the pET-32a empty vector was expressed at 18 kDa after induction. The uninduced pET-32a-Fimb A recombinant protein was not expressed, which is consistent with the expected results.

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

[0083] 9) Using the HIS-tagged antibody as the primary antibody, the seven substances obtained in step 6) were identified by Western blot. The results are as follows: Figure 3As shown in the figure, the results indicate that a reactive band was observed at approximately 32 kDa in the supernatant of the sonicated fragmented Fimb A recombinant protein obtained after IPTG induction, and at approximately 18 kDa in the supernatant and precipitate of the sonicated fragmented pET-32a empty vector after IPTG induction. No reactive band was observed in the uninduced Bacteroides Fimb A recombinant protein. These results demonstrate that the Bacteroides Fimb A recombinant protein was successfully expressed and possesses reactive properties.

[0084] 10) The purified Bacteroides Fimb A recombinant protein obtained in step 8) was identified by Western blot, and the results are as follows: Figure 4 As shown in the figure, the antigen-antibody reaction showed a single band without any other bands, proving that the recombinant Fimb A protein from Bacteroides arthriticum was successfully purified.

[0085] 2. Antigen emulsification

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

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

[0088] 2) Prepare a 5mL centrifuge tube and add equal proportions of antigen and adjuvant. Vortex the tube until a viscous emulsion is formed. To avoid wasting antigen and adjuvant during the degassing process, it is generally advisable to use slightly more antigen and adjuvant than normal. First, drop the emulsified antigen sample one drop at a time onto the surface of distilled water. A fully emulsified sample will appear as oil droplets on the water surface without dispersing, indicating complete emulsification and suitability for immunoassay. If the sample disperses on the water surface, the emulsification is incomplete and the process should be continued following the steps above until the antigen is completely emulsified.

[0089] 3. Immunized animals

[0090] The recombinant Bacteroides Fimb A protein obtained above was used to immunize 6-8 week old female rabbits (weighing 2 kg). The specific steps are as follows:

[0091] (1) After rabbits have adapted to the new environment for one week, blood was collected from the marginal ear vein. The blood volume was about 2 mL as a negative control. After blood collection, the wound was pressed and disinfected with an alcohol swab after 2 minutes. The collected blood should first be placed in a constant temperature incubator at 37℃ and left to stand for 30 minutes to prevent activation of the complement system in the blood. Then it was transferred to an environment at 4℃ and left to stand overnight to allow the serum to separate. The next day, the blood sample that had completely separated into layers was transferred to a centrifuge tube and centrifuged for 10 minutes at 4℃ and 3000 r / min. The supernatant was stored in a freezer at -80℃.

[0092] (2) Basic immunization: Freund's adjuvant (Sigma, SA-F5881) and the above-obtained Bacteroides Fimb A recombinant protein were emulsified according to the method in step 2. 1 mL of the completely emulsified antigen sample with a concentration of 0.5 mg / mL was drawn up with a 2 mL disposable syringe and injected subcutaneously from 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) as described in step 2. After complete emulsification, use a 2mL disposable syringe to draw 1mL of antigen sample with a concentration of 0.5mg / ml and administer it subcutaneously via the back. Avoid the previous injection site during booster immunization. Inject 0.2mL each time, and the protein content of the booster antigen should be 200μg. Booster immunizations are performed again on days 21 and 28. Blood samples are usually collected after the third immunization. Blood is collected from the ear vein 7 days after each booster immunization and before the next immunization. The antibody titer is determined using indirect ELISA to determine the number of booster immunizations. 7 days after the last booster immunization, whole blood is collected from the animal using cardiac blood collection. Serum is collected according to the method described in step (1) above.

[0094] 4. The specific steps for determining antibody titer using the indirect ELISA method are as follows:

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

[0096] (2) Washing: On the second day, discard the coating solution and wash with PBST solution by shaking 3 times, 3 min each time;

[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 with shaking for 3 min each time;

[0098] (4) Primary antibody incubation: Dilute rabbit pre-immunization serum and test antiserum (post-immunization serum) with PBST solution at dilution ratios of 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, etc. Add 100 μL of diluted serum to each well and incubate at 37°C for 1 h. Set up a blank control at the same time. Wash with PBST for 3 min each time.

[0099] (5) Secondary antibody incubation: Commercially available HRP-labeled goat anti-rabbit IgG was diluted with PBST solution at a volume ratio of 1:50000, 100 μL / well, and incubated at 37℃ for 1 h;

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

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

[0102] Before proceeding to the next step, the plate must be washed and the liquid inside the plate patted dry.

[0103] Example 2

[0104] An indirect ELISA kit for detecting Fimb A protein in Bacteroides arthropathica comprises the following components: positive control serum, negative control serum, 96-well ELISA strips, HRP-labeled goat anti-rabbit IgG ELISA secondary antibody, coating buffer, sample dilution buffer, washing buffer, blocking buffer, TMB substrate chromogenic solution (Solepro chromogenic solution, PR1200), and stop solution; wherein the positive control serum is serum from rabbits infected with Bacteroides arthropathica, and the negative control serum is serum from unimmunized and uninfected rabbits.

[0105] Coating solution: Prepare 2000 mL of deionized water with one packet of commercial PBS powder (White Shark, BL601A) to obtain a 0.01 M PBS solution.

[0106] Sample dilution or washing solution: Mix PBS solution and Tween-20 at a volume ratio of 1:2000.

[0107] Blocking solution: 1g of skim milk powder (Biofroxx, 68514-61-4) was used to prepare 20mL of sample dilution solution to obtain skim milk with a mass concentration of 5%.

[0108] TMB substrate developer (commercial Solarbio PR1200 developer).

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

[0110] Microplate preparation method: Using 0.01M phosphate buffer (pH=7.4) as the coating solution, the purified Bacteroides Fimb A recombinant protein prepared in Example 1 was diluted and added to a 96-well microplate at a rate of 100 μl / well, ensuring that the concentration of Bacteroides Fimb A recombinant protein in each well was 0.25 μg / mL. The plate was coated at 4℃ for 12 h. The next day, the coating solution was discarded, and the plate was washed (3 times; each wash used 300 μL of washing buffer per well for 3 min), then patted dry. 200 μL of blocking buffer (5% skim milk) was added to each well, and the plate was incubated at 37℃ for 2 h. The plate was then washed 3 times using the same washing method as before, and patted dry.

[0111] Example 3

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

[0113] 1) The purified Bacteroides 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 100 μL per well of a 96-well microplate strip to ensure that the concentration of Bacteroides Fimb A recombinant protein in each well was 0.25 μg / mL. The strip was coated at 4°C for 12 h.

[0114] 2) Block the 96-well microplate coated overnight by adding 200 μL of 5% skim milk to each well and blocking at 37°C for 2 hours, followed by routine washing with PBST.

[0115] 3) Dilute the serum to be tested, negative serum control and positive serum control separately with PBST buffer according to the optimal dilution ratio of 1:819200 (volume ratio). Add 100 μL of the diluted serum and blank control (PBST buffer) to each well of a 96-well ELISA strip coated with Bacteroides Fimb A protein. Incubate at 37°C for 1 h and then perform routine PBST washing.

[0116] 4) Dilute HRP-labeled goat anti-rabbit IgG at a ratio of 1:30000 (i.e., mix 1 μL of HRP-labeled goat anti-rabbit IgG with 30000 μL of PBST solution), add 100 μL / well to a 96-well microplate, incubate at 37°C for 30 min, and then perform routine PBST washing.

[0117] 5) Remove the TMB substrate colorimetric solution from the 4℃ refrigerator and allow it to warm to room temperature. Add 100μL of TMB substrate colorimetric solution per well and develop the colorimetric solution at 37℃ in the dark for 5-10 minutes.

[0118] 6) Add 100 μL of stop solution to each well to stop the color development process, and then use a microplate reader to read the OD value. 450nm The value of OD in positive serum 450nm The value is denoted as P1, the OD value of negative serum. 450nm The value is denoted as N1, representing the OD value of the serum being tested. 450nm The value is denoted as P, and the OD of the blank control is... 450nm The value is denoted as N. Before proceeding to the next step, the plate must be washed and the liquid inside the plate must be patted dry.

[0119] Judgment Criteria Calculation Method:

[0120]

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

[0122] A P / N value ≥ 0.212 indicates that the serum sample is positive.

[0123] If the P / N value is ≤0.187, the serum sample to be tested is considered negative;

[0124] When the P / N value is less than 0.187, the serum sample is considered suspicious and requires retesting.

[0125] Example 4

[0126] Screening for optimal antigen coating concentration and working concentrations of negative and positive sera.

[0127] The *Bacteroides fibrobacterium* A recombinant protein prepared in Example 1 was coated with the sample at serial dilutions of 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. Simultaneously, *Bacteroides fibrobacterium* negative and positive sera were serially diluted at dilutions of 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200. Goat anti-rabbit enzyme-labeled secondary antibody was diluted 1:50000. The indirect ELISA kit was established 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%, where N is the OD of the negative serum. 450nm P represents positive serum OD 450nm Calculate the optimal concentration for antigen coating and the optimal working concentration for serum.

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

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

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

[0131]

[0132]

[0133] Example 5

[0134] Screening for optimal working concentration of enzyme-labeled antibody

[0135] The purified Fimb A protein was coated at the optimal antigen coating concentration of 0.25 μg / mL. Simultaneously, standard Bacteroides sera and negative and positive sera were diluted at the optimal serum dilution ratio of 1:819200. The enzyme-labeled secondary antibody was diluted according to the manufacturer's instructions at dilutions of 1:5000, 1:10000, 1:20000, 1:30000, and 1:40000. The indirect ELISA kit was established as in Example 2, and the basic operating steps were the same as in Example 3. The P / N ratio was calculated using the square matrix method to determine the optimal working concentration of the enzyme-labeled antibody. The results are shown in Table 2.

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

[0137]

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

[0139] Example 6

[0140] Screening of antigen coating solutions and coating conditions

[0141] Following the optimal experimental conditions of Examples 4 and 5, different coating temperatures and coating times were set (see Table 3 for details). 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 P / N value was calculated using the square matrix method, and the optimal antigen coating solution and coating conditions were calculated. The results are shown in Table 3.

[0142] Table 3 Antigen coating solution and coating conditions

[0143]

[0144]

[0145] The results showed that at 4℃, the highest P / N value was obtained by diluting the antigen with 0.01 mol / L phosphate buffer and coating for 12 h.

[0146] Example 7

[0147] Screening of sealing fluid and sealing conditions

[0148] Following the optimal experimental conditions of Examples 4-6, different blocking solutions and blocking times were set (see Table 4 for details). 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 P / N value was calculated using the square matrix method, and the optimal blocking solution and blocking time were calculated. The results are shown in Table 4.

[0149] Table 4 Optimal sealing solution and sealing conditions

[0150]

[0151] The results show that the optimal sealing conditions are: 5% skim milk, 37°C for 2 hours, resulting in the highest P / N ratio.

[0152] Example 8

[0153] Screening of reaction conditions for TMB substrate chromogenic solution

[0154] 100 μL of TMB substrate chromogenic solution per well, at room temperature and 37°C, with chromogenic times of 5 min, 10 min, 15 min, and 20 min (see Table 5 for details). Each treatment was repeated 3 times, with a blank control included. The indirect ELISA kit was established in the same manner 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 reaction conditions of the TMB substrate chromogenic solution were calculated. The results are shown in Table 5.

[0155] Table 5 Results of the reaction conditions for the colorimetric reagent

[0156]

[0157]

[0158] The results show that the optimal conditions for the color developing solution are 37℃ and 5 minutes of color development.

[0159] Example 9

[0160] Positive and negative cutoff values, repeatability, sensitivity, and specificity tests for indirect ELISA kits

[0161] 1. Using indirect ELISA detection, 30 bovine negative serum samples with known background were tested to determine the negative and positive cutoff values. Blank serum was used as a control. The value was 0.137, and the SD was 0.025. According to the formula for interpreting negative and positive results: The criteria for determining positive and negative results are as follows: a sample P / N value ≥ 0.212 is considered positive, a sample P / N value ≤ 0.187 is considered negative, and a sample P / N value between 0.187 and 0.212 is considered a suspicious sample and requires retesting. If the P / N value remains between these two values, the result is considered positive.

[0162] 2. Using the indirect ELISA detection method and negative / positive criteria for detecting Bacteroides Fimb A protein provided by this invention, positive sera for Bacteroides Fimb. bovine, foot-and-mouth disease virus, brucellosis, and Staphylococcus aureus were detected respectively. (Bacteroides Fimb. bovine strain A198 was purchased from DSMZ GmbH, Germany, DSM20708; foot-and-mouth disease virus positive serum was purchased from the China Institute of Animal Disease Control and Prevention, serial number VIP(P)10121; brucellosis positive serum was purchased from the China Institute of Animal Disease Control and Prevention, serial number VIP(SY)1068; and Staphylococcus aureus positive serum was kindly provided by Professor Wang Jianfa of Heilongjiang Bayi Agricultural Reclamation University). The basic operating steps of the indirect ELISA kit were the same as in Example 3. The positive / negative P / N values ​​were measured, and the results are shown in Table 6.

[0163] Table 6 Results of Specificity Tests

[0164] serum Bacteroides argentis 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 Bacteroides Fimb A protein antibodies provided by this invention is specific.

[0166] 3. Using the indirect ELISA method for detecting Fimb A protein in Bacteroides brevicornu provided by this invention, Bacteroides brevicornu-positive serum was diluted 100-fold initially, and then diluted twice to 1:104857600 to determine the sensitivity of this method. The basic operating steps of the indirect ELISA kit are the same as in Example 3. The P / N value was calculated, and the results are shown in Table 7.

[0167] Table 7 Results of reagent kit sensitivity tests

[0168]

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

[0170] 4. The intra-assay and inter-assay repeatability tests were performed using the indirect ELISA kit for detecting Bacteroides Fimb A protein provided by this invention. Intra-assay repeatability test: 96-well microplates coated from the same batch were used to test three known negative sera and three known positive sera. Inter-assay repeatability test: 96-well microplates coated from different batches were used to test three known negative sera and three known positive sera.

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

[0172] Table 8. Results of intra-batch repeatability tests of the reagent kit

[0173]

[0174] Table 9 Results of inter-batch repeatability tests of the reagent kit

[0175]

[0176] The results show that the CV% values ​​of the kits described in this invention are all less than 10%, proving that the established Bacteroides bovis indirect ELISA antibody detection kit has good repeatability.

[0177] 5. The indirect ELISA kit and PCR identification method for detecting Fimb A protein of Bacteroides arbuscular aureus provided by this invention were used. 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 arbuscular aureus 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 and 89 negative) were tested. The basic operation steps of the indirect ELISA kit were the same as in Example 3. The P / N value was calculated and the results are shown in Table 10.

[0178] Table 10 Results of reagent kit compliance test

[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. In the end, the concordance rate between the indirect ELISA and PCR identification of necrotic bacilli in this invention was 96.3%.

[0181] Example 10

[0182] Indirect ELISA kit for testing clinical samples

[0183] One hundred clinical serum samples from a cattle farm in Heilongjiang Province were tested using the indirect ELISA kit for detecting Bacteroides fibrobacterium A protein antibody provided by this invention. The basic operating steps of the indirect ELISA kit are the same as in Example 3, and the detection results are shown in Table 11.

[0184] Table 11 Results of Clinical Sample Testing

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

[0186] The results show that the kit described in this invention can specifically detect Bacteroides argentiformis, with a positive rate of 3%.

[0187] In summary, this invention demonstrates the application of the Bacteroides Fimb A recombinant protein in the preparation of polyclonal antibodies for detecting Bacteroides Fimb A protein. Furthermore, the recombinant protein of this invention enables the preparation of accurate polyclonal antibodies for detecting Bacteroides Fimb A, and the obtained polyclonal antibodies exhibit good reproducibility. Simultaneously, this invention provides a method for screening for Bacteroides Fimb A infection and limb diseases using polyclonal antibodies prepared from the recombinant protein of this invention, characterized by simple operation, high specificity, high sensitivity, strong reproducibility, and accurate results. Experimental results show that the P / N value of clinically positive bovine serum for Bacteroides Fimb A is >0.212, indicating that the polyclonal antibody has good specificity.

[0188] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An ELISA kit for detecting antibodies against Bacteroides Fimb A protein, comprising individually packaged components: a solid-phase carrier, an enzyme-labeled secondary antibody, a sample diluent, a washing buffer, a chromogenic solution, and a stop solution, characterized in that, The solid-phase carrier is a solid-phase carrier coated with Bacteroides Fimb A protein, and the amino acid sequence of Bacteroides Fimb A protein is shown in SEQ ID NO.1; The method for preparing the solid-phase carrier coated with Bacteroides Fimb A protein includes: mixing 0.01M phosphate buffer and Bacteroides Fimb A protein to obtain a 0.25 μg / mL Bacteroides Fimb A protein solution; adding the Bacteroides Fimb A protein solution to the solid-phase carrier, coating at 4℃ for 12 h, discarding the Bacteroides Fimb A protein solution, adding a 5% (w / w) skim milk solution to the solid-phase carrier, and blocking at 37℃ for 2 h to obtain the solid-phase carrier coated with Bacteroides Fimb A protein.

2. The ELISA kit according to claim 1, characterized in that, The method for preparing the Bacteroides Fimb A protein includes: transforming a recombinant vector containing the Fimb A protein encoding gene into Escherichia coli, inducing expression with isopropyl thiogalactoside, and obtaining a supernatant containing the Fimb A protein; purifying the supernatant containing the Fimb A protein to obtain the Bacteroides Fimb A protein.

3. The ELISA kit according to claim 2, characterized in that, 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 the pET-32a vector.

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

5. The ELISA kit according to any one of claims 1 to 4, characterized in that, The ELISA kit also includes individually packaged standard positive control serum and individually packaged standard negative control serum; the standard positive control serum is animal serum infected with Bacteroides argentis; the standard negative control serum is animal serum not infected with Bacteroides argentis.

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