An Indirect ELISA Detection Kit for Bacteroides argyrophyllin Antibody and Its Application

Abstract

This invention relates to an indirect ELISA detection kit for *Bacteroides argyrophyllinoides* antibodies and its application, belonging to the field of biodetection technology. To address the shortcomings of existing bacterial isolation, identification, and PCR detection methods for *Bacteroides argyrophyllinoides*, and the lack of serological identification methods for this bacterium in current technologies, this invention uses the outer membrane protein Omp1D of *Bacteroides argyrophyllinoides* as the coating antigen in the indirect ELISA detection kit. This enables rapid detection and screening of *Bacteroides argyrophyllinoides* infection in cattle and sheep, featuring simple operation, high sensitivity, accurate results, good specificity, and good reproducibility. Furthermore, the *Bacteroides argyrophyllinoides* antibody indirect ELISA detection kit is characterized by simple preparation method, low cost, and suitability for large-scale production. It provides a rapid, simple, and sensitive diagnostic kit for detecting *Bacteroides argyrophyllinoides* infection in large-scale livestock farming, offering technical support for the prevention and control of *Bacteroides argyrophyllinoides* infection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an indirect ELISA detection kit for Bacteroides aniae and its application. Background Technology

[0002] Foot and hoof diseases are a general term for lesions of the limbs and hooves caused by various adverse factors. Hoof diseases account for more than 90% of all foot and hoof diseases, including foot rot, hoof deformities, laminitis, hoof dermatitis, hoof abscesses, and hoof trauma. Foot rot is particularly harmful among ruminant hoof diseases, causing clinical symptoms such as hoof deformities, lameness, difficulty moving, and loss of appetite. *Bacteroides argentea* is one of the important pathogens causing foot rot in sheep. *Bacteroides argentea* is a Gram-negative, non-spore-forming obligate anaerobic bacillus that can cause foot rot in ruminants and is highly contagious in sheep. In the early stages of the disease, sheep hooves will show slight redness and swelling, with redness visible between the toes or on the coronary band. As the disease progresses, the redness and swelling expand, leading to significant swelling, hoof heat and pain, and lameness. Subsequently, ulcers and necrosis may occur on the hoof skin, resulting in black or brown rotten areas with an unpleasant odor and purulent exudate. If the infection is not controlled in time, the pathogens may enter the bloodstream, causing systemic infection. At this time, sheep will exhibit symptoms such as fever, lethargy, and loss of appetite. In severe cases, it can lead to weight loss, emaciation, and even death. Foot rot not only affects animal health but also has a significant impact on livestock development. For example, it causes direct economic losses such as reduced maternal productivity, increased fattening time, reduced milk production, and decreased fur quality. In the UK, direct related losses amount to £200,000 to £800,000 annually. In countries like Switzerland, the indirect costs of prevention and control are equal to, or even far exceed, the direct costs, severely hindering farm development and resulting in substantial economic losses.

[0003] Currently, the diagnosis of Arthroblastobacterium infection in clinical practice mainly relies on bacterial isolation and identification and PCR diagnostic techniques. However, because Arthroblastobacterium is strictly anaerobic and has stringent requirements for culture gas conditions, bacterial isolation and identification and PCR diagnostic techniques are often at a disadvantage in clinical practice due to competition for survival with other bacterial groups during sample transportation and culture. This results in long detection times and poor stability of test results. At the same time, PCR technology, as a routine diagnostic method, has also shown poor performance in early latent infection of Arthroblastobacterium and in epidemiological surveys involving multiple sample detection. Furthermore, there is currently no serological identification method for Arthroblastobacterium. Therefore, it is necessary to expand research on other diagnostic detection methods for Arthroblastobacterium. Summary of the Invention

[0004] To address the shortcomings of existing bacterial isolation, identification, and PCR detection methods for Bacteroides argentisae, and the lack of serological identification methods for Bacteroides argentisae in current technologies, this invention provides an indirect ELISA detection kit for Bacteroides argentisae antibodies and its application. By using the recombinant protein Omp1D of the Bacteroides argentisae outer membrane as the coating antigen, a simple, convenient, accurate, sensitive, reproducible, and highly specific Bacteroides argentisae antibody detection kit and a method for detecting Bacteroides argentisae antibodies using this kit are established.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0006] The first objective of this invention is to provide an indirect ELISA detection kit for *Bacteroides argyrophyllinoides* antibodies. The kit comprises a solid-phase carrier coated with the recombinant *Bacteroides argyrophyllinoides* protein Omp1D, a diluent, a washing buffer, a standard positive control serum, a standard negative control serum, an enzyme-labeled secondary antibody, a chromogenic solution, and a stop solution. The nucleotide sequence of the *Bacteroides argyrophyllinoides* recombinant protein Omp1D is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The standard positive control serum is serum from rabbits infected with *Bacteroides argyrophyllinoides*, and the standard negative control serum is serum from rabbits that are not immunized or infected with *Bacteroides argyrophyllinoides*.

[0007] In one embodiment of the present invention, the preparation method of the Bacteroides argentifer recombinant protein Omp1D is as follows: Specific primers are designed and synthesized for the nucleic acid sequence corresponding to the Bacteroides argentiferiferiferiferiferiferiferiferotherum Omp1D protein. Using the whole genome DNA of Bacteroides argentiferiferiferotherum as a template, PCR amplification is performed using the primers, and the target gene fragment is recovered. The target gene is ligated into a cloning vector and transformed into competent Escherichia coli cells to obtain positive clones. Plasmids are extracted by bacterial culture. The plasmids and expression vectors are subjected to double enzyme digestion and purification, respectively. Recombinant bacterial culture is obtained by ligation and transformation. IPTG is used to induce expression. The obtained bacterial culture is ultrasonically disrupted to obtain Omp1D protein expressed in precipitate form. After SDS-PAGE and gel purification, the Bacteroides argentiferiferiferiferotherum recombinant protein Omp1D is obtained.

[0008] In one embodiment of the present invention, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4.

[0009] In one embodiment of the present invention, the solid-phase carrier coated with Bacteroides argentiferae recombinant protein Omp1D is a 96-well microplate coated with Bacteroides argentiferae recombinant protein Omp1D. The specific preparation method is as follows: using 0.01M phosphate buffer as the coating solution, the purified recombinant protein Omp1D is diluted to 2μg / mL and added to the microplate at 100μL / well. The plate is then coated at 4℃ for 12h. The next day, the coating solution is discarded, and the plate is washed with PBST. 300μL of 5% skim milk is added as the blocking solution, and the plate is incubated at 37℃ for 2h. The blocking solution is discarded, the plate is washed with PBST, and the plate is dried at room temperature.

[0010] In one embodiment of the present invention, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG, the chromogenic solution is TMB substrate chromogenic solution, the stop solution is a 2M H2SO4 solution, and the diluent and washing solution contain 0.05% Tween-20 PBS buffer by volume.

[0011] A second objective of this invention is to provide the application of the above-mentioned Bacteroides ani antibody indirect ELISA detection kit in non-diagnostic Bacteroides ani antibody detection, characterized in that the application includes the following steps:

[0012] S1. Sample incubation: Add the diluted serum sample to the solid-phase carrier coated with Bacteroides argentis recombinant protein Omp1D, incubate, and then wash with washing buffer to obtain the primary ELISA plate.

[0013] S2. Incubation of the secondary antibody: Add diluted enzyme-labeled secondary antibody to the primary ELISA plate obtained in S1, incubate, and then wash with washing buffer to obtain the intermediate ELISA plate containing the secondary antibody.

[0014] S3, color development: Add the color development solution to the intermediate ELISA plate containing the secondary antibody obtained in S2 and develop the color in the dark to obtain the color-developed ELISA plate;

[0015] S4. Termination of color development: Add the stop solution to the chromogenic ELISA plate obtained in S3 to terminate the color development. Measure the OD value of the diluted serum sample. 450nm The absorbance value at that location;

[0016] S5. Result Interpretation: If the OD of the diluted serum sample is... 450nm A value ≥0.389 is considered positive; if the OD value of the diluted serum sample is... 450nm A value ≤0.312 is considered negative. If the OD value of the diluted serum sample is... 450nm If the value is greater than 0.312 and less than 0.389, the sample is considered suspicious and needs to be retested.

[0017] In one embodiment of the present invention, the serum sample in S1 is diluted by a ratio of 1:12800.

[0018] In one embodiment of the present invention, the incubation temperature of the serum sample in S1 is 37°C and the incubation time is 0.5h.

[0019] In one embodiment of the present invention, the enzyme-labeled secondary antibody in S2 is diluted by a ratio of 1:100000.

[0020] In one embodiment of the present invention, the enzyme-labeled secondary antibody in S2 is incubated at 37°C for 1.5 hours.

[0021] In one embodiment of the present invention, the color development in step S3 is carried out at room temperature for 15 minutes.

[0022] The beneficial effects of this invention are:

[0023] This invention establishes an indirect ELISA detection kit for *Bacteroides argentifer* antibodies using the outer membrane protein Omp1D as the coating antigen. Because the outer membrane protein Omp1D of *Bacteroides argentifer* possesses genetic uniqueness, this kit enables rapid detection and screening of *Bacteroides argentifer* infection in cattle and sheep, featuring simple operation, high speed and sensitivity, accurate results, good specificity and reproducibility. The *Bacteroides argentifer* antibody indirect ELISA detection kit established in this invention was used to detect pathogens such as *Staphylococcus aureus* and *Brucella*, and no cross-reactivity was found, indicating that the kit has high specificity and good accuracy. Furthermore, the *Bacteroides argentifer* antibody indirect ELISA detection kit provided by this invention has the advantages of simple preparation method, low cost, and suitability for large-scale production, providing a rapid, simple, and sensitive diagnostic kit for detecting *Bacteroides argentifer* infection in large-scale livestock farming, and providing technical support for the prevention and control of *Bacteroides argentifer* infection.

[0024] The polyclonal antibodies obtained by immunizing animals in this invention can be used to screen for Bacteroides argentis infection and limb diseases, and are characterized by simple operation, high specificity, high sensitivity, strong reproducibility, and accurate results. Therefore, the polyclonal antibodies or kits containing polyclonal antibodies provided by this invention can be used to detect Bacteroides argentis.

[0025] This invention provides the application of Bacteroides argentiferae Omp1D recombinant protein in the preparation of polyclonal antibodies for detecting Bacteroides argentiferae Omp1D recombinant protein. Furthermore, the recombinant protein described in this invention can be used to prepare polyclonal antibodies that accurately detect Bacteroides argentiferae, and the obtained polyclonal antibodies exhibit good reproducibility. Simultaneously, this invention provides polyclonal antibodies prepared from the recombinant protein described in this invention that can achieve screening for Bacteroides argentiferae infection, featuring simple operation, high specificity, high sensitivity, strong reproducibility, and accurate results. Attached Figure Description

[0026] Figure 1 The image shows the SDS-PAGE results of prokaryotic expression analysis of Bacteroides arthriticus recombinant protein Omp1D. In the image, M is the pre-stained rainbow protein marker, 1 is the whole bacterial cell of uninduced recombinant protein Omp1D disrupted by sonication, 2 is the whole bacterial cell of induced empty vector pET-32a disrupted by sonication, 3 is the whole bacterial cell of induced recombinant protein Omp1D disrupted by sonication, 4 is the supernatant of induced recombinant protein Omp1D disrupted by sonication, and 5 is the precipitate of induced recombinant protein Omp1D disrupted by sonication.

[0027] Figure 2 The image shows the SDS-PAGE analysis results of the purified Bacteroides arthritica recombinant protein Omp1D; where M is the prestained rainbow protein marker and 1 is the purified recombinant protein pET-32a-Omp1D.

[0028] Figure 3 The image shows the results of Western blot identification of purified Bacteroides recombinant protein Omp1D using HIS-tagged antibody as primary antibody; where M is the prestained rainbow protein marker and 1 is the purified recombinant protein pET-32a-Omp1D.

[0029] Figure 4 The image shows the results of Western blot identification of purified Bacteroides recombinant protein Omp1D using a polyclonal antibody as the primary antibody; where M is the prestained rainbow protein marker and 1 is the purified recombinant protein pET-32a-Omp1D.

[0030] Figure 5 The graph shows the results of polyclonal antibody titer determination by indirect ELISA.

[0031] Figure 6 The graph shows the screening results for the optimal working concentration of the enzyme-labeled antibody;

[0032] Figure 7 The graph shows the sensitivity evaluation results of the indirect ELISA detection method provided in Example 3. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described herein are not all embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0035] In the examples below, Bacteroides argentifera strain A198 (DSM20708) was purchased from DSMZ GmbH, Germany.

[0036] In this invention, the nucleotide sequence encoding the recombinant protein Omp1D from Bacteroides arthriticum is shown in SEQ ID NO.1:

[0037]

[0038] In this invention, the amino acid sequence of the Bacteroides recombinant protein Omp1D is shown in SEQ ID NO.2:

[0039] .

[0040] Example 1: Preparation of Bacteroides arthriticum antigen

[0041] 1. Antigen preparation

[0042] The recombinant protein Omp1D from Bacteroides argentisae (purified recombinant protein Omp1D from Bacteroides argentisae) was obtained by the following method:

[0043] (1) Extraction of whole genome DNA of Bacteroides arthriticum

[0044] Whole genome DNA of Bacteroides arthriticum was extracted using a bacterial genome extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0045] (2) Amplification and purification of the Omp1D fragment of Bacteroides arthriticum gene

[0046] Design upstream and downstream primers (Omp1D-F and Omp1D-R) targeting the nucleotide sequence (SEQ ID NO.1) encoding the protein Omp1D. Using the whole genome DNA of Bacteroides argentis obtained in step (1) as a template, the Omp1D gene fragment was amplified by PCR using Omp1D-F and Omp1D-R to obtain the amplification product. Then, the amplification product was purified using the Tiangen Genome Purification Kit to obtain the purified product of the target gene.

[0047] Omp1D-F: 5'-CGggatccATGAAACTTTCACACTTATCATTAGC-3' (SEQ ID NO. 3);

[0048] Omp1D-R: 5'-CCGctcgagTTATTTGCCGTGAGAAATCAACCA-3' (SEQ ID NO. 4).

[0049] The reaction system for the above PCR amplification is as follows: 2.5 μL upstream primer, 2.5 μL downstream primer, 12.5 μL 2×Taq, 3.5 μL ddH2O, and 4 μL Bacteroides genomic DNA; the reaction program for the above PCR amplification is as follows: 94℃ for 2 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for 5 cycles; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for 25 cycles; 72℃ for 5 min.

[0050] (3) Connection

[0051] The purified product of the target gene obtained in step (2) is ligated with the cloning vector pET-32a to obtain the recombinant cloning vector.

[0052] The ligation system consisted of 1 μL of pET-32a, 7.0 μL of purified target gene product, 0.5–1 μL of T4 DNA ligase, and 1 μL of T4 DNA ligase buffer; the ligation conditions were 16°C for 16 h.

[0053] (4) Transformation

[0054] The recombinant cloning vector obtained in step (3) was transformed into E. coli BL21(DE3) competent cells to obtain positive bacterial strains. After shaking culture at 220 rpm and 37℃, bacterial culture was obtained. The plasmid was extracted and sent to a sequencing company (Shanghai Sangon Biotech Co., Ltd.) for sequencing to verify whether the target gene was successfully inserted into the vector. The sequencing results showed that the recombinant plasmid contained a gene encoding the recombinant protein Omp1D with a nucleotide sequence as shown in SEQ ID NO.1.

[0055] The recombinant plasmid and expression vector pET-32a obtained above were digested with restriction endonucleases BamHI and Xhol, respectively (the double digestion system is shown in Table 1, ggatcc is the BamHI restriction site, ctcgag is the Xhol restriction site, the digestion time is 3 h, and the temperature is 37℃) and purified. The sequence fragment of the purified Omp1D gene was ligated with the digestion product of pET-32a plasmid to obtain the recombinant expression vector pET-32a-Omp1D. The recombinant expression vector pET-32a-Omp1D was transformed into E. coli BL21(DE3) by conventional transformation to construct recombinant E. coli expressing Omp1D protein, and cultured in LB liquid medium with shaking at 220 rpm and 37℃.

[0056] Table 1 Double enzyme digestion system

[0057]

[0058] (5) Induction and purification of recombinant proteins

[0059] ① Induction of recombinant proteins:

[0060] The recombinant Escherichia coli culture obtained in step (4) was measured using a spectrophotometer, and the OD was determined. 600nm When the concentration was 0.6–0.8, IPTG was added to a final concentration of 1 mmol / mL, and the mixture was induced at 37°C for 16 h to obtain the induced bacterial culture. The precipitate obtained after centrifugation of the bacterial culture was ultrasonically disrupted at a power of 50 Hz (5 s of sonication followed by a 10 s interval as one cycle). The ultrasonically disrupted mixture was centrifuged again (4°C, 8000 rpm, 20 min) to obtain the Omp1D protein expressed in precipitate form (the induced recombinant protein pET-32a-Omp1D).

[0061] ② Purification of recombinant proteins

[0062] The recombinant protein Omp1D expressed in precipitate form was purified by SDS-PAGE gel excision to obtain purified Bacteroides arthritica recombinant protein Omp1D. The specific method is as follows: After collecting the precipitate, it was resuspended with an appropriate amount of PBS, and protein loading buffer was added. The mixture was heated in a water bath at 100°C for 15 min. A 10% SDS-PAGE protein gel was prepared. A comb was inserted into the upper layer of the gel, and 400 μL of the treated protein sample was added to each gel plate for SDS-PAGE. After electrophoresis, the protein gel was cut off and placed in 0.3 M KCl solution pre-cooled at 4°C for 10 min. At this time, a silvery-white target band appeared. The target band was cut off with a scalpel and then rinsed with PBS for 3 min. The target band was transferred to a clean sealed bag and crushed. The crushed target band was scraped into an EP tube and 500 μL of PBS was added. The PBS was shaken and mixed, and then subjected to repeated freeze-thaw cycles at 4°C and liquid nitrogen for 3 times. After repeated freeze-thaw cycles, the mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, which was the purified recombinant protein Omp1D. The protein concentration was then measured after purification.

[0063] The empty vector pET-32a induced by IPTG, the uninduced recombinant strain pET-32a-Omp1D, and the recombinant strain pET-32a-Omp1D induced by IPTG were ultrasonically disrupted. The supernatant and precipitate were separated and analyzed by SDS-PAGE. The analytical results are as follows: Figure 1 As shown, the results indicate that the recombinant protein Omp1D was successfully expressed in precipitated form at 94 kDa after induction, the empty vector pET-32a was expressed at 18 kDa after induction, and the uninduced recombinant protein pET-32a-Omp1D was not expressed, which is consistent with the expected results.

[0064] SDS-PAGE analysis of the purified Bacteroides recombinant protein Omp1D was performed, and the results are as follows: Figure 2 As shown, the recombinant protein Omp1D from Bacteroides argentifera exhibits a single band at 94 kDa, indicating successful purification of the recombinant protein Omp1D. Protein concentration determination of the purified recombinant protein Omp1D revealed a concentration of 1290 μg / mL.

[0065] Western blot analysis was performed on the purified recombinant protein Omp1D using an HIS-tagged antibody as the primary antibody. The results are as follows: Figure 3 As shown, a single reactive band was observed at approximately 94 kDa, demonstrating that the recombinant protein Omp1D from Bacteroides argentis was successfully expressed and exhibited reactivity.

[0066] Using the previously prepared Bacteroides polyclonal antibody as the primary antibody, the purified Bacteroides recombinant protein Omp1D was identified by Western blot. The results are as follows: Figure 4 As shown, the antigen-antibody reaction result was a single band without any other bands, proving that the Omp1D recombinant protein from Bacteroides argentis was successfully purified.

[0067] 2. Antigen emulsification

[0068] 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:

[0069] Adjust the concentration of the purified antigen (i.e., recombinant protein Omp1D) to 1 mg / mL.

[0070] Choice of immune adjuvants: Freund's adjuvant (Sigma brand adjuvant) is commonly used in immunological experiments, and it is divided into two parts: Freund's complete adjuvant and Freund's incomplete adjuvant. Freund's complete adjuvant is used for primary immunization of animals to stimulate cellular immunity; Freund's incomplete adjuvant stimulates humoral immunity and is therefore usually used for booster immunization.

[0071] Emulsification steps: The antigen and Freund's adjuvant are mixed in a 1:1 ratio during emulsification. For the first immunization, 0.5 mL of antigen at a concentration of 1 mg / mL is mixed with 0.5 mL of Freund's complete adjuvant. After mixing, the antigen concentration is adjusted to 0.5 mg / mL, and the amount of antigen for the first immunization is 500 μg.

[0072] 3. Immunized animals

[0073] Female rabbits aged 6-8 weeks, weighing approximately 2 kg, were used as experimental animals. Purchased from the animal testing center, they were acclimatized in the animal facility for one week before immunization. Recombinant protein Omp1D, emulsified with an equal volume of Freund's complete adjuvant, was administered to the rabbits via subcutaneous injection at multiple sites on the back. The initial immunization dose per rabbit was 1 mL, with 0.2 mL injected at each site, ensuring as much dispersion as possible between injection sites. On days 14 and 28 after the initial immunization, booster immunizations were administered using an equal proportion of emulsified antigen and Freund's incomplete adjuvant (Sigma F5506). Seven days after the final immunization, blood was collected from the marginal ear vein, and antibody titers were determined using an indirect ELISA method. Following the titer determination, whole blood was collected from the animal via cardiac sampling. The collected blood should first be placed in a 37°C incubator and left to stand for about 30 minutes to prevent activation of the complement system in the blood. Then, it should be transferred to a 4°C environment and left to stand overnight to allow the serum to separate. The next day, the completely separated blood sample should be transferred to a centrifuge tube and centrifuged for 10 minutes at 4°C and 3000 rpm. The supernatant should be stored in a -80°C freezer.

[0074] 4. Indirect ELISA method for determining antibody titer

[0075] Indirect ELISA is one of the more commonly used methods for detecting antibodies in immunological experiments. The detection process involves first binding the antigen to a solid-phase carrier, then adding the primary antibody to be tested. After the primary antibody and antigen are fully bound, a horseradish peroxidase-labeled secondary antibody is added. Finally, a colorimetric solution is used for development. The titer of the antibody is determined based on the color intensity or by using an ELISA reader. The specific steps are as follows:

[0076] (1) Antigen coating: The purified Bacteroides recombinant protein Omp1D was diluted to 1 μg / mL with 0.05 M carbonate coating buffer at pH 9.6, and added to a 96-well ELISA plate at a concentration of 100 μL / well. The plate was then 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. The coating buffer was discarded on the second day, and the plate was washed three times with PBST for 3 min each time.

[0077] (3) Blocking: Add 300 μL of 5% skim milk as blocking solution to each well, block at 37°C for 2 h, discard the blocking solution, and wash with PBST for 3 min each time.

[0078] (4) Primary antibody incubation: Dilute rabbit pre-immunization serum and test antiserum 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 of the coated ELISA plate 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.

[0079] (5) Secondary antibody incubation: Dilute commercially available HRP-labeled goat anti-rabbit IgG with PBST solution at a volume ratio of 1:50000, 100 μL / well, incubate at 37℃ for 1 h, discard the secondary antibody liquid, and wash with PBST for 3 min each time.

[0080] (6) Color development: Add 100 μL of TMB substrate color development solution to each well and develop color for 15 min in the dark at room temperature.

[0081] (7) Termination of color development: Add 50 μL of 2 mol / L H2SO4 solution to each well to terminate the color development, and read the OD using a microplate reader. 450nm The value.

[0082] Potency determination criteria: P / N (positive OD) 450nm / negative serum OD 450nm The maximum serum dilution factor at which a value of 2 or higher is taken as the titer of the polyclonal antibody. The titer test results are as follows: Figure 5 As shown, when the serum dilution is 1:102400, the P / N value is still greater than 2, that is, the Omp1D polyclonal antibody titer is 1:102400.

[0083] Example 2: An indirect ELISA detection kit for Bacteroides argyroplasiae antibody

[0084] This embodiment provides an indirect ELISA detection kit for detecting Omp1D antibody against Bacteroides aniae. The kit consists of the following components: a solid-phase carrier coated with recombinant protein Omp1D, diluent, washing buffer, standard positive control serum, standard negative control serum, enzyme-labeled secondary antibody, chromogenic solution, and stop solution.

[0085] Preparation of the solid-phase carrier coated with recombinant protein Omp1D (96-well ELISA plate coated with Bacteroides aniae recombinant protein Omp1D): The purified Bacteroides aniae recombinant protein Omp1D prepared above was diluted with 0.01M phosphate buffer (pH 9.6) as the coating solution and added to the ELISA plate at a rate of 100 μL / well, ensuring that the concentration of Bacteroides aniae recombinant protein Omp1D in each well was 2 μg / mL. Coating was carried out at 4℃ for 12 h. The next day, the coating solution was discarded, and 300 μL / well of PBST was added for washing, for a total of 3 washes, 3 min each time. After washing, the plate was patted dry. 300 μL / well of 5% skim milk was added as the blocking solution, and the plate was incubated at 37℃ for 2 h. The blocking solution was discarded, and 300 μL / well of PBST was added for washing, for a total of 3 washes, 3 min each time. After washing, the plate was patted dry and dried at room temperature until no water droplets were visible to the naked eye. The plate was then packaged, desiccant was added, and the plate was vacuum-sealed.

[0086] Diluent and washing buffer: Both are PBST solutions, specifically PBS solutions with a Tween-20 volume fraction of 0.05% obtained by mixing 1 mL of Tween-20 with 2000 mL of PBS solution.

[0087] Enzyme-labeled secondary antibody: HRP-labeled goat anti-rabbit IgG.

[0088] Developing solution: Ready-to-use TMB substrate developing solution (commercial Solarbio PR1200 developing solution).

[0089] Termination solution: 2M H2SO4 solution, specifically prepared by adding 21.7mL of 98% concentrated sulfuric acid dropwise to 178.3mL of distilled water.

[0090] The standard positive control serum is the serum of rabbits infected with Bacteroides argentis, and the standard negative control serum is the serum of rabbits that are not immunized or infected with Bacteroides argentis.

[0091] Example 3: Method for detecting Bacteroides argalidomide using the kit described in Example 2

[0092] 1. This embodiment, based on the kit provided in Embodiment 2, provides the following detection method:

[0093] Using 0.01M phosphate buffer (pH 9.6) as the coating solution, the purified Bacteroides recombinant protein Omp1D was diluted to 2 μg / mL and added to 100 μL per well of a 96-well ELISA strip, ensuring that the concentration of Bacteroides recombinant protein Omp1D in each well was 2 μg / mL. The strip was coated at 4°C for 12 h. The coating solution was discarded, and the strip was washed with PBST as usual. The 96-well ELISA strip coated overnight was then blocked with 300 μL of 5% skim milk as the blocking solution, and the strip was blocked at 37°C for 2 h. The blocking solution was discarded, and the strip was washed with PBST as usual.

[0094] Dilute the test serum, negative serum control, positive serum control, and blank control (PBST buffer) at a dilution ratio of 1:12800. Add 100 μL of the diluted test serum, negative serum control, positive serum control, and blank control (PBST buffer) to each well of the coated 96-well microplate. Incubate at 37°C for 0.5 h, and then perform routine PBST washing.

[0095] HRP-labeled goat anti-rabbit IgG was diluted at a ratio of 1:100,000 and added to a 96-well microplate at a volume of 100 μL / well. After incubation at 37°C for 1.5 h, the plate was washed with PBST as usual.

[0096] Remove the TMB substrate colorimetric solution from the 4℃ refrigerator and allow it to warm to room temperature. Add the colorimetric solution at a rate of 100μL / well and develop the colorimetric solution at room temperature in the dark for 15 minutes.

[0097] Add 50 μL of stop solution to each well to stop the color development process, and then use a microplate reader to read the OD values. 450nm The value.

[0098] The above-mentioned routine PBST washing refers to adding 300 μL of PBST to each well for washing, for a total of 3 washes, each lasting 3 minutes.

[0099] The calculation method for the test result judgment criteria is as follows:

[0100]

[0101]

[0102] Wherein, N represents negative serum OD. 450nm P represents positive serum OD 450nm , The mean of positive / negative results is given, and SD is the standard deviation of positive / negative results. This is based on the OD values ​​of the obtained diluted samples. 450nm The results are determined using negative and positive cutoff values: if the OD of the diluted sample... 450nmIf the OD value is greater than or equal to the positive threshold, the result is considered positive; if the OD value of the diluted sample is higher... 450nm If the OD value is less than or equal to the negative threshold, the sample is considered negative; if the OD value of the diluted sample is less than or equal to the negative threshold, the sample is considered negative. 450nm If the sample's OD value is between the positive and negative cutoff values, it is considered a suspicious sample and requires retesting. During retesting, if the OD value of the diluted sample is... 450nm If the OD value is greater than or equal to the positive threshold, the result is considered positive; if the OD value of the diluted sample is higher... 450nm If the result is ≤ the negative threshold, it is considered negative. Thirty negative serum samples with known background were tested, and the following results were obtained. SD = 0.077, so the positive threshold is 0.389 and the negative threshold is 0.312.

[0103] If the sample OD 450nm A value ≥0.389 is considered positive.

[0104] If the sample OD 450nm A value ≤ 0.312 is considered negative;

[0105] If the sample OD 450nm If the value is greater than 0.312 and less than 0.389, the sample is considered suspicious and needs to be retested.

[0106] 2. This invention screened the optimal antigen coating concentration, optimal working concentration of negative and positive serum, optimal working concentration of enzyme-labeled antibody, optimal antigen coating solution and coating conditions, optimal blocking solution and blocking conditions, and optimal chromogenic solution action conditions in the above detection methods.

[0107] (1) Screening of optimal antigen coating concentration and optimal working concentration of negative and positive sera

[0108] Based on the detection method provided in this embodiment, the concentration of Bacteroides argentifer recombinant protein Omp1D was diluted to 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL using the coating buffer. Simultaneously, Bacteroides argentifer negative and positive sera were serially diluted at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800 using the dilution buffer. Goat anti-rabbit enzyme-labeled secondary antibody was serially diluted at 1:50000. The P / N ratio was calculated using the square matrix method, where N is the OD of the negative serum. 450nm P represents positive serum OD 450nm .

[0109] 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.

[0110] The screening results are shown in Table 2. When the P / N value is the highest, the optimal antigen coating concentration is 2 μg / mL, and the optimal working concentration of serum is 1:12800.

[0111] Table 2. Screening results for optimal antigen coating concentration and optimal serum dilution.

[0112]

[0113] (2) Screening of the optimal working concentration of enzyme-labeled antibody

[0114] Based on the detection method provided in this embodiment, the purified recombinant protein Omp1D was coated at the optimal antigen coating concentration of 2 μg / mL. Simultaneously, standard Bacteroides sera and negative and positive sera were diluted at the optimal serum dilution factor of 1:12800. The enzyme-labeled secondary antibody was serially diluted according to the instructions at 1:2500, 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:100000, 1:160000, 1:320000, 1:640000, 1:1280000, and 1:2560000. The P / N value was calculated using a checkerboard method. The criterion for determining the optimal working concentration of the enzyme-labeled antibody is: the highest P / N value is considered optimal.

[0115] The screening results are as follows Figure 6 As shown, the P / N value is the largest when the working concentration of the enzyme-labeled antibody is 1:100000, therefore the optimal working concentration of the enzyme-labeled antibody is 1:100000.

[0116] (3) Screening of optimal antigen coating solution and coating conditions

[0117] Based on the detection method provided in this embodiment, the optimal antigen coating concentration, optimal working concentrations of negative and positive sera, and optimal working concentration of enzyme-labeled antibody obtained above were selected. Different coating solutions, coating temperatures, and coating times were set (see Table 3 for details). Each treatment was repeated three times, and the P / N value was calculated using the square matrix method. The criterion for determining the optimal antigen coating solution and coating conditions was: the maximum P / N (positive / negative) value was considered optimal.

[0118] The screening results are shown in Table 3. As can be seen from Table 3, the highest P / N value was obtained when the antigen was diluted with 0.01M phosphate buffer at 4℃ and coated for 12h. Therefore, the optimal antigen coating solution is 0.01M phosphate buffer and the optimal coating condition is 4℃ for 12h.

[0119] Table 3. Screening results of antigen coating solutions and coating conditions

[0120]

[0121] (4) Screening of optimal sealing solution and sealing conditions

[0122] Based on the detection method provided in this embodiment, the optimal antigen coating concentration, optimal working concentrations of negative and positive sera, optimal working concentrations of enzyme-labeled antibodies, and optimal antigen coating solution and coating conditions obtained above were selected. Different blocking solutions, blocking temperatures, and blocking times were set (see Table 4 for details). Each treatment was repeated three times, and the P / N value was calculated using the square matrix method. The criterion for determining the optimal blocking solution and blocking conditions was: the highest P / N (positive / negative) value was considered optimal.

[0123] The screening results are shown in Table 4. As can be seen from Table 4, at 37℃, using 5% skim milk as the blocking solution and blocking for 2 hours yielded the highest P / N value. Therefore, the optimal blocking solution is 5% skim milk, and the optimal blocking condition is 37℃ for 2 hours.

[0124] Table 4. Screening results of sealing fluid and sealing conditions

[0125]

[0126]

[0127] (5) Optimization of optimal reaction conditions for the colorimetric reagent

[0128] Based on the detection method provided in this embodiment, the optimal antigen coating concentration, optimal working concentrations of negative and positive sera, optimal working concentration of enzyme-labeled antibody, optimal antigen coating solution and coating conditions, and optimal blocking solution and blocking conditions were selected. The color development temperature was set to room temperature or 37°C, and the color development time was set to 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min (see Table 5 for details). Each treatment was repeated three times, and the P / N value was calculated using the square matrix method. The criterion for determining the optimal color development solution conditions was: the maximum P / N (positive / negative) value was considered optimal.

[0129] The screening results are shown in Table 5. Under normal temperature conditions, the P / N value obtained after 15 min of color development is the highest. Therefore, the optimal color development condition is 15 min of color development at room temperature.

[0130] Table 5. Screening results of colorimetric conditions

[0131]

[0132] 3. Evaluate the repeatability, sensitivity, specificity, and concordance of the detection using the above-mentioned optimal kit and optimal indirect ELISA method.

[0133] (1) Repeatability evaluation

[0134] The optimal indirect ELISA kit for detecting the Omp1D antibody of Bacteroides argentisi obtained above was used to evaluate the reproducibility of Bacteroides argentisi, specifically through intra-batch and inter-batch repeatability tests.

[0135] Intra-batch repeatability test: Take 96-well microplates coated with recombinant protein Omp1D from the same batch and test 3 known negative sera and 3 known positive sera.

[0136] Inter-batch repeatability test: Take different batches of detachable 96-well microplates coated with recombinant protein Omp1D and test them on 3 known negative sera and 3 known positive sera.

[0137] The basic operating steps of the indirect ELISA kit are the same as the optimal detection method described above. Calculate the average P / N 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 6 and 7.

[0138] As shown in Tables 6 and 7, the CV% values ​​obtained by using the optimal kit are all less than 10%, demonstrating good intra- and inter-batch repeatability.

[0139] Table 6. Results of intra-batch repeatability tests of the reagent kit

[0140]

[0141] Table 7 Results of inter-batch repeatability tests of the reagent kit

[0142]

[0143] (2) Sensitivity assessment

[0144] Using the optimal kit and established optimal detection method provided in this embodiment, positive serum was diluted starting at 12800-fold and then further diluted twofold to 1:26214400 to determine the sensitivity of this method. The OD values ​​of positive serum at different dilutions were calculated. 450nm Values, results are shown below Figure 7 As shown.

[0145] Depend on Figure 7 It can be seen that when the positive serum is diluted to 1:6553600 using the established optimal indirect ELISA method, the P / N value is less than 0.312, and the result is judged as negative. Therefore, the sensitivity of the indirect ELISA detection method established in this embodiment is 1:3276800.

[0146] (3) Specificity evaluation

[0147] Using the optimal kit and established optimal detection method provided in this embodiment, positive sera for Bacteroides aniae, Foot-and-mouth disease, Brucella, and Staphylococcus aureus were detected, and OD was measured. 450nm The values ​​are shown in Table 8.

[0148] As can be seen from Table 8, the optimal reagent kit and optimal detection method provided by the present invention are specific.

[0149] Table 8 Results of Specificity Tests

[0150]

[0151] (4) Compliance test

[0152] Using the optimal kit and established optimal detection method and PCR identification method provided in this embodiment, 20 clinical sheep serum samples with known results were tested (17 positive serum samples and 3 negative serum samples). The PCR identification method was performed according to the method in the literature (Shao Xiqun, Wang Kejian, Chen Lizhi, et al. Establishment of PCR detection method for Bacteroides arthriticum [J]. Special Products Research, 2005, (04): 4-8.). The detection results are shown in Table 9.

[0153] As shown in Table 9, the optimal kit and optimal detection method provided by this invention have a 100% compliance rate, while the compliance rate of the traditional PCR method is 94.1%.

[0154] Table 9. Results of the Compliance Test

[0155]

[0156] Example 4: Clinical samples were tested using the optimal reagent kit and optimal detection method provided in Example 3.

[0157] This embodiment uses the optimal reagent kit and optimal detection method provided in Example 3 to test 19 clinical serum samples from a sheep farm in Heilongjiang Province. The test results are shown in Table 10.

[0158] Table 10 Clinical Sample Testing Results

[0159]

[0160] As shown in Table 10, the optimal kit and optimal detection method provided by the present invention can specifically detect Bacteroides argentiformis, with a positive rate of 5.2%.

[0161] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. An indirect ELISA test kit for Bacteroides capillosus antibody, characterized by, The kit comprises solid phase carriers coated with Bacteroides plecmtum recombinant protein Omp1D, diluent, washing solution, standard positive control serum, standard negative control serum, enzyme-labeled secondary antibody, color developing solution and termination solution; the nucleotide sequence of the Bacteroides plecmtum recombinant protein Omp1D is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the standard positive control serum is serum of a rabbit infected with Bacteroides plecmtum, and the standard negative control serum is serum of a rabbit that has not been immunized or infected with Bacteroides plecmtum.

2. The Bacteroides ovis tumor antibody indirect ELISA test kit according to claim 1, characterized by, The preparation method of the Bacteroides plecmtum recombinant protein Omp1D is as follows: specific primers are designed and synthesized according to the nucleic acid sequence corresponding to the Bacteroides plecmtum Omp1D protein, and the primers are used for PCR amplification with Bacteroides plecmtum whole genome DNA as a template to recover the target gene fragment; the target gene is connected to a cloning vector and transformed into competent E. coli cells to obtain positive clones, and plasmid is obtained by liquid culture and extraction, and the plasmid and the expression vector are double-digested and purified, respectively, and the recombinant bacteria are obtained by ligation and transformation; IPTG is used for induction and expression, the obtained bacteria are ultrasonically broken to obtain the Omp1D protein expressed in the form of a precipitate, and the Bacteroides plecmtum recombinant protein Omp1D is obtained by gel cutting and purification after SDS-PAGE.

3. The indirect ELISA test kit for Bacteroides ovatus antibody according to claim 1, characterized by, The solid phase carrier coated with the Bacteroides plecmtum recombinant protein Omp1D is a 96-well enzyme-labeled plate coated with the Bacteroides plecmtum recombinant protein Omp1D, and the preparation method is as follows: 0.01 M phosphate buffer is used as a coating solution, the purified recombinant protein Omp1D is diluted to 2 μg / mL, 100 μL is added to each well of the enzyme-labeled plate, and coating is performed at 4℃ for 12 h; the next day, the coating solution is discarded, and PBST is used for washing; 300 μL of 5% skimmed milk is added as a blocking solution, and the solution is incubated at 37℃ for 2 h; the blocking solution is discarded, and PBST is used for washing, and the plate is dried at room temperature.

4. The indirect ELISA test kit for Bacteroides ovatus antibody according to claim 1, characterized by, The enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG, the color developing solution is TMB substrate color developing solution, the termination solution is a 2 M H2SO4 solution, and the diluent and the washing solution are both PBS buffer containing 0.05% Tween-20.

Images