ELISA kit for detecting sheep paratuberculosis antibody and application

Abstract

The invention provides an ELISA kit for detecting a sheep paratuberculosis antibody and application, and relates to the field of biological detection.The ELISA kit comprises the following components: a mycobacterium paratuberculosis specific diagnosis antigen (the amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2); coating a buffer solution; a confining liquid; a serum diluent; an enzyme-labeled second antibody diluent; a substrate solution; a stop solution; positive control serum, negative control serum and the like; compared with the prior art, the kit provided by the invention is good in specificity, and the false positive rate is reduced; clinical data show that the positive detection rate 43.78% of the kit is obviously higher than that of an imported kit of ID Vet (6.22%) and IDEXX (3.43%), which indicates that the sensitivity is higher, and infected sheep can be detected earlier and more accurately.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an ELISA kit for detecting sheep paratuberculosis antibodies and its application. Background Art

[0002] Paratuberculosis is caused by Mycobacterium paratuberculosis ( Mycobacterium avium subsp . paratuberculosis MAP (Mesenteric Mycoplasma Enteritis) causes chronic granulomatous enteritis in a variety of animal species. It is a "neglected disease" of ruminants and known as the "invisible killer" of small ruminants. It is characterized by progressive emaciation, persistent diarrhea, granulomatous enteritis, and proliferative inflammation of the mesenteric lymph nodes, ultimately leading to death. It has been recognized by the World Organization for Animal Health (WOAH) as a major global animal health issue and is included in the WOAH List of Diseases, Infections, and Infestations. Current Chinese regulations classify it as a Category III animal disease.

[0003] Paratuberculosis (MAP) is prevalent worldwide and is considered a significant infectious disease affecting both the livestock industry and public health. It is primarily transmitted through the fecal-oral route. MAP persists in soil, water, and forage, leading to prolonged exposure of animals to infection and increasing the risk of disease in susceptible individuals. Humans can become infected with MAP by drinking surface water contaminated with MAP or consuming contaminated animal products, such as meat and dairy. MAP has been reported to cause Crohn's disease in humans and is also associated with rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, multiple sclerosis, and autism. Growing evidence indicates that MAP is a zoonotic pathogen. Currently, there are no effective treatments or commercially available vaccines for the disease. Clinical interventions can only provide temporary relief through symptomatic treatment, and there have been no reports of successful cures for infected individuals. Therefore, the primary strategy for preventing and controlling the disease is early diagnosis, isolation, and elimination.

[0004] However, current diagnostic challenges include hidden clinical symptoms (such as absence of diarrhea, intermittent diarrhea, or only loose stools), delayed onset of characteristic lesions (e.g., typical lesions appear in young individuals, typically over 2 years of age in cattle and 1.5 years in sheep), intermittent intestinal excretion, time-sensitive gold standard pathogen culture (e.g., growth of bacteria over 6 weeks after inoculation for type II MAP and over 6 months for type I MAP), transient and uncertain bacteremia, and low specificity and sensitivity of serum antibody testing. Diagnosis of this disease is challenging, especially early diagnosis. Current consensus suggests that diagnosis of paratuberculosis requires a combination of multiple materials and methods.

[0005] Currently, the specificity of ELISA for detecting paratuberculosis is 48%-92%, and the sensitivity is 50%-70%. According to research, sheep anti-MAP antibodies appear as early as 75 days after infection (sheep animal test in the laboratory) or 8 weeks (reported abroad), and are intermittently or continuously positive in antibody reaction. The difference in ELISA detection results of anti-MAP antibodies is mainly due to the variability of the humoral immunity of infected animals and the antigen in the ELISA kit, including commercial kits. Therefore, it is of great significance to screen early immune response proteins and MAP specific antigens. SUMMARY

[0006] Based on the above background, the present application provides an ELISA kit for detecting sheep paratuberculosis antibodies and application, which can detect MAP infected sheep in the early stage of the disease, and provides technical and data support for the diagnosis and research of sheep paratuberculosis, as follows: An ELISA kit for detecting sheep paratuberculosis antibodies comprises the following components: Mycobacterium paratuberculosis specific detection antigen, the amino acid sequence of the Mycobacterium paratuberculosis specific detection antigen is shown as SEQ ID NO: 1; the nucleotide sequence of the Mycobacterium paratuberculosis specific detection antigen is shown as SEQ ID NO: 2; coating buffer; blocking solution; serum diluent; enzyme-labeled secondary antibody diluent; substrate solution; stop solution; positive control serum and negative control serum.

[0007] Further, the blocking solution is a PBST solution containing 2%-3% BSA; the serum diluent is a PBST solution containing 1% BSA; the enzyme-labeled secondary antibody diluent is a HRP-labeled rabbit anti-sheep IgG antibody, and the dilution factor is 1:5000-1:10000; the substrate solution is a TMB solution, and the stop solution is concentrated sulfuric acid.

[0008] Further, for early detection of sheep paratuberculosis, when in use, the coating concentration of the Mycobacterium paratuberculosis specific detection antigen is 47-63 ng / well; the serum diluent dilutes the serum to be tested by 1:100-200 in volume ratio.

[0009] Further, the coating condition of the Mycobacterium paratuberculosis specific detection antigen is 37℃ incubation for 1-2 hours; and the coating buffer is a carbonate buffer with pH=9.6.

[0010] Furthermore, the blocking time of the blocking solution is 60 minutes; the amount of the blocking solution used is 200 μL / well.

[0011] Furthermore, the serum diluted with the serum diluent is incubated with the coated Mycobacterium paratuberculosis specific detection antigen at 37° C. for 60 minutes.

[0012] Furthermore, the incubation time of the enzyme-labeled secondary antibody was 45 minutes.

[0013] Furthermore, the color development time of the substrate solution is 5-10 minutes, and the color development is carried out under light-proof conditions.

[0014] Furthermore, when the OD of the serum sample to be tested is 450nm When the value is ≤0.489, it is judged as negative; OD 450nm When the value is ≥0.570, it is judged as positive; when 0.489 <OD 450nm When the value was <0.570, it was judged as suspected positive.

[0015] In another aspect, the present invention provides a method for using an ELISA kit for detecting sheep paratuberculosis antibodies, comprising the following steps: S1. Antigen coating: Dilute the paratuberculosis-specific detection antigen protein to 10 μg / mL with coating buffer, add 100 μL / well to a 96-well ELISA plate, and coat at 4°C for 16 h. S2. Blocking: Discard the liquid in the wells, wash three times with 1× PBST and pat dry, add 200 μL / well of blocking solution, and block at 37°C for 1 h; S3. Primary antibody incubation: Discard the liquid in the wells, wash three times with 1× PBST and pat dry, add serum diluent to dilute the test serum, 100 μL / well, and incubate at 37°C for 1 hour; the serum diluent is the primary antibody; S4. Secondary antibody incubation: discard the liquid in the wells, wash three times with 1× PBST and pat dry, add HRP-rabbit anti-goat IgG diluted 1:10000 in serum diluent, and incubate at 37°C for 45 min; S5. Color development and OD measurement 450nm : Discard the liquid in the wells, wash with 1×PBST 5 times, pat dry once, add 100μL / well of substrate solution, incubate in the dark for 10 minutes, stop the color development with stop solution and measure OD 450nm Absorbance value at S6. Result determination: When the OD of the serum sample to be tested 450nm When the value is ≤0.489, it is judged as negative; OD 450nm When the value is ≥0.570, it is judged as positive; when 0.489 <OD 450nm When the value was <0.570, it was judged as suspected positive.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The ELISA kit provided by the present invention uses a specific Mycobacterium paratuberculosis-specific diagnostic antigen, which can more accurately bind to sheep paratuberculosis antibodies, reduce cross-reactions with antibodies of other similar pathogens, and thus improve the specificity of detection. Compared with existing technologies, this kit has good specificity and reduces the false positive rate. Clinical data show that the positive detection rate of this kit is 43.78%, which is significantly higher than the imported kits of ID Vet (6.22%) and IDEXX (3.43%), indicating that it has higher sensitivity and can detect infected sheep earlier and more accurately.

[0017] 2. The ELISA kit provided by the present invention can efficiently detect samples with different antibody levels. Sensitivity test data show that the kit can stably detect strongly positive, moderately positive, and weakly positive sera. In particular, the detection ability of the kit for weakly positive samples is significantly better than that of existing commercial kits (such as ID Vet and IDEXX). It can still clearly distinguish between positive and negative samples near the critical value, and the positive rate of clinical sample detection is much higher than that of the comparative commercial kits.

[0018] 3. The kit provided by the present invention uses a Mycobacterium paratuberculosis-specific diagnostic antigen that can effectively identify low-concentration antibodies produced in the early stages of infection. Experimental data show that the kit can still detect positive signals when the serum is diluted at a multiple of 1:800, indicating that it is highly sensitive to samples with low antibody levels and can detect positive results in the early stages of infection. This provides a reliable tool for early diagnosis, helps to promptly detect infected sheep before the disease spreads on a large scale, and reduces economic losses in the animal husbandry industry. At the same time, because Mycobacterium paratuberculosis is a zoonotic pathogen, controlling animal infection helps reduce the possibility of human infection.

[0019] 4. The positive detection rate of the kit provided by the present invention is significantly higher than that of the imported ID Vet and IDEXX kits. Experiments show that in 466 field sheep serum tests, the positive rate of this kit is 43.78%, while that of ID Vet and IDEXX is only 6.22% and 3.43%, respectively. In addition, in experiments on sheep artificially infected with MAP, the positive detection rate of this kit is 42.86%, far higher than that of ID Vet (10.71%) and IDEXX (8.33%), demonstrating the significant improvement in the sensitivity of this kit.

[0020] 5、The kit provided by the application significantly reduces the risk of false negative, experimental data show that the repeated detection results of weak positive serum show that 6 detections of the kit are positive, while ID Vet and IDEXX appear 1 and multiple false negative or suspected results, which verifies the stability and reliability of the kit provided by the application, and further verifies the sensitivity of the application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 SDS-PAGE results of paratuberculosis specific diagnostic antigen protein induced expression; Figure 2 Paratuberculosis specific diagnostic antigen protein pET28a-MPtb imidazole concentration exploration results; Figure 3 Western blot results of paratuberculosis specific diagnostic antigen protein pET28a-MPtb; Figure 4 Results of diffuse granulomatous lesions of the ileal lamina propria; Figure 5 MAP culture colony morphology. DETAILED DESCRIPTION

[0022] Example 1 Construction scheme of prokaryotic expression vector In this embodiment, focusing on the research of paratuberculosis Mycobacterium tuberculosis specific diagnostic antigen, we selected pET-28a vector as the basis for the experiment, and named the modified vector as pET28a-MPtb for subsequent identification and research.

[0023] The paratuberculosis Mycobacterium tuberculosis specific diagnostic antigen was selected as the target protein for the construction of the prokaryotic expression vector. The nucleotide sequence of the antigen is shown in SEQ ID NO: 2, and the corresponding amino acid sequence is shown in SEQ ID NO: 1.

[0024] The construction scheme is to insert the target protein into the pET-28a prokaryotic expression vector, and add 6His tags at both ends of the target gene. Nde I and Bam H I restriction sites, that is, the construction form is pET-28a+ 6His+ Nde I+ target protein+ Bam H I. S-Tag is a new type of recombinant protein fusion peptide tag.

[0025] The sequence of the upstream primer F used to amplify the target gene is shown in SEQ ID NO: 3, and the sequence of the downstream primer R is shown in SEQ ID NO: 4.

[0026] Example 2 Transformation of the recombinant vector into Escherichia coli BL21-DE3 (1) Add 1 μL of pET28a-MPtb vector to 100 μL of competent bacteria and place on ice for 20 minutes.

[0027] (2) Heat shock at 42°C for 90 seconds, quickly place on ice for 5 minutes, and add 600 μL LB culture medium.

[0028] (3) Shake at 37°C, 180 rpm for 1 hour. After centrifugation, spread the entire plate onto an LB plate containing 50 μg / mL kanamycin (Kana) and invert and culture at 37°C overnight.

[0029] In order to obtain sufficient amount of recombinant protein, the constructed pET28a-MPtb vector was transformed into Escherichia coli BL21-DE3 competent cells. Conversion steps: Mix the plasmid with competent cells: Add 1 μL of pET28a-MPtb vector to 100 μL of competent E. coli BL21-DE3 cells, mix gently, and place on ice for 20 minutes to allow the plasmid to fully adsorb to the cell surface.

[0030] Heat shock treatment: Heat shock the mixture at 42°C for 90 seconds to promote the entry of the plasmid into the cells. Then quickly transfer the centrifuge tube to ice and let it stand for 5 minutes to cool the cells and prevent them from excessive damage.

[0031] Recovery and culture: Add 600 μL of LB culture medium (without antibiotics) to the centrifuge tube, mix gently, and then shake and culture in a shaker at 37°C and 180 rpm for 1 hour to allow the cells to recover and express the resistance protein.

[0032] Spreading and screening: Centrifuge the cultured cell suspension, discard some of the supernatant, resuspend the cells, and spread all of the cells on LB solid medium plates containing 50 μg / mL Kana. Place the plates upside down in a 37°C constant temperature incubator and culture overnight to screen for positive clones successfully transferred into the pET28a-MPtb vector.

[0033] Through the above steps, a prokaryotic expression vector containing the paratuberculosis-specific diagnostic antigen gene was successfully constructed and transformed into Escherichia coli BL21-DE3, laying the foundation for subsequent protein expression and purification.

[0034] Example 3 Expression and Identification of Recombinant Fusion Protein Induced by IPTG Take 250mL and 1000mL conical flasks, sterilize them by high pressure, and place them in a clean bench for ultraviolet irradiation for 30 minutes. Add 50mL of LB medium containing 50μg / mL kanamycin to the 250mL conical flask, take the positive bacteria and add them at a ratio of 1:100, and culture them in a shaking incubator at 37℃ and 220r / min overnight. The next day, take the positive bacteria after shaking and mix them with 50% glycerol at a ratio of 1:1 and freeze them in a -80℃ refrigerator for future use. Add them at a ratio of 1:1000 to 300mL of LB broth containing kanamycin with a final concentration of 50μg / mL, and culture them at 37℃ with shaking until the OD 600nm When the pH value is 0.6-0.8, add IPTG to a final concentration of 1.0 mmol / L and induce at 37°C and 180 rpm for 8 hours. Solubility analysis is performed by the following operation: (1) After induction, centrifuge the bacterial solution at 7000 r / min for 15 min to collect the cells and weigh them; (2) Add 10 mL of Binding Buffer per 1 g of cells and resuspend; (3) Ultrasonic disruption of the resuspended cells in an ice bath for 25 min (150W, 3s on, 5s off); (4) After ultrasonic disruption, centrifuge the cells at 4°C and 12,000 rpm for 15 min to collect the supernatant and precipitate. Add Binding Buffer containing 8 M urea to the precipitate and incubate at 4°C overnight. (5) Take 8 μL of supernatant and 8 μL of precipitate, add 2 μL of protein loading buffer (5×), and boil in a water bath at 100°C for 10 min.

[0035] Perform SDS-PAGE gel electrophoresis analysis: Prepare protein gels according to the instructions of the SDS-PAGE rapid gel preparation kit. To ensure the accuracy and reliability of the experimental results, set up multiple control groups: induced and uninduced E. coli samples, induced and uninduced E. coli containing the pET-30a empty vector, and supernatant and precipitate samples after uninduced ultrasonic disruption of positive bacteria.

[0036] Take 10 μL of the treated sample from each well and add it to the sample. Then perform SDS-PAGE gel electrophoresis. After the electrophoresis, place the protein gel in Coomassie Brilliant Blue dye solution for 20 minutes. Then add destaining solution and change the destaining solution several times and shake until the background is clear. After the destaining is completed, observe the protein gel. Figure 1(M: protein molecular weight standard; 1: whole Escherichia coli without induction; 2: whole Escherichia coli with induction; 3: pET28a empty vector without induction; 4: pET28a empty vector with induction; 5: supernatant of pET28a-MPtb without induction; 6: precipitate of pET28a-MPtb without induction; 7: supernatant of pET28a-MPtb with induction; 8: precipitate of pET28a-MPtb with induction). The size of the paratuberculosis-specific diagnostic antigen protein after induction was 21.6 kDa, which was consistent with the expected size, and coexisted in a soluble form.

[0037] Example 4 Ni column affinity chromatography purification, concentration and concentration determination After optimizing the inducible expression conditions for the recombinant protein, we used a HyPur T Ni-NTA 6FF (His-Tag) pre-packed gravity column produced by Sangon Biotech (Shanghai) Co., Ltd. to purify the recombinant protein. The specific steps are as follows: (1) Carry out experiments based on the optimized induction conditions and finally obtain a supernatant solution containing recombinant protein.

[0038] (2) Filter the supernatant solution using a filter membrane with a pore size of 0.45 μm to remove any impurities that may be present in the solution and prevent them from interfering with subsequent purification steps.

[0039] (3) Wash the purification column with 5 times the column volume of ultrapure water to remove possible impurities and residual substances in the purification column; then balance the purification column with 5 times the column volume of Binding Buffer to make the purification column reach a state suitable for binding to the target protein; slowly add the filtered supernatant solution to the purification column, collect the flow-through at the same time, and apply the flow-through to the column again. Repeat the filtration 3 times to ensure that the target protein can fully bind to the purification column; finally, add the filtered flow-through back to the column and place it in a 4°C environment overnight to allow the target protein to have enough time to bind to the purification column.

[0040] (4) The next day, the flow-through was slowly filtered through the purification column three times, and the purification column was eluted with Wash Buffer containing different concentrations of imidazole. The eluate was collected and reserved for subsequent SDS-PAGE analysis to determine the elution of the target protein.

[0041] (5) Transfer the filtrate containing 250 mM imidazole and the collected filtrate to an ultrafiltration tube. Adjust the centrifuge parameters to: temperature 4°C, speed 3000 r / min, centrifugation time 30 min. When the volume of the solution in the ultrafiltration tube is reduced to 1 mL, stop the centrifugation, add 9 mL of PBS solution to the ultrafiltration tube, continue the centrifugation operation, repeat the ultrafiltration 3 times to complete the replacement of the protein buffer, and finally collect 1 mL of protein concentrate.

[0042] (6) Follow the instructions of the BCA protein quantification kit to detect the protein sample at OD 562nm The standard curve was constructed by measuring the absorbance at OD , and the calculation equation was y = 1079.7x - 12.079 (where x is the protein at OD 562nm The recombinant protein after purification and ultrafiltration was also tested and its absorbance at OD 562nm The absorbance at 1.400 was 1.400, and the result was substituted into the above standard curve formula for calculation. The result showed that the protein concentration after ultrafiltration was 1500 μg / mL.

[0043] (7) The purified and concentrated protein samples were subjected to SDS-PAGE analysis to further verify the purity and molecular weight of the protein. The relevant results are as follows: Figure 2 As shown (M: protein molecular weight standard; 1: protein column; 2-8: 100, 150, 200, 250, 300, 400, 500 mmol / L imidazole elution), from Figure 2 It can be seen that 250mmol / L imidazole concentration is the optimal condition for eluting the target protein, which can obtain paratuberculosis-specific diagnostic antigen protein efficiently and with high purity.

[0044] Example 5 Western bolt analysis of recombinant protein After purification and concentration, the paratuberculosis specific diagnostic antigen protein was diluted and mixed with protein loading buffer at a ratio of 4:1. The protein was then incubated at 100°C in a water bath for 10 minutes and set aside for later use. Western Blot analysis of the Mycobacterium paratuberculosis specific diagnostic antigen protein was performed as follows: (1) SDS-PAGE: Load 10 µL of the prepared protein sample into each well for SDS-PAGE. (Run electrophoresis at 90V for 30 minutes until the protein sample reaches the boundary between the stacking gel and the separating gel, then switch to 120V for 60 minutes). (2) Transfer: After electrophoresis, remove the top layer of concentrated gel and cut the appropriate PVDF membrane according to the size of the separation gel. Arrange them in the order of "sponge-filter paper-PVDF membrane-gel-filter paper-sponge". Place them in a vertical electrophoresis tank with an ice box and transfer solution, and transfer the membrane at a constant current of 250mA for 60 minutes. (3) Blocking: After the transfer is completed, remove the PVDF membrane and wash it on a shaker with 1×TBST for 5 times, each time for 5 minutes. After washing, place the PVDF in blocking solution at 4°C overnight; (4) Primary antibody incubation: The next day, the blocked PVDF membrane was removed and washed with 1x TBST for 5 times, 5 min each time. The PVDF membrane was cut according to the lane position and placed in different antibodies and serum diluted by blocking solution, and incubated at room temperature for 2 h; (5) Secondary antibody incubation: The PVDF membrane was washed with 1x TBST for 5 times, 5 min each time, and then placed in enzyme-labeled secondary antibody diluted by TBST, and incubated at room temperature for 2 h; (6) Color development: The PVDF membrane was washed with 1x TBST for 5 times, 5 min each time, and then ECL color development was added in the dark room. The results were observed using an imaging instrument.

[0045] The results of recombinant protein WB identification are shown in Figure 3 (M: protein molecular weight standard; 1: His mouse tag antibody; 2: paratuberculosis positive sheep serum; 3: sheep negative serum), the paratuberculosis specific diagnostic antigen protein specifically binds to the mouse His tag antibody and the paratuberculosis positive serum at 21.6 kDa, and does not bind to the paratuberculosis negative serum, which not only verifies that the recombinant protein is correctly expressed and has a His tag, and the molecular weight is as expected, but also indicates that it can be recognized by specific antibodies in the paratuberculosis positive serum, has the potential to be used as a paratuberculosis mycobacterium specific diagnostic antigen, has good specificity, and can effectively avoid false positives, providing a strong basis for paratuberculosis diagnosis.

[0046] Example 6 ELISA test Antigen coating: The purified paratuberculosis specific diagnostic antigen protein was diluted to 10 μg / mL with ELISA coating solution, 100 μL / well was added to a 96-well enzyme-labeled plate, and 4°C coating was performed for 16 h; Blocking: Discard the liquid in the wells, wash with 1x PBST for 3 times, and tap dry for the last time. Add blocking solution (2% BSA PBST solution) 200 μL / well, and block at 37°C for 2 h; Primary antibody incubation: Discard the liquid in the wells, wash with 1x PBST for 3 times, and tap dry for the last time. Add diluent (1% BSA PBST solution) to dilute the serum to be tested, 100 μL / well, and incubate at 37°C for 1 h; Secondary antibody incubation: Discard the liquid in the wells, wash with 1x PBST for 3 times, and tap dry for the last time. Add 1:10000 HRP-rabbit anti-sheep IgG diluted with diluent (1% BSA PBST solution), and incubate at 37°C for 45 min; Color development and OD determination 450nm : Discard the liquid in the wells, wash with 1x PBST for 5 times, and tap dry for the last time. Add TMB substrate solution 100 μL / well, incubate at room temperature for 10 min, and terminate the color development after adding the termination solution to determine the OD 450nm absorbance.

[0047] (1) The optimal antigen coating concentration and serum dilution The paratuberculosis specific diagnostic antigen protein was coated with coating solution at 2000 ng / well, 1500 ng / well, 1000 ng / well, 750 ng / well, 500 ng / well, 375 ng / well, 250 ng / well, 186 ng / well, 125 ng / well, 94 ng / well, 63 ng / well, and 47 ng / well, with two repeated wells for each concentration. The primary antibody (goat negative and paratuberculosis goat positive serum) was diluted with antibody diluent at 1:100, 1:200, 1:400, and 1:800, with two repeated wells for each concentration. The OD 450nm value was read by the microplate reader, and the P / N value was calculated (Table 1). The antigen coating concentration and serum dilution at the maximum P / N value were optimal. The serum dilution of 1:200 and the antigen coating concentration of 47 ng were optimal.

[0048] Table 1 Determination of antigen coating concentration and serum dilution

[0049] (2) Determination of optimal coating temperature and time Four different coating temperatures and times were set, and the OD 450nm value was read by the microplate reader, and a table was established for data analysis. The results are shown in Table 2. When the antigen was coated at 37°C for 1 h, the P / N value was maximized at 2.316, and the optimal conditions were determined to be coating at 37°C for 1 h.

[0050] Table 2 Results of optimal antigen coating temperature and time

[0051] (3) Determination of optimal blocking solution Four kinds of blocking solutions were selected, and the OD 450nm value was read by the microplate reader, and a table was established for data analysis. The results are shown in Table 3. When 2% BSA blocking solution was used, the P / N value was maximized at 4.522, and the optimal blocking solution was determined to be 2% BSA.

[0052] Table 3 Results of optimal blocking solution

[0053] (4) Optimization of optimal blocking time According to the above optimized indirect ELISA operation steps, four different blocking times were selected for blocking, and the OD 450nm value was read by the microplate reader, and a table was established for data analysis. The results are shown in Table 4. When the blocking time was 60 min, the P / N value was maximized at 3.017, and the optimal blocking time was determined to be 60 min.

[0054] Table 4 Optimal sealing time results

[0055] (5) Optimal primary antibody incubation time According to the above optimized conditions, set 4 serum incubation times and read the OD value of the microplate reader. 450nm The P / N value was calculated and analyzed. The results are shown in Table 5. When the blocking condition was 60 minutes, the maximum P / N value was 3.151, and the optimal incubation time for the primary antibody was determined to be 60 minutes.

[0056] Table 5 Optimal serum incubation time results

[0057] (6) Optimal dilution of secondary antibody According to the above optimized conditions, set the dilution multiples of 6 enzyme-labeled secondary antibodies and read the OD value of the microplate reader. 450nm The P / N value was calculated and a table was constructed for analysis. The results are shown in Table 6. When the enzyme-labeled secondary antibody was diluted at a multiple of 1:10,000, the maximum P / N value was 5.738, confirming that a dilution multiple of 1:10,000 for the enzyme-labeled secondary antibody was the optimal condition.

[0058] Table 6 Optimal dilution of secondary antibodies

[0059] (7) Optimal secondary antibody incubation time According to the above optimized conditions, set 4 secondary antibody incubation times for incubation. Read the OD value on the microplate reader. 450nm The results are shown in Table 7. When the secondary antibody was incubated for 45 minutes, the maximum P / N value was 4.461, and the incubation time of the enzyme-labeled secondary antibody was determined to be 45 minutes.

[0060] Table 7 Optimal secondary antibody incubation time results

[0061] (8) Optimal color development time results Set 4 color development times for optimization and read the OD value on a microplate reader. 450nm The results are shown in Table 8. The effect is more obvious when the color development time is 5-10 minutes. When the color development time is 10 minutes, the maximum P / N value is 3.134. It is determined that the optimal color development time is 10 minutes.

[0062] Table 8 Optimal color development time results

[0063] (9) Determination of critical value According to the above optimized results, 30 negative serum samples of paratuberculosis sheep were selected for testing and the OD was measured using a microplate reader. 450nm The data were analyzed using a table with the values ​​of 0.3257 and the results are shown in Table 9. After statistical analysis, the mean (X) was 0.3257 and the standard deviation (SD) was 0.0816. Based on the critical value determination principle of "mean ± multiple standard deviation" in statistics, it was calculated that the negative judgment threshold was X + 2SD (0.3257 + 2 × 0.0816 ≈ 0.489) and the positive judgment threshold was X + 3SD (0.3257 + 3 × 0.0816 ≈ 0.570). Therefore, the judgment criteria are as follows: OD 450nm ≤ 0.489 is negative, OD 450nm ≥ 0.570 is positive, 0.489 <OD 450nm <0.570 was considered suspected positive.

[0064] Table 9 Determination of critical values

[0065] (10) Repeatability test ① Intra-batch repeatability test At the same time, three ELISA plates made from the same batch were used to test the serum, and the OD was measured using an ELISA reader. 450nm The data were analyzed using the same method. The coefficient of variation (CV) was calculated using the formula. The results are shown in Table 10. The highest intra-assay coefficient of variation was 18.66%, and the lowest was 8.56%.

[0066] Table 10 Intra-batch repeatability test results

[0067] ②Batch repeatability test At the same time, serum was tested using ELISA plates made from three different batches, and OD was measured using an ELISA reader. 450nm Data analysis was performed using the same values. The coefficient of variation (CV) was calculated according to the formula. The results are shown in Table 11. The highest inter-assay coefficient of variation was 14.61%, and the lowest was 4.96%, both below 15%.

[0068] Table 11 Results of inter-batch repeatability test

[0069] (11) Sensitivity test According to the optimized conditions, the positive serum of paratuberculosis sheep stored in the laboratory was diluted by 7 times and tested, and the OD was measured using a microplate reader. 450nm The results are shown in Table 12. When the serum dilution ratio is 1:800, OD 450nm The value is higher than the positive cut-off value.

[0070] Table 12 Sensitivity test results

[0071] The experimentally developed ELISA was compared with imported commercial kits from ID Vet and IDEXX. Six sera, including strongly positive, moderately positive, and weakly positive for paratuberculosis, as well as three negative sera, were tested six times. The results, as shown in Table 13, show that the ELISA developed in this invention showed positive results in all six tests of the three positive sera. However, the ID Vet and IDEXX assays showed different results for moderately positive and weakly positive sera.

[0072] Table 13 Positive serum test results

[0073] (12) Specificity test Detect 3 other common sheep disease sera (paratuberculosis test is negative), use the negative and positive sera of sheep paratuberculosis in the laboratory as control, and use a microplate reader to measure OD 450nm The results are shown in Table 14. The OD values ​​of the three positive sera for other diseases were 450nm The values ​​were all lower than the negative critical value, while those for paratuberculosis positive serum were higher than the positive critical value.

[0074] Table 14 Specificity test results

[0075] Example 7 Clinical Testing (1) Field samples 466 sheep sera were collected in the field and tested using the ELISA kit of the present invention, along with commercially available kits from ID Vet and IDEXX. The results, as shown in Table 15, show that the positive rate of the method developed in this study was 43.78%, while the results from the ID Vet and IDEXX kits were 6.22% and 3.43%, respectively.

[0076] Table 15 Field clinical sample test results

[0077] (2) Sheep artificially infected with MAP test samples Sera from 112 sheep artificially infected with MAP (2 control sheep + 6 infected sheep, each sampled 14 times) were tested using the ELISA kit of the present invention, along with commercially available kits from ID Vet and IDEXX. The results are shown in Tables 16 and 17: All three methods tested negative for sera collected from the negative control group of the animal experiment at all time periods; for the artificially infected group (6 sheep, pathologically observed), the sera were negative for MAP. Figure 4: Diffuse granulomatous lesions in the lamina propria of the ileum, accompanied by lymphocytes, epithelioid cells, and multinucleated giant cells, HE staining); Pathogen isolation and identification ( Figure 5 MAP culture colony morphology: the colonies appeared papillary, with a smooth surface, cream or light yellow in color, and irregular edges). Tissue nucleic acid testing identified the infection as positive, and animal experiments were successfully established. The ELISA kit of the present invention had a positive rate of 42.86% for 84 sera collected from 6 infected sheep, while the results of ID Vet and IDEXX kits were 10.71% and 8.33%, respectively. The earliest detection by the three detection methods was 75 days after MAP inoculation. Nine serum positivity tests using the ID Vet kit occurred in two sheep. Seven serum positivity tests using the IDEXX kit occurred in only one sheep (one of the sheep that tested positive with ID Vet). Thirty-six serum positivity tests using the ELISA kit of the present invention were distributed in four sheep (including two sheep that tested positive using the other two methods, of which two sheep tested positive continuously starting from 75 days after inoculation).

[0078] Table 16 Test results of artificially infected samples

[0079] Table 17 Detailed results of artificially infected sample testing

Claims

1. An ELISA kit for detecting sheep paratuberculosis antibodies, characterized in that: Contains the following components: A Mycobacterium paratuberculosis specific detection antigen, the amino acid sequence of which is shown in SEQ ID NO: 1; the nucleotide sequence of which is shown in SEQ ID NO: 2; coating buffer; Blocking solution; serum diluent; Enzyme-labeled secondary antibody diluent; Substrate solution; Stop solution; Positive control serum and negative control serum.

2. An ELISA kit for detecting sheep paratuberculosis antibodies according to claim 1, characterized in that, The blocking solution is a PBST solution containing 2%-3% BSA; the serum diluent is a PBST solution containing 1% BSA; the enzyme-labeled secondary antibody diluent is a rabbit anti-goat IgG antibody labeled with HRP, and the dilution multiple is 1:5000-1:10000; the substrate solution is TMB solution, and the stop solution is concentrated sulfuric acid.

3. The use of an ELISA kit for detecting sheep paratuberculosis antibodies according to claim 1 or 2, wherein: For the early detection of sheep paratuberculosis, when in use, the coating concentration of the Mycobacterium paratuberculosis specific detection antigen is 47-63 ng / well; the serum diluent dilutes the serum to be tested at a volume ratio of 1:100-200.

4. The use of an ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, wherein The coating condition of the Mycobacterium paratuberculosis specific detection antigen is incubation at 37° C. for 1-2 hours; and the coating buffer is a carbonate buffer with a pH of 9.

6.

5. The use of an ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, wherein: The blocking time of the blocking solution was 60 minutes; the amount of the blocking solution used was 200 μL / well.

6. The use of an ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, wherein: The serum diluted with the serum diluent is incubated with the coated Mycobacterium paratuberculosis specific detection antigen at 37° C. for 60 minutes.

7. The use of an ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, wherein: The incubation time for enzyme-labeled secondary antibody was 45 minutes.

8. The use of the ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, characterized in that, The color development time of the substrate solution is 5-10 minutes, and the color development is carried out under light-proof conditions.

9. The use of the ELISA kit for detecting sheep paratuberculosis antibodies as claimed in claim 3, characterized in that: When the OD of the serum sample to be tested 450nm When the value is ≤0.489, it is judged as negative; OD 450nm When the value is ≥0.570, it is judged as positive; when 0.489<OD 450nm When the value was < 0.570, it was judged as suspected positive.

10. The method for using the ELISA kit for detecting sheep paratuberculosis antibodies according to claim 1 or 2, wherein: The steps include: S1. Antigen coating: Dilute the paratuberculosis-specific detection antigen protein to 10 μg / mL with coating buffer, add 100 μL / well to a 96-well ELISA plate, and coat at 4°C for 16 h. S2. Blocking: Discard the liquid in the wells, wash three times with 1× PBST and pat dry, add 200 μL / well of blocking solution, and block at 37°C for 1 h; S3. Primary antibody incubation: Discard the liquid in the wells, wash three times with 1× PBST and pat dry, add serum diluent to dilute the test serum, 100 μL / well, and incubate at 37°C for 1 hour; the serum diluent is the primary antibody; S4. Secondary antibody incubation: discard the liquid in the wells, wash three times with 1× PBST and pat dry, add HRP-rabbit anti-goat IgG diluted 1:10000 in serum diluent, and incubate at 37°C for 45 min; S5. Color development and OD measurement 450nm : Discard the liquid in the wells, wash with 1×PBST 5 times, pat dry once, add 100μL / well of substrate solution, incubate in the dark for 10 minutes, stop the color development with stop solution and measure OD 450nm Absorbance value at S6. Result determination: When the OD of the serum sample to be tested 450nm When the value is ≤0.489, it is judged as negative; OD 450nm When the value is ≥0.570, it is judged as positive; when 0.489 < OD 450nm When the value was < 0.570, it was judged as suspected positive.

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