An elisa kit for detecting antibody of paratuberculosis of sheep and application thereof

Abstract

The application provides an ELISA kit for detecting ovine paratuberculosis antibodies and application, relates to the field of biological detection, and the ELISA kit comprises the following components: paratuberculosis mycobacterium specific diagnostic antigen (amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as 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; compared with prior art, the kit provided by the application has good specificity and reduces false positive rate; clinical data shows that the positive detection rate of the kit is 43.78%, which is significantly higher than that of imported kits of ID Vet (6.22%) and IDEXX (3.43%), indicating that the kit has higher sensitivity and can detect infected sheep earlier and more accurately.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to an ELISA kit for detecting antibodies against sheep paratuberculosis and its application. Background Technology

[0002] Paratuberculosis is caused by Mycobacterium paratuberculosis (Mycobacterium paratuberculosis) Mycobacterium avium subsp . paratuberculosis Mesenteric enteritis (MAP) causes chronic granulomatous enteritis in various animals and is considered a "neglected disease" in ruminants, often referred to as a "silent killer" of small ruminants. It is characterized by progressive weight loss, persistent diarrhea, granulomatous enteritis, and mesenteric lymphadenitis, ultimately leading to death. It is recognized as a major global health issue by the World Organisation for Animal Health (WOAH) and is listed in the WOAH List of Diseases, Infections and Corresponding Infections. Current Chinese regulations classify it as a Class III animal disease.

[0003] Paratuberculosis (MAP) is widespread globally and is considered a significant infectious disease affecting both livestock farming and public health. It is primarily transmitted via the fecal-oral route. MAP can survive for extended periods in soil, water, and feed, leading to prolonged exposure in animals and increasing the likelihood of infection in susceptible individuals. Humans can become infected by drinking MAP-contaminated surface water or consuming contaminated meat, dairy products, or other animal-derived products. 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. Increasing evidence suggests that MAP is a zoonotic pathogen. Currently, there are no specific treatments or commercially available vaccines for MAP. Clinical interventions only provide temporary symptomatic relief, and there are no reports of successfully curing infected individuals. Therefore, the primary strategy for controlling this disease is early diagnosis, early isolation, and culling.

[0004] However, current diagnostic challenges for this disease include the insidious nature of clinical symptoms (e.g., no diarrhea, intermittent diarrhea, or only loose stools), the delayed development of characteristic lesions (e.g., typical lesions appear in cattle over 2 years old and sheep over 1.5 years old after infection), the intermittent nature of intestinal bacterial shedding, the time-sensitive nature of gold standard pathogen culture (e.g., bacterial growth more than 6 weeks after type II MAP inoculation and more than 6 months after type I MAP inoculation), the transient and uncertain nature of bacteremia, and the low specificity and sensitivity of serum antibody testing. Diagnosing this disease is quite difficult, and early diagnosis is even more challenging. The current consensus is that diagnosing paratuberculosis requires a combination of various 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 the antibody reaction is positive intermittently or continuously. The difference in the 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:

[0007] An ELISA kit for detecting sheep paratuberculosis antibodies comprises the following components:

[0008] 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;

[0009] Coating buffer;

[0010] Blocking solution;

[0011] Serum diluent;

[0012] Enzyme-labeled secondary antibody diluent;

[0013] Substrate solution;

[0014] Termination solution;

[0015] Positive control serum and negative control serum.

[0016] 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 termination solution is concentrated sulfuric acid.

[0017] 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.

[0018] Further, the coating condition of the paratuberculosis Mycobacterium specific detection antigen is 37℃ incubation for 1-2 hours; the coating buffer is a carbonate buffer with pH=9.6.

[0019] Further, the blocking time of the blocking solution is 60 minutes; the use amount of the blocking solution is 200 μL / well.

[0020] Further, the serum diluted by the serum diluent is incubated with the coated paratuberculosis Mycobacterium specific detection antigen at 37℃ for 60 minutes.

[0021] Further, the incubation time of the enzyme-labeled secondary antibody is 45 minutes.

[0022] Further, the color developing time of the substrate solution is 5-10 minutes, and the color developing is carried out under light shielding condition.

[0023] Further, when the OD 450nm value of the serum sample to be detected is ≤0.489, it is determined as negative; when the OD 450nm value is ≥0.570, it is determined as positive; and when 0.489<OD 450nm <0.570, it is determined as suspected positive.

[0024] In another aspect, the present application provides a use method of an ELISA kit for detecting sheep paratuberculosis antibody, comprising the following steps:

[0025] S1, antigen coating: the paratuberculosis specific detection antigen protein is diluted to 10 μg / mL with coating buffer, 100 μL / well is added to a 96-well enzyme-labeled plate, and 4℃ coating is carried out for 16 hours;

[0026] S2, blocking: the liquid in the hole is discarded, 1×PBST is washed for 3 times and dried, 200 μL / well of blocking solution is added, and 37℃ blocking is carried out for 1 hour;

[0027] S3, primary antibody incubation: the liquid in the hole is discarded, 1×PBST is washed for 3 times and dried, the serum to be detected is diluted with serum diluent, 100 μL / well is added, and 37℃ incubation is carried out for 1 hour; the serum diluent is a primary antibody;

[0028] S4, secondary antibody incubation: the liquid in the hole is discarded, 1×PBST is washed for 3 times and dried, 1:10000 HRP-rabbit anti-sheep IgG diluted by serum diluent is added, and 37℃ incubation is carried out for 45 minutes;

[0029] S5, color development and determination of OD 450nm : the liquid in the hole is discarded, 1×PBST is washed for 5 times, once dried, 100 μL / well of substrate solution is added, incubated in the dark for 10 minutes, and the OD 450nm value is determined after the color development is terminated by the termination solution.

[0030] S6, result determination: when the OD value of the serum sample to be tested is less than or equal to 0.489, it is determined to be negative; when the OD value is greater than or equal to 0.570, it is determined to be positive; when 0.489 < OD value < 0.570, it is determined to be suspected positive. 450nm 450nm 450nm

[0031] Compared with the prior art, the present application has the beneficial effects that:

[0032] 1. The ELISA kit provided by the present application uses specific Myco. paratuberculosis specific diagnostic antigens, which can more accurately bind to sheep paratuberculosis antibodies, reduce cross-reactions with other similar pathogen antibodies, and improve the specificity of detection. Compared with the prior art, the kit has good specificity and reduces the false positive rate; clinical data shows that the positive detection rate of the kit is 43.78%, which is significantly higher than that of 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.

[0033] 2. The ELISA kit provided by the present application can efficiently detect samples with different antibody levels. Sensitivity test experimental data shows that the kit can stably detect strongly positive, moderately positive and weakly positive serum, especially the detection ability of weakly positive samples is significantly better than that of existing commercial kits (such as ID Vet and IDEXX), and it can still clearly distinguish positive and negative samples near the critical value, and the positive rate of clinical sample detection is much higher than that of the compared commercial kits.

[0034] 3. The kit provided by the present application uses Myco. paratuberculosis specific diagnostic antigens that can effectively recognize low-concentration antibodies produced in the early stage of infection. Experimental data shows that the kit can still detect positive signals when the serum dilution multiple is 1:800, indicating that it has high sensitivity to low antibody level samples and can detect positive results in the early stage of infection, providing a reliable tool for early diagnosis and helping to detect infected sheep in time when the disease has not yet spread on a large scale, reducing the economic loss of animal husbandry; at the same time, because Myco. paratuberculosis is a zoonotic pathogen, controlling animal infection helps to reduce the possibility of human infection.

[0035] ​​​4、The positive detection rate of the kit provided by the application is significantly higher than that of the ID Vet and IDEXX imported kits, experiments show that in the detection of 466 field sheep sera, the positive rate of the kit is 43.78%, while the positive rates of the ID Vet and IDEXX are only 6.22% and 3.43% respectively; in addition, in the sheep experiment artificially infected with MAP, the positive detection rate of the kit is 42.86%, which is much higher than that of the ID Vet (10.71%) and the IDEXX (8.33%), proving that the kit has significantly improved sensitivity.

[0036] 5、The kit provided by the application significantly reduces the risk of false negative, experimental data shows that the repeated detection results of weak positive serum show that the kit is positive in 6 times, while the ID Vet and the IDEXX appear false negative or suspected results for 1 time and multiple times respectively, verifying the stability and reliability of the kit provided by the application, and further verifying the sensitivity of the application. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 SDS-PAGE results of paratuberculosis specific diagnostic antigen protein induced expression;

[0038] Figure 2 Paratuberculosis specific diagnostic antigen protein pET28a-MPtb imidazole concentration exploration results;

[0039] Figure 3 Western blot results of paratuberculosis specific diagnostic antigen protein pET28a-MPtb;

[0040] Figure 4 Ileum lamina propria diffuse granulomatous lesion results;

[0041] Figure 5 MAP culture colony morphology. DETAILED DESCRIPTION

[0042] Example 1 Construction scheme of prokaryotic expression vector

[0043] In this embodiment, the research on paratuberculosis Mycobacterium tuberculosis specific diagnostic antigen is focused on, and the pET-28a vector is selected as the basis for the experiment to construct a suitable prokaryotic expression vector. In order to facilitate subsequent description and research identification, the modified vector is named pET28a-MPtb.

[0044] The paratuberculosis Mycobacterium tuberculosis specific diagnostic antigen is 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.

[0045] The construction scheme is to insert the target protein into the pET-28a prokaryotic expression vector, and add a 6His tag, and introduce Nde Iand Bam H I enzyme digestion sites at both ends of the target gene, 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.

[0046] The sequence of the upstream primer F used for amplifying 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.

[0047] Example 2: Transformation of recombinant vector into E. coli BL21-DE3

[0048] (1) Add 1 μL of pET28a-MPtb vector to 100 μL of competent bacteria, and place on ice for 20 min.

[0049] (2) Heat shock at 42°C for 90 s, quickly place in ice for 5 min, and add 600 μL of LB culture solution.

[0050] (3) 37°C, 180 r / min shaking for 1 h, after centrifugation, all were coated on LB plate containing 50 μg / mL kanamycin (Kana), 37°C inverted culture overnight.

[0051] In order to obtain sufficient amount of recombinant protein, the constructed pET28a-MPtb vector was transformed into E. coli BL21-DE3 competent cells,

[0052] Transformation steps:

[0053] Plasmid and competent cells were mixed: 1 μL of pET28a-MPtb vector was added to 100 μL of competent E. coli BL21-DE3 cells, and gently mixed and placed on ice for 20 minutes to allow the plasmid to be fully adsorbed to the cell surface.

[0054] Heat shock treatment: the mixture was heat shocked at 42°C for 90 seconds to promote the plasmid to enter the cell interior, and then the centrifuge tube was quickly transferred to ice for 5 minutes to cool the cells and prevent excessive damage.

[0055] Recovery and culture: 600 μL of LB culture solution (without antibiotics) was added to the centrifuge tube, gently mixed, and then placed in a shaking incubator at 37°C, 180 r / min for 1 hour to recover the cells and express the resistant protein.

[0056] Coating and screening: centrifuge the cell suspension after culture, resuspend the cells after discarding part of the supernatant, and coat them all on LB solid medium plates containing 50 μg / mL Kana, and place the plates in a 37°C constant temperature incubator overnight to culture, to screen out positive clones successfully transformed with pET28a-MPtb vector.

[0057] By the above steps, the prokaryotic expression vector containing the paratuberculosis specific diagnostic antigen gene is successfully constructed, and it is transformed into E. coli BL21-DE3, which lays a foundation for subsequent protein expression and purification.

[0058] Example 3 IPTG induced expression of recombinant bacteria fusion protein

[0059] Take 250 mL and 1000 mL conical flasks for high pressure sterilization, and place them in the ultraclean bench for ultraviolet irradiation for 30 min, add 50 mL of LB medium containing 50 μg / mL kanamycin to the 250 mL conical flask, take the positive bacteria at a ratio of 1:100, and culture in a shaking incubator at 37°C, 220 r / min overnight. Take the positive bacteria after shaking (the remaining bacteria are mixed with 50% glycerol at a ratio of 1:1 and stored in a -80°C refrigerator for standby) every other day, add 300 mL of LB broth containing 50 μg / mL kanamycin to the 300 mL conical flask, and shake culture at 37°C, 220 r / min until the OD 600nm is 0.6-0.8, add IPTG with a final concentration of 1.0 mmol / L, and induce at 37°C, 180 r / min for 8 h. Perform solubility analysis by the following operations:

[0060] (1) Centrifuge the bacteria at 7000 r / min for 15 min after induction, and weigh the bacteria;

[0061] (2) Add 10 mL of Binding Buffer to each 1 g of bacteria for resuspension;

[0062] (3) Resuspend the bacteria in an ice bath for 25 min (150 w, work for 3 s and stop for 5 s);

[0063] (4) Centrifuge the bacteria after ultrasonic disruption at 4°C, 12000 r / min for 15 min to collect the supernatant and precipitate, and add Binding Buffer containing 8M urea to the precipitate at 4°C overnight;

[0064] (5) Take 8 μL of supernatant and precipitate, add 2 μL of protein loading buffer (5x), and boil in a water bath at 100°C for 10 min.

[0065] SDS-PAGE gel electrophoresis analysis: according to the SDS-PAGE gel preparation kit instructions to prepare protein gel. To ensure the accuracy and reliability of the experimental results, set up multiple control groups: E. coli induced and uninduced samples, E. coli containing pET-30a empty vector induced and uninduced samples, and positive bacteria uninduced supernatant and precipitate samples after ultrasonic disruption.

[0066] The above treated sample, 10 μL per well, was added, and then SDS-PAGE gel electrophoresis was carried out. After electrophoresis, the protein gel was stained in coomassie brilliant blue staining solution for 20 min, and then destaining solution was added. By replacing the destaining solution several times and shaking, the background was clear. After destaining, the protein gel was observed. The results are shown in Figure 1 (M: protein molecular weight marker; 1: E. coli whole bacteria uninduced; 2: E. coli whole bacteria induced; 3: pET28a empty vector uninduced; 4: pET28a empty vector induced; 5: pET28a-MPtb uninduced disruption supernatant; 6: pET28a-MPtb uninduced disruption precipitate; 7: pET28a-MPtb induced disruption supernatant; 8: pET28a-MPtb induced disruption precipitate), the size of the induced Mycobacterium tuberculosis specific diagnostic antigen protein is 21.6 kDa, which is consistent with the expected size, and exists in soluble form.

[0067] Example 4 Ni column affinity chromatography purification, concentration and concentration determination

[0068] After completing the optimization of the induction expression conditions of the recombinant protein, we selected HyPur T Ni-NTA 6FF (His-Tag) preloaded gravity column produced by Shenguo Bioengineering (Shanghai) Co., Ltd. to purify the recombinant protein, and the specific operation steps are as follows:

[0069] (1) According to the optimized induction conditions, the supernatant solution containing the recombinant protein was finally obtained.

[0070] (2) The above supernatant solution was filtered using a filter membrane with a pore size of 0.45 μm to remove impurities that may exist in the solution and avoid interference with the subsequent purification steps.

[0071] (3) Wash the purification column with 5 column volumes of ultrapure water to remove any impurities and residues that may be present in the purification column; then equilibrate the purification column with 5 column volumes of Binding Buffer to make the purification column suitable for binding the target protein; slowly add the filtered supernatant to the purification column, collect the flow-through at the same time, and put the flow-through back onto the column. Repeat this filtration process 3 times to ensure that the target protein can fully bind to the purification column; finally, add the filtered flow-through back into the column and place it in a 4°C environment overnight to allow the target protein sufficient time to bind to the purification column.

[0072] (4) The next day, the flow-through solution was slowly filtered through the purification column three times. The purification column was eluted with Wash Buffer containing different concentrations of imidazole. The eluent was collected and reserved for subsequent SDS-PAGE analysis to determine the elution status of the target protein.

[0073] (5) Transfer the filtrate containing 250mM imidazole and the filtrate collected thereafter to an ultrafiltration tube. Adjust the centrifuge parameters as follows: temperature 4℃, speed 3000r / min, centrifugation time 30min. When the volume of the solution in the ultrafiltration tube is reduced to 1mL, stop the centrifugation, add 9mL 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 1mL of protein concentrate.

[0074] (6) Follow the instructions of the BCA protein quantification kit to detect the protein sample at OD. 562nm A standard curve was constructed using the absorbance at OD, yielding the calculation equation y = 1079.7x - 12.079 (where x is the absorbance of the protein at OD). 562nm The absorbance at OD200 is denoted as y; y represents the protein concentration (μg / mL, R² = 0.9953). The purified ultrafiltration recombinant protein was then analyzed, and its absorbance at OD200 was also measured. 562nm The absorbance at the point was 1.400. Substituting this into the above standard curve formula, the result showed that the protein concentration after ultrafiltration was 1500 μg / mL.

[0075] (7) SDS-PAGE analysis was performed on the purified and concentrated protein sample 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 chromatography; 2-8: elution with 100, 150, 200, 250, 300, 400, 500 mmol / L imidazole), from... Figure 2 It can be seen that a concentration of 250 mmol / L imidazole is the optimal condition for eluting the target protein, which can obtain paratuberculosis-specific diagnostic antigen protein efficiently and with high purity.

[0076] Example 5 Western bolt analysis of recombinant protein

[0077] After dilution, the purified concentrated paratuberculosis specific diagnostic antigen protein was mixed with protein loading buffer at a ratio of 4:1, and then incubated at 100°C for 10 min for standby. The paratuberculosis mycobacterium specific detection antigen protein was detected by Western Blot according to the following operation:

[0078] (1) SDS-PAGE: The prepared protein sample was loaded into each well at 10 µL for SDS-PAGE. (90V electrophoresis for 30 min to move the protein sample to the boundary between the concentrated gel and the separation gel, and then transfer to 120V electrophoresis for 60 min);

[0079] (2) Membrane transfer: After electrophoresis, the upper concentrated gel was removed, and the appropriate PVDF membrane was cut according to the size of the separation gel. In the order of "sponge-filter paper-PVDF membrane-gel-filter paper-sponge", it was placed in a vertical electrophoresis tank with ice box and membrane transfer solution, and transferred at 250mA constant current for 60 min;

[0080] (3) Blocking: After membrane transfer, the PVDF membrane was taken out, and 1×TBST was shaken for 5 min, 5 times. After washing, the PVDF was placed in the blocking solution at 4°C overnight;

[0081] (4) Primary antibody incubation: The next day, the blocked PVDF membrane was washed with 1×TBST for 5 min, 5 times. According to the lane position, the PVDF membrane was cut and placed in different antibodies and sera diluted with blocking solution, and incubated at room temperature for 2 h;

[0082] (5) Secondary antibody incubation: The PVDF membrane was washed with 1×TBST for 5 min, 5 times, and then placed in enzyme-labeled secondary antibody diluted with TBST, and incubated at room temperature for 2 h;

[0083] (6) Color development: The PVDF membrane was washed with 1×TBST for 5 min, 5 times, and then ECL color development was added in the dark room, and the results were observed using an imaging instrument.

[0084] The results of WB identification of recombinant protein are as follows Figure 3The specific diagnostic antigen protein of paratuberculosis was specifically combined with the mouse His-tag antibody and the paratuberculosis positive serum of sheep at 21.6 kDa, and was not combined with the paratuberculosis negative serum of sheep, which verified that the recombinant protein was correctly expressed and had a His tag, the molecular weight was consistent with the expectation, and the specific diagnostic antigen protein of paratuberculosis could be recognized by the specific antibody in the paratuberculosis positive serum of sheep, had the potential to be used as a specific diagnostic antigen of Mycobacterium paratuberculosis, had good specificity, could effectively avoid false positives, and provided a strong basis for the diagnosis of paratuberculosis.

[0085] Example 6 ELISA test

[0086] 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;

[0087] Blocking: Discard the liquid in the wells, wash 3 times with 1 × PBST, and tap dry after the last time. Add blocking solution (2% BSA PBST solution) 200 μL / well, and block at 37°C for 2 h;

[0088] Primary antibody incubation: Discard the liquid in the wells, wash 3 times with 1 × PBST, and tap dry after the last time. Add the diluent (1% BSA PBST solution) to dilute the serum to be tested, 100 μL / well, and incubate at 37°C for 1 h;

[0089] Secondary antibody incubation: Discard the liquid in the wells, wash 3 times with 1 × PBST, and tap dry after the last time. Add 1:10,000 HRP-rabbit anti-sheep IgG diluted with diluent (1% BSA PBST solution), and incubate at 37°C for 45 min;

[0090] Color development and OD determination 450nm : Discard the liquid in the wells, wash 5 times with 1 × PBST, and tap dry after the last time. Add substrate TMB solution 100 μL / well, incubate at room temperature in the dark for 10 min, and determine the OD after the color development is terminated by the termination solution 450nm The absorbance value.

[0091] (1) Optimal antigen coating concentration and serum dilution factor

[0092] Coated with coating solution, paratuberculosis specific diagnostic antigen protein according to 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, 47 ng / well, each concentration set two repeated wells. Dilute the primary antibody (goat negative and paratuberculosis goat positive serum) with antibody diluent at 1:100, 1:200, 1:400, 1:800, each concentration set two repeated wells. Read the OD 450nm value to calculate the P / N value (Table 1), the antigen coating concentration and serum dilution multiple with the maximum P / N value are the best, and the serum dilution multiple 1:200 and the antigen coating concentration 47 ng are the best.

[0093] Table 1 Determination of antigen coating concentration and serum dilution

[0094]

[0095] (2) Determination of optimal coating temperature and time

[0096] Set four different coating temperatures and times, read the OD 450nm value and calculate the P / N value to establish a table analysis. The results are shown in Table 2. When the antigen is coated at 37℃ for 1h, the P / N value is the largest, which is 2.316, and the optimal conditions are determined as coating at 37℃ for 1h.

[0097] Table 2 Results of optimal antigen coating temperature and time

[0098]

[0099] (3) Determination of the best blocking solution

[0100] Select four kinds of blocking solution to read the OD 450nm value to establish a table analysis. The results are shown in Table 3. When using 2% BSA blocking solution, the P / N value is the largest, which is 4.522. It is determined that 2% BSA blocking solution is the best.

[0101] Table 3 Results of the best blocking solution

[0102]

[0103] (4) Optimization of the best blocking time

[0104] According to the above optimized indirect ELISA operation steps, select four different blocking times for blocking, read the OD 450nmP / N value calculation and table analysis. The results are shown in Table 5. Under the condition of blocking for 60 min, the maximum P / N value is 3.151, and the optimal incubation time of the primary antibody is determined to be 60 min.

[0105] Table 4 Results of the optimal blocking time

[0106]

[0107] (5) Optimal incubation time of the primary antibody

[0108] According to the above optimization conditions, four serum incubation times were set, and the OD of the enzyme marker was read 450nm P / N value calculation and table analysis. The results are shown in Table 5. Under the condition of blocking for 60 min, the maximum P / N value is 3.151, and the optimal incubation time of the primary antibody is determined to be 60 min.

[0109] Table 5 Results of the optimal serum incubation time

[0110]

[0111] (6) Optimal dilution ratio of the secondary antibody

[0112] According to the above optimization conditions, six dilution ratios of the enzyme marker secondary antibody were set, and the OD of the enzyme marker was read 450nm P / N value calculation and table analysis. The results are shown in Table 6. When the dilution ratio of the enzyme marker secondary antibody is 1:10 000, the maximum P / N value is 5.738, and the dilution ratio of the enzyme marker secondary antibody is determined to be 1:10 000 as the optimal condition.

[0113] Table 6 Optimal dilution ratio of the secondary antibody

[0114]

[0115] (7) Optimal incubation time of the secondary antibody

[0116] According to the above optimization conditions, four secondary antibody incubation times were set for incubation. The OD of the enzyme marker was read 450nm P / N value calculation and table analysis. The results are shown in Table 7. When the secondary antibody is incubated for 45 min, the maximum P / N value is 4.461, and the incubation time of the enzyme marker secondary antibody is determined to be 45 min.

[0117] Table 7 Results of the optimal secondary antibody incubation time

[0118]

[0119] (8) Optimal color development time results

[0120] Four color development times were set for optimization, and the OD of the enzyme marker was read 450nmThe data was analyzed according to the P / N value. The results are shown in Table 8. The effect was more obvious when the color development was 5-10 min. When the color development was 10 min, the maximum P / N value was 3.134, and the optimal color development time was determined to be 10 min.

[0121] Table 8 Results of optimal color development time

[0122]

[0123] (9) Determination of critical value

[0124] According to the above-mentioned optimized results, 30 batches of paratuberculosis sheep negative serum were selected for testing, and the OD 450nm value was established to analyze the data, and the results are shown in Table 9. The average value (X) was 0.3257, and the standard deviation (SD) was 0.0816. According to the critical value determination principle of "average value ± multiple standard deviation" in statistics, the negative determination threshold was calculated as X + 2SD (0.3257 + 2 × 0.0816 ≈ 0.489), and the positive determination threshold was calculated as X + 3SD (0.3257 + 3 × 0.0816 ≈ 0.570). Therefore, the determination criteria are as follows: OD 450nm ≤ 0.489 is negative, OD 450nm ≥ 0.570 is positive, and 0.489 < OD 450nm < 0.570 is suspected positive.

[0125] Table 9 Determination of critical value

[0126]

[0127] (10) Repetitive test

[0128] ① Intra-batch repeatability test

[0129] Three enzyme-coated plates prepared at the same time were used to detect the serum, and the OD 450nm value was determined by using an enzyme-labeled instrument. The data was analyzed according to the P / N value. According to the formula, the coefficient of variation (CV) was calculated. The results are shown in Table 10. The highest value of intra-batch coefficient of variation was 18.66%, and the lowest value was 8.56%.

[0130] Table 10 Results of intra-batch repeatability test

[0131]

[0132] ② Inter-batch repeatability test

[0133] Three enzyme-coated plates prepared at the same time were used to detect the serum, and the OD 450nmThe data was analyzed according to the formula to calculate the coefficient of variation (CV). The results are shown in Table 11, the highest value of the batch-to-batch variability coefficient is 14.61%, and the lowest value is 4.96%, both of which are lower than 15%.

[0134] Table 11 Results of batch repeatability test

[0135]

[0136] (11) Sensitivity test

[0137] According to the optimized conditions, the laboratory preserved paratuberculosis goat positive serum was diluted by 7 times for detection, and the OD 450nm value was determined by using an enzyme label instrument. The results are shown in Table 12, when the serum dilution multiple is 1:800, the OD 450nm value is higher than the positive critical value.

[0138] Table 12 Results of sensitivity test

[0139]

[0140] The ELISA established by the test was compared with the imported commercial kit of ID Vet and IDEXX, and 6 sera of strong positive, medium positive, weak positive and 3 negative were detected for 6 times. The results are shown in Table 13, the 6 times detection of 3 positive sera by the ELISA established by the application showed positive, and the detection of medium positive and weak positive sera by ID Vet and IDEXX showed different results.

[0141] Table 13 Test results of positive serum

[0142]

[0143] (12) Specificity test

[0144] The serum of 3 other common goat diseases (paratuberculosis detection is negative) was detected, and the negative and positive serum of laboratory goat paratuberculosis was used as a control, and the OD 450nm value was determined by using an enzyme label instrument for data analysis. The results are shown in Table 14, the OD 450nm value of 3 other disease positive sera is lower than the negative critical value, and the paratuberculosis positive serum is higher than the positive critical value.

[0145] Table 14 Results of specificity test

[0146]

[0147] Example 7 Clinical detection

[0148] (1) Field samples

[0149] Forty-six sheep serum samples were collected in the field and simultaneously detected using the ELISA kit of this invention, along with imported commercial kits ID Vet and IDEXX. The results are shown in Table 15: the positive rate of the method established in this study was 43.78%, while the positive rates of ID Vet and IDEXX kits were 6.22% and 3.43%, respectively.

[0150] Table 15 Results of field clinical sample testing

[0151]

[0152] (2) Samples from sheep artificially infected with MAP test

[0153] Serum samples from 112 sheep (2 control sheep + 6 infected sheep, all sampled 14 times) artificially infected with MAP were simultaneously detected using the ELISA kit of this invention and imported commercial kits from ID Vet and IDEXX. The results are shown in Tables 16 and 17: Serum samples from the negative control group (2 control sheep + 6 infected sheep, 14 sampling times each) were all negative using the three methods; for the artificially infected group (6 sheep, pathological observation... Figure 4 Diffuse granulomatous lesions of the ileum lamina propria, accompanied by lymphocytes, epithelioid cells and multinucleated giant cells (HE staining).

[0154] Pathogen isolation and identification Figure 5 MAP culture colony morphology: The colonies are papillary in appearance, smooth in surface, cream-colored or pale yellow, with irregular edges. Tissue nucleic acid detection confirmed infection, and animal experiments were successfully established. The ELISA kit of this invention showed a positive rate of 42.86% in 84 serum samples from 6 infected sheep, compared to 10.71% for ID Vet and 8.33% for IDEXX kits. All three methods detected positive results as early as 75 days after MAP inoculation. The ID Vet kit showed 9 positive serum samples in 2 sheep; the IDEXX kit showed 7 positive serum samples in only 1 sheep (one of the sheep that tested positive with ID Vet); the ELISA kit of this invention showed 36 positive serum samples in 4 sheep (including 2 sheep that tested positive with the other two methods, with 2 sheep showing continuous positivity starting 75 days after inoculation).

[0155] Table 16 Detection Results of Artificially Infected Samples

[0156]

[0157] Table 17 Detailed Results of Artificial Infection Sample Detection

[0158]

Claims

1. An ELISA kit for detecting antibodies against sheep paratuberculosis, characterized in that, It contains the following components: A specific detection antigen for Mycobacterium paratuberculosis, the amino acid sequence of which is shown in SEQ ID NO: 1; Coating buffer; Sealing liquid; Serum diluent; Enzyme-labeled secondary antibody dilution solution; Substrate solution; Termination solution; Positive control serum and negative control serum; The coating concentration of the Mycobacterium paratuberculosis-specific detection antigen is 47-63 ng / well; The serum diluent is used to dilute the serum to be tested at a volume ratio of 1:100 to 1:200; The enzyme-labeled secondary antibody dilution solution contains HRP-labeled rabbit anti-sheep IgG antibody, with a dilution ratio of 1:5000-1:10000; The result determination criteria for the kit are as follows: when the OD of the serum sample to be tested is... 450nm A value ≤ 0.489 is considered negative; OD 450nm A value ≥ 0.570 is considered positive; a value < 0.489 is considered positive. 450nm A value < 0.570 is considered a suspected positive result.

2. The ELISA kit for detecting antibodies against ovine paratuberculosis as described in 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 substrate solution is a TMB solution, and the termination solution is concentrated sulfuric acid.

3. An ELISA kit for detecting antibodies against ovine paratuberculosis as described in claim 1 or 2, characterized in that, The sealing time of the sealing liquid is 60 minutes, and the usage is 200 μL / well.

4. An ELISA kit for detecting antibodies against ovine paratuberculosis as described in claim 1 or 2, characterized in that, The coating conditions for the Mycobacterium paratuberculosis-specific detection antigen are incubation at 37°C for 1-2 hours; the coating buffer is a carbonate buffer with pH=9.

6.

5. An ELISA kit for detecting antibodies against ovine paratuberculosis as described in claim 1 or 2, characterized in that, The incubation time for the enzyme-labeled secondary antibody is 45 minutes.

6. An ELISA kit for detecting antibodies against ovine paratuberculosis as described in claim 1 or 2, characterized in that, The color development time of the substrate solution is 5-10 minutes, and the color development is carried out under light-protected conditions.

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