CN120779028A - ELISA kit for detecting sheep paratuberculosis antibodies and its application - Google Patents

Abstract

The invention provides an ELISA kit for detecting sheep paratuberculosis antibody and application thereof, relating to the field of biological detection, wherein the ELISA kit comprises an antigen for diagnosis of paratuberculosis specific for sheep (the amino acid sequence is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2), a coating buffer solution, a sealing solution, a serum diluent, an enzyme-labeled secondary antibody diluent, a substrate solution, a termination solution, positive control serum, negative control serum and the like, compared with the prior art, the kit provided by the invention has good specificity, the false positive rate is reduced, clinical data shows that the positive detection rate of the kit is obviously higher than that of an import kit of ID Vet (6.22%) and IDEXX (3.43%), which shows that the sensitivity is higher, and the infected sheep can be detected earlier and more accurately.

Description

ELISA kit for detecting sheep paratuberculosis antibody and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an ELISA kit for detecting sheep paratuberculosis antibodies and application thereof.

Background

The paratuberculosis (Paratuberculosis) is chronic granulomatous enteritis caused by mycobacterium paratuberculosis (Mycobacterium avium subsp, paratuberculosis, MAP) of various animals, is a disease which is ignored by ruminants, and is called as a small ruminant 'stealth killer'. It is characterized by progressive wasting, refractory diarrhea, granulomatous enteritis and mesenteric lymphadenitis in diseased animals, ultimately leading to death. The world animal health organization (World Organisation for ANIMAL HEALTH, WOAH) recognizes a global significant problem in the field of animal health and is also listed in the list of WOAH epidemic diseases, infections and infections, which are classified as three animal epidemic diseases by the current regulations in our country.

Paratuberculosis is widely prevalent worldwide and is considered an important infectious disease affecting the economics and public health of animal husbandry. The disease is transmitted mainly by the faecal-oral route. MAP survives in soil, water and forage for a long period of time, resulting in animals being in an infection exposure state for a long period of time, thereby increasing the chances of illness in susceptible animals. Humans may be infected with MAP once drinking MAP contaminated surface water, or eating 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 diseases such as rheumatoid arthritis, hashimoto thyroiditis, type I diabetes, multiple sclerosis and autism in humans, and more evidence suggests that MAP is a zoonotic agent. The disease has no specific therapeutic drugs and commercial vaccines at present, clinical intervention can only temporarily relieve symptoms through symptomatic treatment, and no report of successful cure of infected individuals is yet seen. Therefore, the primary strategy for preventing the disease is early diagnosis, early isolation and elimination.

However, the present diagnosis of the disease has the advantages of hidden clinical symptoms (such as no diarrhea, intermittent diarrhea or just fecal disfigurement, etc.), characteristic pathological changes hysteresis (such as young infection, typical pathological changes occurring over the age of 2 years and over the age of 1.5 years of sheep), intermittent intestinal tract bacterial discharge, timeliness of gold standard pathogen culture (such as over 6 weeks after II type MAP inoculation, over half a year of I type MAP growth), transient and uncertain bacteremia, low specificity and sensitivity of serum antibody detection, etc. Diagnosis of the disease is rather difficult and early diagnosis is more difficult. It is currently agreed that the diagnosis of paratuberculosis requires the use of a variety of materials in combination with a variety of methods.

At present, the specificity of ELISA detection of paratuberculosis is 48% -92%, and the sensitivity is 50% -70%. Sheep anti-MAP antibodies were studied to appear at the earliest 75d (the laboratory sheep animal test) or 8 weeks (reported abroad) post-infection, as intermittent or continuous antibody reaction positives. Differences in ELISA detection results for anti-MAP antibodies are mainly due to humoral immunity of infected animals and variability of antigen in ELISA kits, including commercial kits. Therefore, screening for proteins that produce an immune response early and MAP-specific antigens is of great importance.

Disclosure of Invention

Based on the background, the invention provides an ELISA kit for detecting sheep paratuberculosis antibody and application thereof, and the kit can detect MAP infected sheep at early stage of disease, provides technical and data support for diagnosis and research of sheep paratuberculosis, and specifically comprises the following steps:

An ELISA kit for detecting sheep paratuberculosis antibody, which comprises the following components:

The specific detection antigen of the mycobacterium paratuberculosis has an amino acid sequence shown in SEQ ID NO. 1; the nucleotide sequence of the specific detection antigen of the mycobacterium paratuberculosis is shown as SEQ ID NO. 2;

coating buffer solution;

a sealing liquid;

serum dilutions;

enzyme-labeled secondary antibody diluent;

a substrate solution;

A stop solution;

Positive control serum and negative control serum.

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

Further, the kit is used for early detection of sheep paratuberculosis, when the kit is used, the coating concentration of the paratuberculosis specific detection antigen is 47-63 ng/hole, and the serum diluent dilutes serum to be detected according to the volume ratio of 1:100-200.

Further, the coating condition of the antigen for specific detection of the mycobacterium paratuberculosis is 37 ℃ for 1-2 hours, and the coating buffer is carbonate buffer with pH=9.6.

Further, the blocking time of the blocking liquid was 60 minutes, and the amount of the blocking liquid used was 200. Mu.L/well.

Further, the serum diluted by the serum dilution is incubated with the coated mycobacterium paratuberculosis specific detection antigen at 37 ℃ for 60 minutes.

Further, the incubation time of the enzyme-labeled secondary antibody was 45 minutes.

Further, the substrate solution is developed for 5 to 10 minutes, and the development is performed under a dark condition.

Further, when the OD _450nm value of the serum sample to be tested is less than or equal to 0.489, the sample is judged to be negative, when the OD _450nm value is more than or equal to 0.570, the sample is judged to be positive, and when the OD _450nm value is less than 0.489 and less than 0.570, the sample is judged to be suspected positive.

On the other hand, the invention provides a use method of an ELISA kit for detecting sheep paratuberculosis antibodies, which comprises the following steps:

S1, antigen coating, namely diluting paratuberculosis specificity detection antigen protein to 10 mug/mL by using a coating buffer solution, adding 96-well ELISA plates into 100 mug/well, and coating for 16h at 4 ℃;

s2, sealing, namely discarding the liquid in the hole, washing 3 times by using 1 XPBST, drying by beating, adding 200 mu L/hole of sealing liquid, and sealing for 1h at 37 ℃;

S3, primary antibody incubation, namely discarding liquid in the hole, washing 3 times by using 1 XPBST, beating to dry, adding serum diluent to dilute serum to be tested, and incubating for 1h at 37 ℃ at 100 mu L/hole, wherein the serum diluent is primary antibody;

S4, secondary antibody incubation, namely discarding liquid in a hole, washing 3 times by using 1 XPBST, beating, adding HRP-rabbit anti-sheep IgG of 1:10000 diluted by serum diluent, and incubating for 45min at 37 ℃;

S5, developing and measuring OD _450nm, namely discarding liquid in the hole, washing for 5 times by using 1 XPBST, performing one-time shooting, adding 100 mu L/hole of substrate solution, incubating for 10 minutes in a dark place, and measuring the absorbance value at the OD _450nm after the stop solution stops developing;

S6, judging the result that the serum sample to be tested is negative when the OD _450nm value is less than or equal to 0.489, positive when the OD _450nm value is more than or equal to 0.570, and suspected positive when the OD _450nm value is less than 0.570.

Compared with the prior art, the invention has the beneficial effects that:

1. The ELISA kit provided by the invention uses specific diagnosis antigen of the paratuberculosis mycobacterium, can be combined with the paratuberculosis antibody of sheep more accurately, reduces cross reaction with other similar pathogen antibodies, improves detection specificity, has good specificity and reduces false positive rate compared with the prior art, and clinical data show that the positive detection rate of the kit is 43.78% which is obviously higher than ID Vet (6.22%) and IDEXX (3.43%) and is an import kit, which shows that the sensitivity is higher and the infected sheep can be detected earlier and more accurately.

2. The ELISA kit provided by the invention can be used for efficiently detecting samples with different antibody levels, and the sensitivity test experimental data show that the kit can be used for stably detecting strong positive, medium positive and weak positive serum, especially the detection capability of the kit on weak positive samples is obviously better than that of the conventional commercial kit (such as ID Vet and IDEXX), positive and negative samples can still be clearly distinguished near a critical value, and the detection positive rate of clinical samples is far higher than that of the commercial kit for comparison.

3. The kit provided by the invention adopts the specific diagnosis antigen of the mycobacterium paratuberculosis to effectively identify low-concentration antibodies generated in early infection, experimental data show that the kit can still detect positive signals when the serum dilution is 1:800, and has high sensitivity to samples with low antibody level, positive results can be detected at the early stage of infection, a reliable tool is provided for early diagnosis, and is beneficial to timely finding infected sheep when the diseases are not spread in a large scale, so that the economic loss of animal husbandry is reduced, and meanwhile, because the mycobacterium paratuberculosis is a zoonotic agent, the control of animal infection is beneficial to reducing the possibility of human infection.

4. The positive detection rate of the kit provided by the invention is obviously higher than ID Vet and IDEXX import kits, experiments show that in 466 wild sheep serum detection, the positive rate of the kit is 43.78%, while ID Vet and IDEXX are only 6.22% and 3.43% respectively, in addition, in sheep experiments artificially infected with MAP, the positive detection rate of the kit is 42.86%, which is far higher than ID Vet (10.71%) and IDEXX (8.33%), thus proving that the kit has obvious improvement in sensitivity.

5. The kit provided by the invention obviously reduces the risk of false negative, experimental data show that repeated detection results of weak positive serum show that 6 times of detection of the kit are positive, and ID Vet and IDEXX respectively show 1 time and multiple times of false negative or suspected results, so that the stability and reliability of the kit provided by the invention are verified, and the sensitivity of the kit is further verified.

Drawings

FIG. 1 shows SDS-PAGE results of induction expression of paratuberculosis specific diagnostic antigen proteins;

FIG. 2 shows the results of the concentration search for the paratuberculosis specific diagnostic antigen protein pET28a-MPtb imidazole;

FIG. 3 shows the results of the paratuberculosis specific diagnostic antigen protein pET28a-MPtb Western blot;

FIG. 4 shows the results of diffuse granulomatous lesions of the ileum lamina propria;

FIG. 5 shows the morphology of MAP cultured colonies.

Detailed Description

EXAMPLE 1 construction scheme for prokaryotic expression vectors

In this example, focusing on the specific diagnostic antigen related study of Mycobacterium paratuberculosis, we selected to experiment on the basis of pET-28a vector in order to construct an adapted prokaryotic expression vector, and named pET28a-MPtb for convenience of subsequent explanation and study identification.

And (3) selecting a specific diagnosis antigen of the mycobacterium paratuberculosis as a target protein, and constructing a prokaryotic expression vector. The nucleotide sequence of the antigen is shown as SEQ ID NO. 2, and the corresponding amino acid sequence is shown as SEQ ID NO. 1.

The construction scheme is that a target protein is inserted into a pET-28a prokaryotic expression vector, a 6His tag is added, nde I and BamH I enzyme cutting sites are respectively introduced at two ends of a target gene, and the construction form is pET-28 a+6His+Nde I+target protein+BamH I. S-Tag is a novel recombinant protein fusion peptide Tag.

The sequence of the upstream primer F for amplifying the target gene is shown as SEQ ID NO. 3, and the sequence of the downstream primer R is shown as SEQ ID NO. 4.

EXAMPLE 2 transformation of recombinant vector into E.coli BL21-DE3

(1) Mu.L of pET28a-MPtb vector was added to 100. Mu.L of competent bacteria and left on ice for 20min.

(2) Heat-shock at 42 ℃ for 90s, rapidly placing in ice for 5min, and adding 600 mu L of LB culture solution.

(3) Shaking at 37℃and 180 r/min for 1h, centrifuging and then spreading all on LB plates containing 50. Mu.g/mL kanamycin (Kana), and culturing overnight at 37℃in an inverted state.

In order to obtain enough recombinant protein, the constructed pET28a-MPtb vector is transformed into competent cells of escherichia coli BL21-DE3,

The conversion step:

The plasmid is mixed with competent cells, 1 mu L of pET28a-MPtb vector is added into 100 mu L of competent E.coli BL21-DE3 cells, and the mixture is placed on ice for 20 minutes after being gently mixed, so that the plasmid can be fully adsorbed on the cell surface.

Heat shock treatment the mixture was heat shocked at 42 ℃ for 90 seconds to force the plasmid into the cell interior, followed by rapid transfer of the centrifuge tube into ice and resting for 5 minutes to cool the cells and prevent excessive damage thereto.

Resuscitator and culture, namely adding 600 mu L of LB culture solution (without antibiotics) into a centrifuge tube, gently mixing, and placing into a shaking table at 37 ℃ and 180 r/min for shaking culture for 1 hour to resuscitate cells and express resistant proteins.

Coating and screening, namely centrifuging the cultured cell suspension, discarding part of supernatant, re-suspending cells, coating the whole on an LB solid medium plate containing 50 mug/mL Kana, inversely placing the plate in a 37 ℃ constant temperature incubator for overnight culture, and screening positive clones successfully transferred into the pET28a-MPtb vector.

Through the steps, a prokaryotic expression vector containing the paratuberculosis specific diagnostic antigen gene is successfully constructed and transformed into escherichia coli BL21-DE3, so that a foundation is laid for subsequent protein expression and purification.

Example 3 IPTG identification of expression of recombinant fusion proteins induced

Taking 250mL and 1000mL conical flask for autoclaving, placing in an ultra-clean bench for ultraviolet irradiation for 30min for standby, adding 50mL LB culture medium containing 50 mug/mL kanamycin into the 250mL conical flask, taking positive bacteria 1:100 ratio, adding the positive bacteria, and culturing overnight at 37 ℃ and 220r/min in a shaking incubator. The positive bacteria 1:1000 obtained after shaking after taking every other day (the rest bacterial liquid is frozen in a refrigerator at-80 ℃ for standby after being mixed with 50% glycerol 1:1) are added into 300mL of LB broth containing kanamycin with the final concentration of 50 mug/mL, IPTG with the final concentration of 1.0mmol/L is added when the final concentration of 37 ℃ is cultured until OD _600nm is 0.6-0.8 in a shaking way, and induction is carried out for 8 hours at 37 ℃ and 180 r/min. The solubility analysis was performed by the following operations:

(1) The induced bacterial liquid is 7000r/min, centrifuged for 15min to collect bacterial cells and weighed;

(2) 10mL Binding Buffer is added to each 1g thallus for re-suspension;

(3) The resuspended thalli are crushed by ice bath for 25min (150 w, work is stopped for 3s for 5 s);

(4) Centrifuging the thallus subjected to ultrasonic crushing at 4 ℃ for 15min at 12000r/min, collecting supernatant and precipitate, adding Binding Buffer containing 8M urea into the precipitate, and standing overnight at 4 ℃;

(5) 8. Mu.L of each of the supernatant and pellet was added to 2. Mu.L of protein loading buffer (5X), and the mixture was boiled in a water bath at 100℃for 10min.

SDS-PAGE gel electrophoresis analysis was performed to prepare the protein gel according to the instructions for rapid preparation of the kit from the SDS-PAGE gel. In order to ensure the accuracy and reliability of the experimental results, a plurality of groups of control groups are established, namely an escherichia coli induced and uninduced sample, an escherichia coli induced and uninduced sample containing a pET-30a empty vector, and a supernatant and a sediment sample after the positive bacteria uninduced ultrasonic disruption.

The treated samples were loaded at 10. Mu.L per well, followed by SDS-PAGE gel electrophoresis. After electrophoresis, the albumin glue is placed in coomassie brilliant blue dye solution for dyeing for 20min, then a decolorizing solution is added, and the decolorizing solution is replaced for a plurality of times and oscillated for decolorizing until the background is clear. After the decoloration is completed, the protein glue is observed. The results are shown in FIG. 1 (M: protein molecular mass standard; 1: whole Escherichia coli is not induced; 2: whole Escherichia coli is induced; 3: pET28a empty vector is not induced; 4: pET28a empty vector is induced; 5: pET28 a-MPtb. Mu. Induced disruption supernatant; 6: pET28 a-MPtb. Mu. Induced disruption precipitation; 7: pET28 a-MPtb. Mu. Induced disruption supernatant; 8: pET28 a-MPtb. Mu. Induced disruption precipitation), the size of the induced paratuberculosis-specific diagnostic antigen protein is 21.6kDa, which is consistent with the expected size, and coexists in a soluble form.

EXAMPLE 4 Ni column affinity chromatography purification concentration and concentration determination

After optimizing recombinant protein induced expression conditions, hyPur T Ni-NTA 6FF (His-Tag) preloaded gravity column produced by biological engineering (Shanghai) Co., ltd was selected for purification of recombinant protein, and the specific operation steps are as follows:

(1) And carrying out experiments according to the optimized induction conditions to finally obtain a supernatant solution containing the recombinant protein.

(2) The supernatant solution is filtered by a filter membrane with the pore diameter of 0.45 μm to remove impurities possibly existing in the solution, and the impurities are prevented from interfering with the subsequent purification steps.

(3) Washing the purification column with 5 times of ultrapure water to remove impurities and residual substances possibly existing in the purification column, balancing the purification column with 5 times of Binding Buffer to ensure that the purification column is in a state suitable for Binding target proteins, slowly adding the filtered supernatant into the purification column, collecting the penetrating fluid, loading the penetrating fluid on the column again, repeatedly filtering for 3 times to ensure that the target proteins can be fully bound with the purification column, and finally, adding the filtered penetrating fluid into the column again, and standing in an environment of 4 ℃ for overnight to ensure that the target proteins are sufficiently bound with the purification column.

(4) The next day, the flow-through was further slowly filtered 3 times from the purification column, eluting the purification column with a Wash Buffer containing different concentrations of imidazole, and collecting the eluate for subsequent SDS-PAGE analysis to determine the elution profile of the target protein.

(5) Transferring the filtrate containing 250mM imidazole and collected after the imidazole into an ultrafiltration tube, adjusting parameters of a centrifuge to be the temperature of 4 ℃ and the rotating speed of 3000r/min, centrifuging for 30min, stopping centrifuging when the volume of the solution in the ultrafiltration tube is reduced to 1mL, adding 9mL of PBS solution into the ultrafiltration tube, continuing centrifuging operation, repeating ultrafiltration for 3 times, completing replacement of protein buffer solution, and finally collecting 1mL of protein concentrate.

(6) The standard curve was constructed by measuring the absorbance of protein samples at OD _562nm, according to BCA protein quantification kit instructions, resulting in the calculation equation y= 1079.7x-12.079 (where x is the absorbance of the protein at OD _562nm; y is the protein concentration in μg/mL, r2= 0.9953). The purified and ultrafiltered recombinant protein was tested, and the absorbance at OD _562nm was also measured to be 1.400, which was substituted into the standard curve formula to calculate, and the result showed that the protein concentration after ultrafiltration was 1500. Mu.g/mL.

(7) SDS-PAGE analysis is carried out on the purified and concentrated protein sample to further verify the information of the purity, the molecular weight and the like of the protein, the related results are shown in a figure 2 (M: protein molecular mass standard; 1: protein column chromatography; 2-8:100, 150, 200, 250, 300, 400, 500 mmol/L imidazole elution), and the concentration of 250mmol/L imidazole is the optimal condition for eluting the target protein, so that the paratuberculosis specific diagnostic antigen protein can be obtained efficiently and highly purity.

EXAMPLE 5 Western bolt analysis of recombinant proteins

After the purified and concentrated paratuberculosis specific diagnostic antigen protein is diluted, the protein is uniformly mixed with a protein loading buffer solution according to a ratio of 4:1, and the mixture is left for standby in a water bath at 100 ℃ for 10 min. Western Blot detection of antigen proteins for specific detection of Mycobacterium paratuberculosis was performed as follows:

(1) SDS-PAGE prepared protein samples were subjected to SDS-PAGE at 10. Mu.L per well. (90V electrophoresis for 30min to limit the gel concentration and gel separation, and then 120V electrophoresis for 60 min);

(2) Transferring membrane, namely cutting off the upper concentrated gel after electrophoresis, and cutting out a proper PVDF membrane according to the size of the separation gel. The materials are placed in the order of sponge-filter paper-PVDF membrane-gel-filter paper-sponge. Placing the membrane into a vertical electrophoresis tank with an ice box and a membrane transferring liquid, and transferring the membrane for 60min at a constant flow rate of 250 mA;

(3) After membrane transfer is completed, taking out the PVDF membrane, washing the PVDF membrane for 5 times by a1 XTBST shaking table for 5min each time, and placing the PVDF membrane in a sealing liquid at 4 ℃ overnight after washing is completed;

(4) Taking out the PVDF membrane after sealing the membrane in the next day, washing the PVDF membrane with a1 XTBST shaking table for 5 times, each time for 5 minutes, cutting the PVDF membrane according to the positions of lanes, respectively placing the PVDF membrane in different antibodies and serum diluted by sealing liquid, and incubating the PVDF membrane with the shaking table at room temperature for 2 hours;

(5) The second antibody incubation, namely washing the PVDF membrane with a1 XTBST shaker for 5 times and 5min each time, then placing the PVDF membrane in the enzyme-labeled second antibody diluted by TBST, and incubating the PVDF membrane with the shaking shaker at room temperature for 2h;

(6) Color development PVDF membrane was washed 5 times with 1 XTBE shaking table for 5min each time, ECL was added dropwise to the dark room for color development, and the results were observed using an imager.

The recombinant protein WB identification result is shown in figure 3 (M: protein molecular mass standard; 1: his murine tag antibody; 2: paratuberculosis sheep positive serum; 3: sheep negative serum), paratuberculosis specific diagnosis antigen protein is specifically combined with murine His tag antibody and sheep paratuberculosis positive serum at 21.6kDa, and is not combined with sheep paratuberculosis negative serum, thus not only verifying that the recombinant protein is correctly expressed and has His tag, the molecular weight accords with expectations, but also indicating that the recombinant protein can be identified by specific antibody in sheep paratuberculosis positive serum, has potential as paratuberculosis specific diagnosis antigen of paratuberculosis mycobacteria, has good specificity, can effectively avoid false positives, and provides powerful basis for paratuberculosis diagnosis.

Example 6 ELISA test

Antigen coating, namely diluting the purified paratuberculosis specificity diagnosis antigen protein to 10 mug/mL by ELISA coating liquid, adding 96-well ELISA plate into 100 mug/well, and coating for 16h at 4 ℃;

Blocking, namely, discarding the liquid in the hole, washing 3 times by using 1 XPBST, and finally, beating and drying. Blocking solution (PBST solution with 2% BSA) was added at 200 μl/well and blocked at 37 ℃ for 2h;

The primary antibody is incubated, the liquid in the hole is discarded, the liquid is washed 3 times by 1 XPBST, and finally the liquid is beaten dry. Diluting the serum to be tested by adding a diluent (PBST solution containing 1% BSA), and incubating at 37 ℃ for 1h with 100 mu L/hole;

Secondary antibody incubation, namely discarding the liquid in the hole, washing 3 times by using 1 XPBST, and finally beating and drying. HRP-rabbit anti-sheep IgG diluted 1:10000 in diluent (PBST solution containing 1% BSA) was added and incubated at 37 ℃ for 45min;

Color development and OD _450nm. The well liquid was discarded, washed 5 times with 1 XPBST and finally dried by pipetting. 100 mu L/well of substrate TMB solution is added, incubated at room temperature and in dark place for 10min, and the absorbance at OD _450nm is measured after the stop solution is used for stopping color development.

(1) Antigen coating concentration and serum optimal dilution

The paratuberculosis specific diagnostic antigen proteins were coated with coating fluid 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, 47 ng/well, two replicate wells were set for each concentration. Primary antibodies (sheep negative and paratuberculosis sheep positive serum) were diluted 1:100, 1:200, 1:400, 1:800 with antibody dilutions, with two duplicate wells per concentration. And (3) reading the OD _450nm value of the enzyme-labeled instrument to calculate a P/N value (table 1), wherein the antigen coating concentration and the serum dilution factor are optimal when the P/N value is maximum, and the effect is optimal when the serum dilution factor is 1:200 and the antigen coating concentration is 47 ng.

TABLE 1 determination of antigen coating concentration and serum dilution

(2) Determination of optimal coating temperature and time

Setting 4 different coating temperatures and times, reading the OD _450nm value of the enzyme label instrument, calculating the P/N value and establishing a table for analysis. As shown in Table 2, when the antigen was coated at 37℃for 1h, the P/N value was 2.316 at the maximum, and the coating at 37℃was determined as the optimal condition for coating for 1 h.

TABLE 2 optimal antigen coating temperature and time results

(3) Determination of optimal sealing liquid

Four types of sealing liquid are selected to read the value of the microplate reader OD _450nm so as to establish a table analysis. As a result, the P/N value was 4.522 at the maximum when a 2% BSA blocking solution was used, as shown in Table 3. The blocking solution was determined to be optimal for 2% BSA.

TABLE 3 optimal sealing fluid results

(4) Optimization of optimal closing time

And according to the optimized indirect ELISA operation steps, four different closing time periods are selected for closing, and the OD _450nm value of the microplate reader is read to establish table data analysis. As a result, as shown in Table 4, when the closing time period was 60 minutes, the P/N value was 3.017 at the maximum, and the optimal closing time was 60 minutes.

TABLE 4 optimal seal time results

(5) Optimal first antibody incubation time

And setting 4 serum incubation times according to the optimized conditions, and reading the OD _450nm value of the microplate reader to calculate the P/N value and perform tabulation analysis. The results are shown in Table 5, and the maximum P/N value is 3.151 under the condition of closing for 60min, and the optimal time for primary antibody incubation is determined to be 60min.

Table 5 best serum incubation time results

(6) Optimal dilution factor of secondary antibody

And setting dilution multiples of 6 enzyme-labeled secondary antibodies according to the optimized conditions, reading the OD _450nm value of the enzyme-labeled instrument, and calculating the P/N disease tabulation analysis. As shown in Table 6, the P/N value was 5.738 at the maximum when the dilution factor of the second antibody was 1:10,000, and the dilution factor of the second antibody was 1:10,000.

TABLE 6 optimal dilution of secondary antibody

(7) Optimal secondary antibody incubation time

According to the optimized conditions, 4 secondary antibody incubation times are set for incubation. And (5) reading the OD _450nm value of the microplate reader, calculating P/N and establishing a table for analysis. The results are shown in Table 7, and when the secondary antibody is incubated for 45min, the P/N value is 4.461 at maximum, and the incubation time of the enzyme-labeled secondary antibody is determined to be 45min.

TABLE 7 optimal secondary antibody incubation time results

(8) Optimum development time results

4 Developing times are set for optimization, and the reading OD _450nm value of the enzyme label instrument is read for data analysis. As shown in Table 8, the effect was more remarkable when the color was developed for 5 to 10 minutes, and when the color was developed for 10 minutes, the P/N value was 3.134 at the maximum, and the optimal development time was determined to be 10 minutes.

TABLE 8 optimal color development time results

(9) Determination of the critical value

According to the optimized result, 30 parts of negative serum of the paratuberculosis sheep are selected for test, and the OD _450nm value is measured by using an enzyme-labeled instrument to establish table analysis data, and the result is shown in table 9. The mean value (X) was 0.3257 and Standard Deviation (SD) was 0.0816 by statistical analysis. According to the critical value determination principle of mean value plus or minus multiple standard deviation in statistics, the negative judgment threshold value is calculated to be X+2SD (0.3257 +2×0.0816 ≡0.489), and the positive judgment threshold value is calculated to be X+3SD (0.3257 +3×0.0816 ≡0.570). Therefore, the judgment standard is that OD _450nm is less than or equal to 0.489 and is negative, OD _450nm is more than or equal to 0.570 and is positive, and OD _450nm less than 0.489 and is less than 0.570 and is suspected positive.

TABLE 9 determination of critical values

(10) Repeatability test

① Within-batch repeatability test

Serum was detected at the same time using 3 elisa plates made in the same batch, and the OD _450nm values were measured using an elisa instrument for data analysis. Coefficient of Variation (CV) is calculated according to the formula. The results are shown in Table 10, where the highest value of the variation coefficient in the lot is 18.66% and the lowest value is 8.56%.

TABLE 10 within-batch repeatability test results

② Batch-to-batch repeatability test

Serum was assayed at the same time using 3 different batches of enzyme-labeled plates and the OD _450nm values were measured using an enzyme-labeled instrument for data analysis. Coefficient of Variation (CV) is calculated according to the formula. As shown in Table 11, the highest value of the variation coefficient between batches was 14.61%, the lowest value was 4.96%, and the variation coefficient between batches was lower than 15%.

TABLE 11 results of the batch-to-batch repeatability test

(11) Sensitivity test

According to the optimized conditions, the positive serum of the paratuberculosis sheep stored in a laboratory is diluted by 7 times and then detected, and the OD _450nm value is measured by using an enzyme-labeled instrument. The results are shown in Table 12, with a serum dilution of 1:800, the OD _450nm value was above the positive threshold.

TABLE 12 sensitivity test results

The ELISA established by the test is compared with ID Vet and IDEXX imported commercial kits, and 6 times of serum are respectively detected for strong positive, medium positive, weak positive and 3 negative of paratuberculosis. The results are shown in Table 13, and the ELISA established by the invention shows positive results for 6 tests of 3 positive serum, and ID Vet shows different results for positive and weak positive serum in the pair of IDEXX.

TABLE 13 results of positive serum tests

(12) Specificity test

3 Kinds of common sheep serum (negative in paratuberculosis detection) are detected, negative and positive serum of laboratory sheep paratuberculosis is used as a control, and an enzyme-labeled instrument is used for measuring OD _450nm value for data analysis. As shown in Table 14, the OD _450nm of the 3 other positive sera were below the negative threshold and the paratuberculosis positive sera were above the positive threshold.

TABLE 14 specificity test results

Example 7 clinical testing

(1) Wild sample

466 Parts of sheep serum are collected in the wild, and the ELISA kit is adopted to detect together with ID Vet and IDEXX imported commercial kits. The results are shown in Table 15, and the established method positive rate of the study is 43.78%, and the detection results of ID Vet and IDEXX kit are 6.22% and 3.43%, respectively.

TABLE 15 detection results of field clinical samples

(2) Sheep artificial infection MAP test sample

Sheep were artificially infected with 112 parts of MAP (2 control sheep+6 infected sheep, each 14 samples) sheep serum and detected simultaneously with ID Vet and IDEXX imported commercial kits using the ELISA kit of the invention. The results are shown in tables 16 and 17, and the serum collected from the sheep in the negative control group of animal test in all time periods was detected as negative by 3 methods, for the artificially infected group (6 sheep, through pathological observation (FIG. 4: ileum lamina propria diffuse granulomatous lesions, accompanied by lymphocyte, epithelia-like cell and multinucleated megacell, HE staining);

Pathogen isolation and identification (FIG. 5: MAP cultured colony morphology: colony appearance is nipple-shaped, surface is smooth, cream-colored or light yellow, edge is irregular), tissue nucleic acid detection is identified as infection, animal experiments are successfully established, the positive rate of 84 sera of 6 infected sheep is 42.86% by using the ELISA kit of the invention, the positive rate of ID Vet and the detection result of IDEXX kit are respectively 10.71% and 8.71%, 3 methods detection is found at the earliest after MAP inoculation, 9 times of serum positives of the ID Vet kit appear in 2 sheep, 7 times of serum positives appear in 1 sheep (one sheep positive in ID Vet detection), 36 times of serum positives of the ELISA kit of the invention are distributed in 4 sheep (including 2 sheep positive in other two methods detection), and 2 sheep are continuous positive from 75d after inoculation).

TABLE 16 detection results of artificially infected samples

TABLE 17 detailed results of detection of artificially infected samples

Claims (10)

1. An ELISA kit for detecting sheep paratuberculosis antibodies, comprising 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 antibody as claimed in claim 1, characterized in that the confining buffer 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-sheep IgG antibody containing HRP labeling, and the dilution factor is 1:5000-1:10000; the substrate solution is TMB solution, and the stop buffer is concentrated sulfuric acid. 3. The application of the ELISA kit for detecting sheep paratuberculosis antibody as claimed in claim 1 or 2, characterized in that, for the early detection of sheep paratuberculosis, when used, the coating concentration of the Mycobacterium paratuberculosis specific detection antigen is 47-63ng/well; the serum to be tested is diluted by the serum diluent in a volume ratio of 1:100-200. 4. Use of the ELISA kit for detecting sheep paratuberculosis antibodies according to claim 3, characterized in that the coating conditions for the Mycobacterium paratuberculosis specific detection antigen are incubation at 37°C for 1-2 hours; and the coating buffer is a carbonate buffer with a pH of 9.6. 5. Use of the ELISA kit for detecting sheep paratuberculosis antibodies according to claim 3, characterized in that the blocking time of the blocking solution is 60 minutes; and the amount of the blocking solution used is 200 μL/well. 6. Use of the ELISA kit for detecting sheep paratuberculosis antibodies according to claim 3, characterized in that 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 of the enzyme-labeled secondary antibody is 45 minutes. 8. Use of the ELISA kit for detecting sheep paratuberculosis antibodies according to 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 dark conditions. 9. The use of an ELISA kit for detecting ovine paratuberculosis antibodies as claimed in claim 3, wherein when the OD _450nm value of the serum sample to be tested is ≤0.489, it is judged as negative; when the OD _450nm value is ≥0.570, it is judged as positive; when 0.489<OD _450nm value<0.570, it is judged as a suspected positive. 10. A method for using an ELISA kit for detecting sheep paratuberculosis antibodies according to claim 1 or 2, characterized in that it comprises 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 determination of OD _{450 nm} : Discard the liquid in the wells, wash five times with 1× PBST, pat dry once, add 100 μL/well of substrate solution, incubate in the dark for 10 min, stop color development with stop solution, and measure the absorbance at OD _{450 nm} ; S6. Result determination: When the OD _450nm value of the serum sample to be tested is ≤0.489, it is determined to be negative; when the OD _450nm value is ≥0.570, it is determined to be positive; when 0.489 < OD _450nm value < 0.570, it is determined to be suspected positive.