An indirect elisa antibody detection kit for bovine paratuberculosis mycobacterium and application thereof

Abstract

The application discloses a Mycobacterium avium subsp. paratuberculosis (MAP) indirect ELISA antibody detection kit and application thereof, and belongs to the technical field of microorganism detection. The kit contains a microplate coated with a recombinant protein MAP 625472 composed of three proteins MAP1272c, MAP0862 and MAP2154c in series. Experiments prove that the indirect ELISA method based on the recombinant protein MAP 625472 has high accuracy, high sensitivity and high specificity. In the detection of clinical serum, the positive coincidence rate of the kit is 96.7%, the negative coincidence rate is 95.1%, and the sensitivity is higher than that of an IDvet paratuberculosis antibody detection kit, indicating that the MAP indirect ELISA antibody detection kit can replace foreign products for the detection of bovine PTB, and provides an effective technical means for the prevention and control of bovine PTB in China.

Description

TECHNICAL FIELD

[0001] The present application relates to a bovine Mycobacterium paratuberculosis indirect ELISA antibody detection kit and its application. The present application belongs to the technical field of microorganism detection. BACKGROUND

[0002] Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogenic bacteria causing paratuberculosis (PTB), also known as Johne's disease (JD), which causes huge economic losses to the livestock industry. MAP is transmitted and endangers public health safety through milk and feces.

[0003] In recent years, important progress has been made in the diagnosis of mycobacteria, however, the diagnosis of PTB has been seriously affected by other mycobacterial infections because they have many homologous antigens, all have slow growth, difficult bacterial isolation, acid-fastness, and other characteristics, and the infection of other mycobacteria affects the progress of MAP infection (Karuppusamy S et al., 2019; Raffo E et al., 2020; Raffo E et al., 2017). Therefore, the development and optimization of sensitive and specific MAP detection technology can more effectively control the spread of MAP, which will have important significance for reducing the prevalence of PTB in livestock herds (Moyano R D et al., 2021).

[0004] Currently, the main PTB diagnosis for cattle includes bacterial culture or acid-fast staining, PCR, pathological detection and ELISA antibody detection. Bacterial culture or acid-fast staining, PCR, pathological detection have the disadvantages of complicated operation, low sensitivity and specificity, and are not suitable for large-scale detection of groups. ELISA antibody detection is simple to operate, sensitive and specific, but although there are commercial PTB serological diagnostic products at present, such as IDvet paratuberculosis antibody detection kit and IDEXX Mycobacterium paratuberculosis antibody detection kit, the price is expensive, the step of absorbing non-specific reaction by adding Mycobacterium smegmatis antigen is needed, and there is a certain difference in detecting all infected animals (Costanzo G et al., 2012; Li L et al., 2017). Therefore, it is urgent to develop a MAP ELISA antibody detection method with independent intellectual property rights and can be popularized.

[0005] The application utilizes MAP specific antigens MAP0862, MAP2154c and MAP1272c, obtains high-purity coated antigens through prokaryotic expression and affinity chromatography purification, establishes a rapid, sensitive and specific MAP indirect ELISA antibody detection method through screening of raw materials and optimization of methods, fills the domestic product blank, and provides strong technical support for effective prevention and control of bovine PTB. SUMMARY

[0006] The application aims to provide a bovine Mycobacterium paratuberculosis indirect ELISA antibody detection kit and application thereof.

[0007] In order to achieve the above-mentioned purpose, the following technical means are adopted in the application:

[0008] The application selects seven specific antigen genes of MAP reported in the early stage, namely MAP0847, MAP1272c, MAP3184, MAP3812c, MAP3890, MAP0862 and MAP2154c, obtains seven kinds of recombinant proteins through prokaryotic expression and affinity chromatography purification. Seven kinds of recombinant proteins are respectively used as coated antigens, and 50 MAP clinical positive bovine serum samples and 50 MAP clinical negative bovine serum samples are used to evaluate the antigenicity of the seven kinds of recombinant proteins. The results show that the sensitivities of MAP0847, MAP1272c, MAP3184, MAP3812c, MAP3890, MAP0862 and MAP2154c are 84%, 94%, 88%, 92%, 92%, 94% and 96% respectively, and the specificities are 94%, 100%, 96%, 96%, 98%, 100% and 100% respectively.

[0009] The three proteins of MAP1272c, MAP0862 and MAP2154c with high sensitivity and strong specificity are used for tandem fusion expression, purification and antigenicity analysis. The results show that the recombinant protein MAP 625472 (78.2 kDa) with high purity and good antigenicity is obtained, which is used as the coated antigen for MAP indirect ELISA.

[0010] Through optimization and screening of the concentration of the recombinant protein MAP 625472 coated antigen, the blocking solution, the sample diluent, the serum dilution, the enzyme-labeled secondary antibody protective solution and dilution, the color development time, and determination of the positive and negative critical values, the MAP indirect ELISA detection method is established, the Cutoff value of which is 0.300, the sensitivity is 96.67%, the specificity is 93.48% (AUC=0.975); the batch and batch variation coefficients are between 0.73%-6.38% and 0.49%-6.5% respectively; the coincidence rate test result shows that: the positive coincidence rate of the MAP indirect ELISA antibody detection method and the IDvet paratuberculosis antibody detection kit is 96.7%, the negative coincidence rate is 95.1%, the total coincidence rate is 95.2%, and the sensitivity is better than that of the IDvet kit; the assembled MAP indirect ELISA antibody detection kit is used to detect 11,127 clinical bovine serum samples from Xinjiang, Heilongjiang, Shandong, Shanghai, Liaoning and Guangdong. The results show that the positive rates of MAP antibodies are 5.4%, 9.7%, 21.3%, 7.6%, 68.2% and 7.6% respectively, and the coincidence rate of 186 MAP antibody positive bovine and MAP-IS900 nucleic acid positive is 91.40%.

[0011] On the basis of the above research, the application provides a bovine Mycobacterium avium subsp. paratuberculosis (MAP) indirect ELISA antibody detection kit, the kit contains a microplate coated with a recombinant protein MAP 625472 composed of three proteins MAP1272c, MAP0862 and MAP2154c in series, and the amino acid sequence of the recombinant protein MAP 625472 is shown as SEQ ID NO. 21.

[0012] Preferably, the coating buffer solution used for preparing the microplate coated with the recombinant protein MAP 625472 is CB solution, the coating concentration of the recombinant protein MAP 625472 is 10 μg / mL, and the coating condition is 4℃ for 16-20 h; the blocking solution is a PBS solution containing 5% w / w gelatin and 5% w / w trehalose.

[0013] Preferably, the kit further contains MAP negative control, MAP positive control, MAP sample diluent, enzyme-labeled secondary antibody, 10x concentrated washing solution, color developing solution A, color developing solution B and stop solution.

[0014] Preferably, the enzyme-labeled secondary antibody is goat anti-bovine IgG-HRP enzyme conjugate, the protective solution of the enzyme-labeled secondary antibody is a PB solution containing 5% v / v rabbit serum, and the dilution of the enzyme-labeled secondary antibody is 1:30,000; the MAP sample diluent is a PBS solution containing 5% v / v horse serum and 0.1% w / w casein; and the 10x concentrated washing solution is a 10x PBST solution.

[0015] Preferably, when the kit is used for detecting bovine paratuberculosis mycobacterium, the dilution of the serum to be detected is 1:40, the incubation temperature is 25°C, the incubation time of the serum to be detected is 35 min±1 min, the incubation time of the enzyme-labeled secondary antibody is 30 min±1 min, and the color development time is 10 min.

[0016] Further, the present application also provides application of the bovine paratuberculosis mycobacterium indirect ELISA antibody detection kit in preparation of a reagent for detecting bovine paratuberculosis mycobacterium,

[0017] Preferably, when the kit is used for detecting bovine paratuberculosis mycobacterium, the following steps are performed:

[0018] (1) Preparation of reagents

[0019] a) The reagents and micro-well plates are warmed to room temperature before use and are stored at 2-8°C after use;

[0020] b) Preparation of washing solution: dilute the 10x concentrated washing solution with distilled water or deionized water at a ratio of 1:10, and mix well to obtain the working concentration of the washing solution;

[0021] c) Preparation of samples: take the required number of sample dilution plates, and dilute the samples to be detected with the sample diluent according to a certain gradient;

[0022] (2) Operation steps of the indirect ELISA kit

[0023] a) Sample addition: take out the micro-well plates coated with the recombinant protein MAP 625472, add 100 μL of undiluted negative control and positive control to two holes respectively, mark the positive control as PC1 and PC2, and mark the negative control as NC1 and NC2, and add 100 μL of the diluted sample to be detected to each hole; cover the plate with a sealing film, and incubate at 25°C for 35 min±1 min;

[0024] b) Plate washing: shake off the liquid in the holes, fill each hole with the working concentration of the washing solution, and wash 5 times, and shake off the last time and pat dry;

[0025] c) Addition of enzyme-labeled secondary antibody: add 100 μL of enzyme-labeled secondary antibody to each hole, cover the plate with a sealing film, and incubate at 25°C for 30 min±1 min;

[0026] d) Washing the plate: Wash the plate according to step b) above, and pat it dry;

[0027] e) Colorimetric reaction: Before use, take equal volumes of colorimetric solution A and colorimetric solution B and mix them thoroughly. Add 100 μL to each well, cover with a sealing film, and react at 25°C in the dark for 10 min.

[0028] f) Termination and reading: Add 50 μL of termination solution to each well and read the OD value at 450 nm wavelength using an ELISA reader. Read the value within 10 min after termination.

[0029] g) Judgment of experimental validity

[0030] Positive control mean (PC) PC =(PC1+PC2) / 2

[0031] The mean of negative controls (NC) NC =(NC1+NC2) / 2

[0032] Criteria for determining the validity of the experiment: 0.8 ≤ PC ≤1.9, NC ≤0.2;

[0033] If the test is invalid, repeat the test once by following the above steps;

[0034] (3) Judgment of experimental results: The calculation of sample S / P = (sample OD value - NC) ) / (PC - NC If the S / P ratio is ≥0.3, the sample is judged to be antibody positive; if the S / P ratio is <0.3, the sample is judged to be antibody negative.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] The indirect ELISA method based on recombinant protein MAP 625472 established in this invention exhibits high accuracy (AUC=0.9875), high sensitivity (96.67%), and high specificity (93.48%) for selected cutoff values ​​(>0.3000). In detecting clinical serum, the positive concordance rate with the IDvet paratuberculosis antibody detection kit was 96.7%, and the negative concordance rate was 95.1%. Furthermore, in the sensitivity test using clinical positive serum, half of the serum samples showed higher sensitivity than the IDvet paratuberculosis antibody detection kit. This indicates that the MAP indirect ELISA antibody detection method based on recombinant protein 625472 of this invention can replace foreign products for the detection of bovine PTB, providing a powerful technical means for the effective prevention and control of bovine PTB in my country. Attached Figure Description

[0037] Figure 1 PCR amplification of MAP0862, MAP2154c and MAP1272c;

[0038] Wherein, 1: DNA Marker; 2: MAP0862 gene fragment; 3: MAP2154c gene fragment; 4: MAP1272c gene fragment;

[0039] Figure 2 SDS-PAGE was used to identify the expression of the 625472 protein;

[0040] Wherein, 1: pET22b-625472-BL21 uninduced; 2: pET22b-625472-BL21 induced; 3: supernatant after sonication after pET22b-625472-BL21 induction; 4: precipitate after sonication after pET22b-625472-BL21 induction; M: protein molecular weight marker;

[0041] Figure 3 SDS-PAGE identification for affinity chromatography purification of 625472 protein;

[0042] Wherein, 1: protein molecular weight marker; 2: 100 mM imidazole elution; 3: 150 mM imidazole elution; 4: 200 mM imidazole elution;

[0043] Figure 4 Antigenicity analysis of recombinant protein MAP 625472;

[0044] Among them, A: MAP negative serum; B: MAP positive serum; C: anti-GST tag protein polyclonal antibody; D: anti-MAP0862 protein polyclonal antibody; E: anti-MAP2154c protein polyclonal antibody; F: anti-MAP1272c protein polyclonal antibody;

[0045] Figure 5 Critical value analysis for the MAP indirect ELISA detection method;

[0046] Among them, A: ROC data analysis of MAP clinical positive and negative serum; B: ROC curve of MAP clinical positive and negative serum;

[0047] Figure 6 For frequency histograms;

[0048] Among them, A: frequency histogram of S / P values ​​at a 1:320 dilution; B: frequency histogram of S / P values ​​at a 1:640 dilution; C: frequency histogram of S / P values ​​at a 1:1,280 dilution; D: frequency histogram of S / P values ​​at a 1:2,560 dilution.

[0049] Figure 7 For Levry-Jenings control charts;

[0050] A: Levry-Jenings control chart at a 1:320 dilution; B: Levry-Jenings control chart at a 1:640 dilution; C: Levry-Jenings control chart at a 1:1,280 dilution; D: Levry-Jenings control chart at a 1:2,560 dilution. Detailed Implementation

[0051] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become clearer as a result. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0052] The experimental materials and their sources involved in the above embodiments are as follows:

[0053] 1. Bacterial strains and laboratory animals

[0054] Mycobacterium tumefaciens strain ATCC11728 and MAP K-10 were purchased from ATCC; Mycobacterium bovis AN5 (CVCC291) was purchased from the China Institute of Veterinary Drug Control; Mycoplasma bovis strain from Hubei was isolated and preserved by the Animal Mycoplasma Disease Innovation Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; Escherichia coli BL21 (DE3) was preserved in our laboratory; 12-month-old healthy cattle (whose antibodies were negative by Brucella antibody detection kit, Foot-and-mouth disease antibody detection kit, BVDV antibody detection kit, Mycobacterium paratuberculosis antibody detection kit, Mycoplasma bovis ELISA antibody detection kit, Mycobacterium bovis ELISA antibody detection kit, and Escherichia coli BL21 whole cell protein ELISA detection method) were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0055] 2. Test kit

[0056] Brucella antibody test kit, foot-and-mouth disease antibody test kit, and bovine mycoplasma ELISA antibody test kit were purchased from Harbin Guosheng Biotechnology Co., Ltd.; bovine mycobacterium ELISA antibody test kit was purchased from Wuhan Keqian Animal Biotechnology Co., Ltd.; and bovine viral diarrhea p80 antibody test kit and paratuberculosis antibody test kit were purchased from IDvet, France.

[0057] 3. Reagents and Instruments

[0058] Sheep anti-bovine IgG-HRP enzyme conjugate was purchased from Jackson Biopharmaceuticals; bovine viral diarrhea / mucosal disease inactivated vaccine was purchased from Huawit (Jiangsu) Biopharmaceutical Co., Ltd.; MONTANIDE ISA 201 VG was purchased from Seppic Biopharmaceuticals, France; and ELX800 microplate reader was purchased from BioTeK, USA.

[0059] Example 1: Screening for Mycobacterium paratuberculosis antigens

[0060] This embodiment uses seven previously reported MAP specific antigen genes: MAP0847, MAP1272c, MAP3184, MAP3812c, MAP3890, MAP0862, and MAP2154c. PCR amplification was performed using PrimeSTAR Max DNA polymerase and the primers listed in Table 1, with the MAP K-10 genome as a template. After double digestion of the PCR products, ligation was performed using T4 DNA ligase with the pET22b vector treated with the same enzyme. Finally, the obtained recombinant plasmid was transformed into DH5α, and the plasmid was extracted and identified by double enzyme digestion. Correct recombinant plasmids were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing verification.

[0061] Table 1 Primers

[0062]

[0063]

[0064] Seven recombinant proteins were obtained through prokaryotic expression and affinity chromatography purification. The antigenicity and specificity of these seven recombinant proteins were evaluated using 50 clinically positive and 50 clinically negative bovine serum samples for MAP as coating antigens.

[0065] (1) Evaluation of the antigenicity of seven MAP proteins by clinical negative / positive MAP serum showed that the sensitivities of MAP0847, MAP1272c, MAP3184, MAP3812c, MAP3890, MAP0862 and MAP2154c were 84% (42 / 50), 94% (47 / 50), 88% (44 / 50), 92% (46 / 50), 92% (46 / 50), 94% (47 / 50) and 96% (48 / 50), respectively, and the specificities were 94% (47 / 50), 100% (50 / 50), 96% (48 / 50), 96% (48 / 50), 98% (49 / 50), 100% (50 / 50) and 100% (50 / 50), respectively.

[0066] (2) Specificity test results showed that MAP0862, MAP2154c, and MAP1272c antigens showed no cross-reactivity with bovine serum positive for Mycoplasma bovis, Mycobacterium tumefaciens, Bovine viral diarrhea virus, Mycobacterium bovis, Escherichia coli BL21, and recombinant Escherichia coli pET22b-BL21. The construction method of recombinant Escherichia coli pET22b-BL21 was as follows: the empty pET22b vector was transformed into BL21 competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated at 37°C for 12-16 h. Single colonies were picked and cultured in 5 mL of LB liquid medium (containing 100 μg / mL ampicillin) at 37°C for 10-12 h, which yielded recombinant Escherichia coli pET22b-BL21.

[0067] Tandem fusion expression, purification, and antigenicity analysis of three proteins, MAP1272c, MAP0862, and MAP2154c, which exhibit high sensitivity and specificity.

[0068] Example 2: Construction, expression, purification, and antigenicity analysis of recombinant protein MAP 625472

[0069] method:

[0070] 1. Construction of prokaryotic expression vectors

[0071] (1) Amplification of the target gene

[0072] Using MAP K-10 genomic DNA as a template, the MAP0862, MAP2154, and MAP1272c genes were amplified using the primers and PrimerSTAR Max polymerase listed in Table 2. The amplification cycle parameters were: 98℃ pre-denaturation for 10 s, 98℃ denaturation for 10 s, 65℃ annealing for 5 s, and 72℃ extension for 10 s, followed by a final extension at 72℃ for 1 min. The amplification products were detected by 1% agarose gel electrophoresis, and the PCR products were purified using a gel extraction kit.

[0073] Table 2 Primers

[0074]

[0075] (2) Construction of recombinant plasmids

[0076] The pET22b vector was double-digested with NdeI and XhoI, and the digested products were purified by 1% agarose gel electrophoresis and gel extraction kit. The MAP0862, MAP2154c, and MAP1272c genes were ligated into the pET22b vector using a recombinant method. The reaction conditions were as follows: 1 μL of MAP0862 PCR product, 1 μL of MAP2154c PCR product, 1 μL of MAP1272c PCR product, 2 μL of double-digested pET22b vector, and 5 μL of SoSoo Mix Plus. The reaction was carried out at 50℃ for 15 min, and the mixture was transformed into DH5α competent cells. The correctly identified plasmid was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing verification, yielding the recombinant plasmid pET22b-625472.

[0077] 2. Expression and purification of recombinant protein MAP 625472

[0078] The correctly sequenced recombinant plasmid pET22b-625472 was transformed into competent BL21 expression cells. The cells were evenly spread on solid LB agar plates (containing 100 μg / mL ampicillin). A single colony was picked and inoculated into 5 mL of LB liquid medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C and 220 rpm. This bacterial culture was then transferred 1:100 to LB liquid medium (containing 100 μg / mL ampicillin) and incubated at 37°C and 180 rpm until OD500. 600nm When the value is 0.6-0.8, add IPTG to a final concentration of 1 mM and induce for 4 hours. Centrifuge to collect the bacterial cells, discard the supernatant, suspend the bacterial solution in Buffer A, and sonicate for 30 min, sonicating for 3 s and then pausing for 5 s.

[0079] Resuspend the centrifuged bacterial pellet in denaturing buffer B until completely dissolved. Place the dissolved pellet in a dialysis bag and dialyze to renature with buffer C, changing the dialysis buffer every 24 hours. Centrifuge the dialyzed protein at 15,000 rpm for 30 minutes at 4°C.

[0080] The supernatant after centrifugation was purified using a Glutathione Sepharose 6 Fast Flow resin column, and unbound proteins were washed away with 40 mL of Buffer D and 40 mL of Buffer E, respectively. The target protein was eluted with 5 mL of 20 mM Tris-HCl-10 mM reduced glutathione-pH 8.0, and then concentrated and desalted using an ultrafiltration tube.

[0081] 3. Antigenicity analysis of recombinant protein MAP 625472

[0082] 1) Transfer: The purified recombinant protein MAP 625472 was identified by SDS-PAGE and then transferred onto an NC membrane.

[0083] 2) Blocking: Immerse the NC membrane in PBS solution containing 5% (V / V) fish skin glue and incubate at 37°C for 2 h.

[0084] 3) Washing: Wash the membrane 3 times with PBST, 3-5 minutes each time.

[0085] 4) Primary antibody incubation: Bovine MAP positive serum (1:40), bovine MAP negative serum (1:40), MAP0862 mouse positive serum (1:1,000), MAP2154c mouse positive serum (1:1,000), MAP1272c mouse positive serum (1:1,000), and GST mouse positive serum (1:1,000) were diluted with PBS according to the above ratios. Six NC membranes were then immersed in the diluted positive and negative sera and incubated at 37°C for 1 h.

[0086] 5) Washing: Wash the membrane 3 times with PBST, 3-5 minutes each time.

[0087] 6) Secondary antibody incubation: Dilute HRP-labeled anti-bovine IgG and HRP-labeled anti-mouse IgG with PBST at a ratio of 1:10,000 (V / V) and dilute them at a ratio of 1:20,000. Immerse the NC membrane in the corresponding diluted secondary antibody solution and incubate at 37°C for 1 h.

[0088] 7) Washing: Wash the membrane 3 times with PBST, 3-5 minutes each time.

[0089] 8) Color development: Develop color according to the DAB color development kit instructions, rinse the NC membrane with water, take a picture to record the color, and then store it in PBS.

[0090] 9) Judgment: After DAB color development, a positive result is a clear colored band at the 78.2kDa position on the NC membrane, and a negative result is no colored band on the NC membrane.

[0091] result:

[0092] 1. PCR amplification of the target gene

[0093] Using MAP K-10 genomic DNA as a template, the MAP0862, MAP2154, and MAP1272c genes were amplified by PCR. The PCR products were detected by 1% agarose gel electrophoresis. The results showed that the amplified products were consistent with the expected sizes, approximately 927 bp, 552 bp, and 639 bp, respectively. (See details...) Figure 1 .

[0094] 2. Expression of recombinant protein MAP 625472

[0095] After the correctly identified pET22b-625472 recombinant plasmid was transformed into the BL21 expression strain, and induced at 37℃ for 4 h, approximately 78.2 kDa of protein expression was observed, consistent with the expected protein size. Figure 2 The amino acid sequence of the recombinant protein MAP 625472 obtained is shown in SEQ ID NO.21.

[0096] 3. Purification of recombinant protein MAP 625472

[0097] The recombinant protein MAP 625472, after induced expression, was purified by denaturation, renaturation, and affinity chromatography to obtain high-purity recombinant protein MAP 625472 (78.2 kDa). Figure 3 ).

[0098] 4. Antigenicity analysis of recombinant protein MAP 625472

[0099] Western blot results showed that a specific band of 78.2 kDa appeared on the NC membrane of MAP-positive serum, mouse polyclonal antibodies against MAP0862 protein, mouse polyclonal antibodies against MAP2154c protein, and mouse polyclonal antibodies against MAP1272c protein, while no band was observed on the NC membrane of MAP-negative serum and mouse polyclonal antibodies against GST-tagged protein. These results indicate that the recombinant protein MAP 625472 has good antigenicity and did not affect the antigenicity of the three proteins MAP0862, MAP2154c, and MAP1272c. Figure 4 ).

[0100] Example 3: Establishment and application of an indirect ELISA antibody detection kit for Mycobacterium paratuberculosis.

[0101] method:

[0102] 1. Operation steps of MAP indirect ELISA detection method

[0103] 1) Coating: Dilute the purified recombinant protein MAP 625472 to 10 μg / mL with CB (Carbonate-Bicarbonate Buffer), 100 μL per well, and incubate at 4℃ for 16-20 h.

[0104] 2) Washing: Pour out the liquid in the wells, add 1×PBST, 300 μL / well, wash 5 times, and pat dry.

[0105] 3) Blocking: Add blocking solution, 150 μL / well, and incubate at 37℃ for 3-4 h.

[0106] 4) Washing: The washing method is the same as step 2).

[0107] 5) Add sample: Add a certain amount of diluted test sample, 100 μL / well, and incubate at 37℃ for 35 min.

[0108] 6) Washing: The washing method is the same as step 2).

[0109] 7) Add enzyme-labeled antibody: Add 100 μL of freshly diluted goat anti-bovine IgG-HRP enzyme conjugate to each well and incubate at 37℃ for 30 min.

[0110] 8) Washing: The washing method is the same as step 2).

[0111] 9) Add substrate and colorimetric solution: Add 100 μL of substrate solution to each reaction well and react at 37°C for 10 min.

[0112] 10) Termination: Add 50 μL of stop solution to each well to terminate the reaction. Read the OD value at 450 nm using a microplate reader (within 30 min after termination) and record the results.

[0113] 2. Optimization of reaction conditions for MAP indirect ELISA

[0114] 2.1 Screening of optimal coating solution and optimal coating conditions

[0115] The affinity-purified recombinant protein MAP 625472 was diluted to 10 μg / mL using three coating buffers: CB (Carbonate-Bicarbonate Buffer), PB (Phosphate-Buffered Saline), and TB (Tris-Buffered Saline). 100 μL of each buffer was added to each well of a 96-well ELISA plate. The plates were incubated under three conditions: 37°C for 4 h, 25°C for 16–20 h, and 4°C for 16–20 h. Bovine MAP-positive serum (three replicates) and bovine MAP-negative serum (three replicates) with known background were analyzed using the indirect ELISA method described above. The maximum P / N ratio (OD value of positive serum) was recorded. 450nm Mean / Negative Serum OD 450nm The coating buffer and coating conditions corresponding to the average value are taken as the optimal coating buffer and optimal coating conditions.

[0116] 2.2 Screening of blocking solutions

[0117] The purified recombinant protein MAP 625472 was diluted to 10 μg / mL with the optimal coating buffer, 100 μL per well, and incubated at 4°C for 16–20 h. The liquid in the wells was discarded, and the cells were washed five times with 1×PBST washing buffer and blotted dry. Blocking was performed with PBS solutions containing 5% w / w gelatin + 5% w / w trehalose, 1% w / w ovalbumin + 5% w / w trehalose, and 5% v / v rabbit serum + 5% w / w trehalose, respectively. Bovine MAP-positive serology (three replicates) and bovine MAP-negative serology (three replicates) with known background were analyzed using the indirect ELISA method described above. The maximum P / N value (OD value of positive serum) was recorded. 450nm Mean / Negative Serum OD 450nm The sealing liquid corresponding to the average value is taken as the optimal sealing liquid.

[0118] 2.3 Determination of the optimal antigen coating concentration and the optimal dilution of the serum to be tested

[0119] 1) Dilute the recombinant protein MAP 625472 at a concentration of 2 mg / mL to 50 μg / mL, 10 μg / mL, 5 μg / mL and 1 μg / mL coating working solutions respectively with the best coating buffer. After dilution, add 100 μL / well to the microplate and incubate at 4℃ for 16-20 h.

[0120] 2) Pour out the liquid from the wells, wash 5 times with 1×PBST washing buffer, and pat dry. Add the optimal blocking buffer to block the microplate at 150 μL / well, and incubate at 37℃ for 3-4 h.

[0121] 3) Discard the sealing solution and dry it at room temperature (10-30℃) and humidity ≤25% for 18-22 hours.

[0122] 4) Clinical positive and negative sera with known backgrounds were diluted at ratios of 1:10, 1:20, 1:40 and 1:80 and added to coated plates with different coating working solutions, 100 μL / well, and incubated at 37°C for 35 min.

[0123] 5) Following the indirect ELISA method described above, test bovine MAP-positive serology (three replicates) and MAP-negative serology (three replicates) with known background, and take the maximum P / N value (OD value of positive serum). 450nm Mean / Negative Serum OD 450nm The optimal concentration is the antigen coating concentration corresponding to the average value and the dilution concentration of the serum to be tested.

[0124] 2.4 Screening of Sample Diluents

[0125] 1) Dilute the purified recombinant protein MAP 625472 to 10 μg / mL with the optimal coating buffer, 100 μL per well, and incubate at 4℃ for 16-20 h.

[0126] 2) Pour out the liquid from the well, wash 5 times with 1×PBST washing solution for 30 seconds each time, and pat dry.

[0127] 3) Block the microplate with the optimal blocking solution, 150 μL / well, at 37℃ for 3-4 h.

[0128] 4) Discard the sealing solution and dry it at room temperature (10-30℃) and humidity ≤25% for 18-22 hours.

[0129] 5) Four sample diluents were used: 5% w / w gelatin + 0.1% w / w casein, 5% v / v horse serum + 0.1% w / w casein, 1% w / w ovalbumin + 0.1% w / w casein, and 5% v / v rabbit serum + 0.1% w / w casein in PBS solution. Bovine MAP positive and negative sera with known background were diluted to the optimal dilution.

[0130] 6) Add diluted serum samples (100 μL / well) to the coated plate and incubate at 37°C for 35 min. Perform indirect ELISA on bovine MAP-positive serum (three replicates) and negative serum (three replicates) with known background, and record the maximum P / N value (OD value of positive serum). 450nm Mean / Negative Serum OD 450nm The sample diluent corresponding to the average value is taken as the optimal sample diluent.

[0131] 2.5 Screening of enzyme-labeled secondary antibody protective solution

[0132] Goat anti-bovine IgG-HRP enzyme conjugates were diluted 1:30,000 using PB solutions containing 0.1% w / w ovalbumin and 5% v / v rabbit serum, respectively. The diluted goat anti-bovine IgG-HRP enzyme conjugates were then stored at 37°C and -20°C for 10 days. Destructive testing was performed on days 1, 3, 5, 7, and 10 as a reference for the performance of the enzyme-labeled secondary antibody protective solution. A control group was also included, stored at 4°C. Both the experimental and control groups were tested using the optimized indirect ELISA conditions described above. Bovine MAP-positive serology (three replicates) and bovine MAP-negative serology (three replicates) with known background were analyzed according to the indirect ELISA method described above. The P / N ratio (OD of positive serum) under different storage conditions was used as a reference. 450nm Mean / Negative Serum OD 450nm The stability of the average value was used to determine the optimal enzyme-labeled secondary antibody protection solution.

[0133] 2.6 Determination of enzyme-labeled secondary antibody dilution

[0134] Following the established optimal ELISA conditions, goat anti-bovine IgG-HRP enzyme conjugate was diluted 1:10,000, 1:20,000, 1:30,000, and 1:40,000 using the optimal enzyme-labeled secondary antibody protective solution. Bovine MAP-positive serology (three replicates) and MAP-negative serology (three replicates) with known background were then analyzed using the indirect ELISA method described above. The maximum P / N value (OD value of positive serum) was recorded. 450nm Mean / Negative Serum OD 450nm The optimal working concentration is the enzyme-labeled secondary antibody dilution corresponding to the average value.

[0135] 2.7 Determination of the optimal incubation time and temperature for the reagent kit

[0136] Following the optimal ELISA conditions determined above, at 37℃ and 25℃, the detection results were compared for different primary antibody incubation times + secondary antibody incubation times + display times (35 min + 30 min + 15 min, 35 min + 30 min + 10 min, 60 min + 30 min + 15 min, 60 min + 30 min + 10 min). Referring to the indirect ELISA method described above, bovine MAP-positive serology (three replicates) and MAP-negative serology (three replicates) with known background were detected, using the maximum P / N value (OD value of positive serum). 450nm Mean / Negative Serum OD 450nm The optimal incubation time is determined based on the principle of average value.

[0137] 3. Determination of the cutoff value for the MAP indirect ELISA detection method

[0138] 3.1 MAP Clinical Serum

[0139] 147 clinically positive MAP serum samples were collected (positive for MAP antibodies using the IDvet paratuberculosis antibody detection kit and positive for nucleic acid in MAP-PCR of rectal scrapings), and 138 clinically negative MAP serum samples were collected (negative for MAP antibodies using the IDvet paratuberculosis antibody detection kit and negative for nucleic acid in MAP-PCR of rectal scrapings). The sources of the clinical serum are shown in Table 3.

[0140] Table 3. Statistical table of serum sources for MAP clinical trials

[0141]

[0142] 3.2 Establishment of the MAP indirect ELISA detection method

[0143] The following methods were used to test 285 clinical serum samples from patients with MAP.

[0144] 1) Coating: Dilute the purified recombinant protein MAP 625472 to 10 μg / mL with CB coating buffer, 100 μL per well, and incubate at 4℃ for 16-20 h.

[0145] 2) Washing: Pour out the liquid in the wells, add 1×PBST, 300 μL / well, wash 5 times, and pat dry.

[0146] 3) Blocking: Add 150 μL of PBS solution containing 5% w / w gelatin + 5% w / w trehalose to each well and incubate at 37℃ for 3-4 h.

[0147] 4) Washing: The washing method is the same as step 2).

[0148] 5) Add the serum to be tested: Dilute the serum to be tested with PBS solution containing 5% v / v horse serum + 0.1% w / w casein at a ratio of 1:40, 100 μL / well, and incubate at 25℃ for 35 min.

[0149] 6) Washing: The washing method is the same as step 2).

[0150] 7) Add enzyme-labeled secondary antibody: Dilute goat anti-bovine IgG-HRP enzyme conjugate 1:30,000 with PB solution containing 5% v / v rabbit serum, 100 μL / well, and incubate at 25℃ for 30 min.

[0151] 8) Washing: The washing method is the same as step 2).

[0152] 9) Add colorimetric solutions: Add 50 μL of colorimetric solution A per well and 50 μL of colorimetric solution B per well in sequence, and incubate at 25℃ for 10 min.

[0153] 10) Add stop solution: Add 100 μL of 2 M sulfuric acid to each well, take a reading at a wavelength of 450 nm (the reading should be completed within 10 min after adding the stop solution), and record the result.

[0154] 11) S / P value calculation: S / P = (OD of clinical sample) 450nm Value – Negative control serum OD 450nm (mean) / (positive control serum OD) 450nm Mean value - negative control serum OD 450nm (mean).

[0155] 12) Critical value determination: based on the clinical MAP serum sample OD 450nm Based on the measurement results, the S / P value of each sample was calculated, and ROC data analysis of the S / P values ​​of the test results was performed using GraphPad Prism 9.0 software to obtain the optimal critical value.

[0156] 4. Determination of sensitivity criteria for the MAP indirect ELISA detection method

[0157] 4.1 Usage and Judgment of the Trial Reagent Kit of the Present Invention

[0158] (1) Preparation of reagents

[0159] a) Before use, bring the reagents and microplates to room temperature, and after use, return them to 2-8℃.

[0160] b) Preparation of washing solution: Dilute the 10× concentrated washing solution with distilled water or deionized water at a ratio of 1:10 (e.g., take 100 mL of concentrated solution and add 900 mL of distilled water or deionized water), and mix thoroughly to obtain the washing solution of working concentration.

[0161] c) Sample preparation: Take the required number of sample dilution plates and use sample diluent to serially dilute the samples to be tested according to a certain gradient. Note that you should not dilute the positive and negative controls.

[0162] (2) Indirect ELISA kit operation steps

[0163] a) Sample addition: Remove the coated plate and add 100 μL of undiluted negative control and positive control (two wells each, labeled PC1 and PC2 for positive control and NC1 and NC2 for negative control). Add 100 μL of diluted test sample to each well of the test sample. Cover with sealing film and incubate at 25°C for 35 min (±1 min).

[0164] b) Washing the plate: Shake off the liquid in the holes, fill each hole with the working concentration of washing solution and wash 5 times. Shake off the last time and pat dry.

[0165] c) Add enzyme-labeled secondary antibody: Add 100 μL of goat anti-bovine IgG-HRP enzyme conjugate to each well, cover with sealing film, and incubate at 25℃ for 30 min (±1 min).

[0166] d) Wash the plate: Wash the plate according to step b) above, and pat it dry.

[0167] e) Colorimetric reaction: Before use, take equal volumes of colorimetric solution A and colorimetric solution B and mix them thoroughly. Add 100 μL to each well, cover with a sealing film, and react at 25°C in the dark for 10 min.

[0168] f) Termination and reading: Add 50 μL of stop solution to each well and read the OD value at 450 nm wavelength using an ELISA reader (read the value within 10 min after termination).

[0169] g) Judgment of experimental validity

[0170] Positive control mean (PC) PC =(PC1+PC2) / 2

[0171] The mean of negative controls (NC) NC =(NC1+NC2) / 2

[0172] Criteria for determining the validity of the experiment: 0.8 ≤ PC ≤1.9, NC ≤0.2.

[0173] If the test is invalid, the operation in the test is questionable, and the test should be repeated according to the operating instructions.

[0174] (3) Judgment of experimental results: The calculation of sample S / P = (sample OD value - NC) ) / (PC - NC If the S / P ratio is ≥0.3, the sample is judged to be antibody positive; if the S / P ratio is <0.3, the sample is judged to be antibody negative.

[0175] 4.2 Determination of the Limit of Detection

[0176] Sensitivity control sera were diluted at ratios of 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560, and 1:5,120. Repeatability was validated 10 times using three laboratory-prepared batches of the product (20200301, 20200302, and 20200303). The S / P value of serum samples at different dilutions was calculated according to the kit's determination method. The maximum dilution factor at which the serum dilution (S / P value ≥ 0.3) still showed a positive result was used as the limit of detection for this kit.

[0177] 4.3 Determination of sensitivity testing standards for reagent kits

[0178] Using the Lvery-Jennings control chart method, statistical analysis was performed on the data of sensitivity control sera at dilutions of 1:320, 1:640, 1:1,280, and 1:2,560, based on the data in section 4.2. Frequency analysis was performed on the test data using SPSS 18.0, and histograms were plotted to analyze the distribution characteristics of the test data. Simultaneously, based on OD... 450nm Average value ( Levery-Jennings control charts were plotted using the standard deviation (SD) and the standard deviation (SD) to determine the sensitivity testing criteria for the kit.

[0179] 5. Evaluation of the effectiveness of the MAP indirect ELISA detection method

[0180] 5.1 Sensitivity Test

[0181] Bovine MAP strong positive serum (bovine MAP positive serum diluted 2-fold), bovine MAP positive serum (bovine MAP positive serum diluted 8-fold), bovine MAP weak positive serum (bovine MAP positive serum diluted 16-fold), 5 bovine MAP positive serum samples (IDvet paratuberculosis antibody detection kit detected positive antibody and rectal scraping MAP-PCR detected positive nucleic acid), and 5 bovine MAP negative serum samples (IDvet paratuberculosis antibody detection kit detected negative antibody and rectal scraping MAP-PCR detected negative nucleic acid) were diluted with sample diluent at ratios of 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560, and 1:5,120. Following the operating steps in 4.1, the three batches of MAP indirect ELISA antibody detection kits (20200301, 20200302, and 20200303) developed in this invention were used to determine the limit of detection (LOD) for clinical samples.

[0182] 5.2 Specificity Test

[0183] Five samples of bovine MAP-negative serum, one sample of bovine mycoplasma-positive serum, one sample of bovine mycobacterium-positive serum, one sample of mycobacterium praephedrine-positive serum, one sample of bovine viral diarrhea virus-positive serum, one sample of Escherichia coli BL21(DE3)-positive bovine serum, and one sample of recombinant Escherichia coli pET22b-BL21(DE3)-positive bovine serum were diluted at a ratio of 1:40 using sample diluent. Following the operating procedure in 4.1, the specificity of the three batches of MAP indirect ELISA antibody detection kits (20200301, 20200302, and 20200303) prepared in this invention was determined.

[0184] 5.3 Repeatability Test

[0185] Bovine MAP strong positive serum, bovine MAP positive serum, bovine MAP weak positive serum, and bovine MAP negative serum were diluted at a ratio of 1:40 using sample diluent. Following the operating procedure in 4.1, three batches of the MAP indirect ELISA antibody detection kits (20200301, 20200302, and 20200303) developed in this invention were used for detection. Five kits were taken from each batch, and each kit was used to test the samples four times. The inter-batch and intra-batch repeatability of each batch of kits was statistically analyzed, and the inter-batch and intra-batch coefficients of variation were calculated.

[0186] 5.4 Comparative test with commercially available kits

[0187] (1) Compliance rate test

[0188] Sixty-one clinical serum samples were diluted 1:40 with sample diluent and tested using the MAP indirect ELISA antibody detection kit of this invention, following the procedure in section 4.1. Simultaneously, a commercially available IDvet paratuberculosis antibody detection kit was used for testing. The positive concordance rate, negative concordance rate, and overall concordance rate between the MAP antibody indirect ELISA kit and the commercial kit were statistically analyzed and calculated.

[0189] (2) Sensitivity comparison test

[0190] Ten clinical serum samples that tested positive with the commercial IDvet paratuberculosis antibody detection kit were selected and diluted at ratios of 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560 and 1:5,120. These samples were then tested using the MAP indirect ELISA antibody detection kit of this invention and the commercial IDvet paratuberculosis antibody detection kit, respectively, and the sensitivity of the two kits was compared.

[0191] 6. Assembly and application of the indirect ELISA antibody detection kit for Mycobacterium paratuberculosis.

[0192] Using the MAP indirect ELISA antibody detection kit developed in the pilot phase of this invention, and following the operating steps in 4.1, bovine serum samples from various regions (975 from Xinjiang, 394 from Shandong, 7,222 from Heilongjiang, 432 from Liaoning, 184 from Shanghai, 1,200 from Guangdong, and 720 from Henan) were tested to analyze the prevalence of pTB in cattle herds. Rectal scrapings from 45 MAP antibody-positive cattle from Xinjiang, 60 from Heilongjiang, and 81 from Liaoning were collected and amplified using MAP-IS900 PCR. The concordance rate between the MAP indirect ELISA antibody detection kit and MAP nucleic acid positivity was calculated.

[0193] result:

[0194] 1. Optimization of MAP indirect ELISA conditions

[0195] 1.1 Screening of optimal coating solution and optimal coating conditions

[0196] Protein 625472 was diluted to 10 μg / mL using CB, PB, and TB coating buffers at 37℃, 25℃, and 4℃, respectively. The results, obtained using an indirect ELISA method, showed that the P / N ratio was highest when using CB buffer and incubation at 4℃ for 16-20 h (see Table 4). These results indicate that CB buffer is the optimal coating buffer, and the optimal incubation condition is 4℃ for 16-20 h.

[0197] Table 4. Detection results of coating solution and coating condition screening (OD) 450nm average value)

[0198]

[0199] 1.2 Optimization of antigen coating concentration and serum dilution

[0200] Recombinant 625472 protein was diluted with CB coating buffer to 50 μg / mL, 10 μg / mL, 5 μg / mL, and 1 μg / mL, respectively, and coated at 4℃ for 16–20 h. The protein was then incubated with different dilutions of test serum (1:10, 1:20, 1:40, 1:80) and detected according to the indirect ELISA procedure. The results showed that the P / N value was highest when the antigen coating concentration was 10 μg / mL and the test serum dilution was 1:40 (see Table 5). These results indicate that the optimal antigen coating concentration is 10 μg / mL, and the optimal serum dilution is 1:40.

[0201] Table 5. Detection results of optimized antigen coating concentration and serum dilution (OD) 450nm average value)

[0202]

[0203] 1.3 Screening of blocking solutions

[0204] Recombinant protein MAP 625472 was diluted to 10 μg / mL using CB coating buffer and coated at 4℃ for 16-20 h. Blocking was performed using 1% w / w ovalbumin + 5% w / w trehalose, 5% w / w gelatin + 5% w / w trehalose, and 5% v / v rabbit serum + 5% w / w trehalose, respectively. The test serum was diluted 1:40, and detection was performed according to the indirect ELISA procedure. The results showed that the P / N value was highest with the blocking buffer of 5% w / w gelatin + 5% w / w trehalose. Therefore, 5% w / w gelatin + 5% w / w trehalose was selected as the optimal blocking buffer (see Table 6).

[0205] Table 6. Detection results of screening different blocking solutions (OD) 450nm average value)

[0206]

[0207] 1.4 Screening of Sample Diluents

[0208] Recombinant protein MAP 625472 was diluted to 10 μg / mL using CB coating buffer and coated at 4℃ for 16-20 h. It was then blocked with 5% w / w gelatin + 5% w / w trehalose. Serum samples were diluted 1:40 with PBS solutions containing 5% w / w gelatin + 0.1% w / w casein, 5% v / v rabbit serum + 0.1% w / w casein, 1% w / w ovalbumin + 0.1% w / w casein, and 5% v / v horse serum + 0.1% w / w casein. Detection was performed according to the indirect ELISA procedure. Results showed that the P / N ratio was highest with PBS solution containing 5% v / v horse serum + 0.1% w / w casein. Therefore, PBS solution containing 5% v / v horse serum + 0.1% w / w casein was selected as the optimal sample dilution (Table 7).

[0209] Table 7. Detection results (OD) of different sample dilution solutions 450nm average value)

[0210]

[0211] 1.5 Screening of enzyme-labeled secondary antibody protective solution

[0212] Recombinant protein MAP 625472 was diluted to 10 μg / mL using CB coating solution and coated at 4℃ for 16-20 h. Blocking was performed using 5% w / w gelatin + 5% w / w trehalose. Serum samples were diluted 1:40 with PBS containing 5% v / w horse serum + 0.1% w / w casein. The enzyme-labeled secondary antibody was diluted with PB solution containing 5% v / w rabbit serum and PB solution containing 0.1% w / w ovalbumin, respectively. Detection was performed according to the indirect ELISA procedure. Results showed that the enzyme stability was best when the enzyme-labeled secondary antibody protection solution was PB solution containing 5% v / w rabbit serum. Therefore, the optimal enzyme-labeled secondary antibody protection solution was determined to be PB solution containing 5% v / w rabbit serum (Table 8).

[0213] Table 8. Results of the destructive test of different enzyme-labeled secondary antibody protective solutions (OD). 450nm average value)

[0214]

[0215] 1.6 Determination of enzyme-labeled secondary antibody dilution

[0216] The recombinant protein MAP 625472 was diluted to 10 μg / mL using CB coating solution and coated at 4℃ for 16-20 h. It was then blocked with PBS solution containing 5% gelatin and 5% trehalose. The serum to be tested was diluted 1:40 with PBS solution containing 5% horse serum and 0.1% casein. The enzyme-labeled secondary antibody was diluted with PB solution containing 5% rabbit serum at ratios of 1:10,000, 1:20,000, 1:30,000, and 1:40,000, respectively. The detection was performed according to the indirect ELISA procedure. The results showed that the P / N value was the highest when the enzyme-labeled secondary antibody was diluted 1:30,000. Therefore, the optimal dilution ratio of the enzyme-labeled secondary antibody was determined to be 1:30,000 (Table 9).

[0217] Table 9. Detection results of different dilutions of enzyme-labeled secondary antibody (OD) 450nm average value)

[0218]

[0219] 1.7 Optimization of incubation time and incubation temperature

[0220] The recombinant protein MAP 625472 was diluted to 10 μg / mL using CB coating solution and coated at 4℃ for 16-20 h. It was then blocked with PBS solution containing 5% w / w gelatin and 5% w / w trehalose. The serum to be tested was diluted 1:40 with PBS solution containing 5% v / v horse serum and 0.1% w / w casein. The enzyme-labeled secondary antibody was diluted 1:30,000 with PB solution containing 5% v / v rabbit serum. Incubation times for the primary antibody + secondary antibody + color development were set at 37℃ and 25℃ for four groups: 35 min + 30 min + 15 min, 35 min + 30 min + 10 min, 60 min + 30 min + 15 min, and 60 min + 30 min + 10 min, respectively. The results showed that the optimal incubation time for the kit was: 35 min of primary antibody incubation, 30 min of secondary antibody incubation, and 10 min of color development at 25℃ (Table 10).

[0221] Table 10. Detection results of incubation time and incubation temperature of the kit (OD) 450nm average value)

[0222]

[0223] 2. Determination of the cutoff value for the MAP indirect ELISA detection method

[0224] The established MAP indirect ELISA method was used to detect 150 clinically positive bovine MAP serum samples and 138 clinically negative bovine MAP serum samples (Tables 11-14). ROC data analysis was performed on the S / P values ​​of the 288 clinical serum samples using GraphPad Prism 9.0 software. The optimal cut-off value was determined based on the best sensitivity and specificity at 95% CI and the area under the curve (AUC) at 95% CI. The analysis results showed that a cut-off value of 0.300 for the MAP indirect ELISA method was the optimal critical point for data analysis. Figure 5 ).

[0225] Table 11 Detection results of MAP clinically positive serum (OD) 450nm value)

[0226]

[0227]

[0228]

[0229] Table 12 Detection results of MAP clinically positive serum (S / P value)

[0230]

[0231]

[0232] Table 13 Detection results of MAP clinically negative serum (OD) 450nm value)

[0233]

[0234]

[0235] Table 14 Detection results of MAP clinically negative serum (S / P value)

[0236]

[0237]

[0238] 3. Determination of sensitivity criteria for the MAP indirect ELISA detection method

[0239] 3.1 Determination of the minimum detection limit of the reagent kit

[0240] Three batches of reagent kits (20200301, 20200302, and 20200303) were tested using sensitivity control sera diluted at ratios of 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560, and 1:5,120. The results showed that at a dilution of 1:2,560, all three batches were positive (S / P value ≥ 0.3), and at a dilution of 1:5,120, all three batches were negative (S / P value < 0.3). Therefore, the limit of detection for sensitivity of the reagent kits was determined to be 1:2,560 (see Tables 15-20).

[0241] Table 15 Detection results of the lowest detection limit (OD) for reagent kit batch 20200301 450nm value)

[0242]

[0243] Table 16 Detection results of the lowest detection limit (OD) for reagent kit batch 20200302 450nm value)

[0244]

[0245] Table 17 Detection results of the lowest detection limit (OD) for reagent kit batch 20200303 450nm value)

[0246]

[0247] Table 18. Detection results of the lowest detection limit (S / P value) for reagent kit batch 20200301

[0248]

[0249] Table 19. Detection results of the lowest detection limit (S / P value) for reagent kit batch 20200302

[0250]

[0251] Table 20. Detection results of the lowest detection limit (S / P value) for reagent kit batch 20200303

[0252]

[0253] 3.2 Determination of sensitivity testing standards for reagent kits

[0254] The detection results of the three batches of reagent kit sensitivity control serum at dilutions of 1:320, 1:640, 1:1,280, and 1:2,560 were statistically analyzed using SPSS 18.0 software (Table 21). Frequency analysis and histogram plots were also performed on the detection data. The results showed that the data conformed to a normal distribution and had good stability. Figure 6 ).

[0255] The average S / P value for a 1:320 dilution was 1.108, with a standard deviation of 0.058. The values ​​for all 29 dilutions ranged from 0.992 to 1.224. ±2SD, 95.5% confidence limit) Figure 7 A), accounting for 96.6% of the total, is in line with the following: ±2SD is the general principle for warning limits, therefore the sensitivity standard for a 1:320 dilution is set as: 0.90 ≤ S / P < 1.25.

[0256] The average S / P value for a 1:640 dilution was 0.877, with a standard deviation of 0.062. The values ​​for all 29 dilutions ranged from 0.754 to 1.000. ±2SD, 95.5% confidence limit) Figure 7 B), accounting for 96.6% of the total, is in line with the following: ±2SD is the general principle for warning limits, therefore the sensitivity standard for a 1:640 dilution is set as: 0.70 ≤ S / P < 1.05.

[0257] The average S / P value for a 1:1,280 dilution was 0.624, with a standard deviation of 0.056, and 29 values ​​falling between 0.511 and 0.737. ±2SD, 95.5% confidence limit) Figure 7 C), accounting for 96.6% of the total, is in line with the following: ±2SD is the general principle for warning limits, therefore the sensitivity standard for a 1:1,280 dilution is set as: 0.45 ≤ S / P < 0.75.

[0258] The average S / P value for a 1:2,560 dilution was 0.415, with a standard deviation of 0.049, and 29 values ​​falling between 0.316 and 0.514. ±2SD, 95.5% confidence limit) Figure 7 D), accounting for 96.6% of the total, is in line with the following: ±2SD is the general principle for warning limits, therefore the sensitivity standard for a 1:2,560 dilution is set as: 0.30 ≤ S / P < 0.55.

[0259] Table 21. Statistical analysis of sensitivity repeatability test results for three batches of reagent kits at different dilutions (S / P values)

[0260]

[0261] 4. Evaluation of the effectiveness of the MAP indirect ELISA detection method

[0262] 4.1 Sensitivity Test

[0263] Three batches of kits were used to test bovine MAP strongly positive serum, bovine MAP positive serum, bovine MAP weakly positive serum, MAP positive serum from 5 infected animals, and MAP negative serum from 5 known animals. The results showed that the limits of detection (LODs) for all three kits were 1:2,560 for strongly positive serum samples, 1:640 for positive serum samples, and 1:320 for weakly positive serum samples. All MAP positive serum samples from known infected animals were positive, with the LOD for known infected animal serum 1 being 1:2,560, for known infected animal serums 2 and 3 being 1:640, and for known infected animal serums 4 and 5 being 1:320. All MAP negative serum samples from known animals were negative (see Tables 22-27 for details).

[0264] Table 22 Sensitivity test results (OD) of kit batch 20200301 450nm value)

[0265] Table 23 Sensitivity test results (S / P values) of kit batch 20200301

[0266]

[0267]

[0268] Note: A positive result is defined as S / P ≥ 0.3; a negative result is defined as S / P < 0.3.

[0269] Table 24 Sensitivity test results (OD) of kit batch 20200302 450nm value)

[0270]

[0271] Table 25 Sensitivity test results (S / P values) of kit batch 20200302

[0272]

[0273] Note: A positive result is defined as S / P ≥ 0.3; a negative result is defined as S / P < 0.3.

[0274] Table 26 Sensitivity test results (OD450nm value) of kit batch 20200303

[0275]

[0276]

[0277] Table 27 Sensitivity test results (S / P values) of batch 20200303 reagent kit

[0278]

[0279] Note: A positive result is defined as S / P ≥ 0.3; a negative result is defined as S / P < 0.3.

[0280] 4.2 Specificity Test

[0281] Three batches (20200301, 20200302, and 20200303) of bovine MAP indirect ELISA antibody detection kits were used to detect five bovine MAP negative serum, bovine mycoplasma positive serum, bovine mycobacterium positive serum, bovine mycobacterium pratibacterium positive serum, bovine viral diarrhea virus positive serum, bovine coli BL21(DE3) positive serum, and recombinant bovine coli pET22b-BL21(DE3) positive serum. All test results were negative. See Tables 28 and 29 for details.

[0282] Table 28 Specificity test results (OD) of three batches of reagent kits 450nm value)

[0283]

[0284] Table 29 Specificity test results (S / P values) of three batches of reagent kits

[0285]

[0286]

[0287] 4.3 Intra-batch repeatability test

[0288] Five different bovine MAP indirect ELISA antibody detection kits from the same batch (20200301, 20200302, and 20200303) were used to detect bovine MAP strongly positive, positive, weakly positive, and negative serum samples. Each kit was tested four times. The intra-batch coefficient of variation (CVA) of the three batches of kits was calculated. The results showed that the CVA for positive samples in 20200301, 20200302, and 20200303 were between 1.26% and 5.68%, 0.73% and 4.76%, and 1.14% and 6.38%, respectively. The results for negative samples were all negative. These results indicate that the kit has good intra-batch reproducibility.

[0289] 4.4 Inter-batch repeatability test

[0290] Three different batches of the test kit (20200301, 20200302, and 20200303) were used to test serum samples with strong positive, positive, weak positive, and negative MAP results. Each kit was tested four times. The inter-batch coefficient of variation for positive samples ranged from 0.49% to 6.50%, and all negative samples were negative, indicating that the kit has good inter-batch repeatability.

[0291] 4.5 Compliance test with commercially available kits

[0292] Sixty-one clinical serum samples were tested using the pilot-scale MAP indirect ELISA antibody detection kit and the commercial IDvet paratuberculosis antibody detection kit, respectively. The results showed that the positive concordance rate with the commercial IDvet kit was 96.7%, the negative concordance rate was 95.1%, and the overall concordance rate was 95.2%. The statistical results are shown in Table 30.

[0293] Table 30 Statistical results of the conformity rate between three batches of pilot-scale products and the IDvet reagent kit

[0294]

[0295] 4.6 Sensitivity comparison test with commercially available kits

[0296] Ten clinically positive serum samples were tested using both the pilot-scale MAP indirect ELISA antibody detection kit and the IDvet paratuberculosis antibody detection kit, and their sensitivity was compared. The results showed that the MAP indirect ELISA antibody detection kit had significantly higher sensitivity than the IDvet paratuberculosis antibody detection kit for serum samples 1, 3, 4, 8, and 9. For serum samples 2, 5, 6, 7, and 10, the sensitivity of both kits was consistent. These results indicate that the MAP indirect ELISA antibody detection kit has higher sensitivity than the IDvet paratuberculosis antibody detection kit. (Tables 31-34)

[0297] Table 31. Sensitivity test results of clinical positive serum for pilot-scale products (OD) 450nm value)

[0298]

[0299] Table 32 Sensitivity test results of pilot-scale products in clinical positive serum (S / P value)

[0300]

[0301] Table 33 Sensitivity test results of clinically positive serum using the IDvet kit (OD) 450nm value)

[0302]

[0303] Table 34. Sensitivity test results of IDvet kit for clinically positive serum (S / P value)

[0304]

[0305] 5. Assembly and application of the indirect ELISA antibody detection kit for Mycobacterium paratuberculosis

[0306] 5.1 Reagent Kit Assembly

[0307] The components and finished product box of the assembled "Mycobacterium paratuberculosis indirect ELISA antibody detection kit" are shown in Table 35.

[0308] Table 35 Reagent Kit Composition

[0309]

[0310] The MAP microplate is a microplate coated with recombinant protein MAP 625472, the amino acid sequence of which is shown in SEQ ID NO.21. The MAP sample dilution buffer is a PBS solution containing 5% v / v horse serum and 0.1% w / w casein; the 10× concentrated wash buffer is a 10× PBST solution.

[0311] 5.2 Application of the reagent kit

[0312] The assembled MAP indirect ELISA antibody detection kit was used to detect bovine serum samples from 975 samples in Xinjiang, 394 samples in Shandong, 7,222 samples in Heilongjiang, 432 samples in Liaoning, 184 samples in Shanghai, 1,200 samples in Guangdong, and 720 samples in Henan. Statistical results showed that the MAP antibody positivity rates in each province were 5.4% in Xinjiang, 9.7% in Heilongjiang, 68.2% in Liaoning, 21.3% in Shandong, 11.2% in Henan, 7.6% in Shanghai, and 6.7% in Guangdong. Among the MAP antibody-positive bovine samples (45 in Xinjiang, 60 in Heilongjiang, and 81 in Liaoning), the MAP-IS900 PCR amplification nucleic acid positivity rates were 88.89%, 93.33%, and 91.36%, respectively, with an overall concordance rate of 91.40% (Table 36).

[0313] Table 36 Positive concordance rate between MAP indirect ELISA antibody detection kit and MAP-IS900 PCR

[0314]

[0315] 6. Summary

[0316] (1) The optimization results of the MAP indirect ELISA detection method showed that the optimal reaction conditions of the kit were as follows: the coating buffer was CB solution, the antigen coating concentration was 10 μg / mL, and the coating condition was 4℃ for 16-20 h; the blocking solution was PBS solution containing 5% w / w gelatin + 5% w / w trehalose; the enzyme-labeled secondary antibody protection solution was PB solution containing 5% v / v rabbit serum, and the optimal enzyme-labeled secondary antibody dilution was 1:30,000; the optimal sample dilution solution was PBS solution containing 5% v / v horse serum + 0.1% w / w casein, and the optimal dilution of the serum to be tested was 1:40; the optimal incubation time of the kit was 25℃, 35 min for primary antibody, 30 min for secondary antibody, and 10 min for color development.

[0317] (2) The MAP indirect ELISA antibody detection method based on recombinant protein MAP 625472 has a cutoff value of 0.300, sensitivity of 96.67%, and specificity of 93.48% (AUC=0.975).

[0318] (3) The limit of detection for the established MAP indirect ELISA antibody detection method for sensitive control serum is 1:2,560; at the same time, the sensitivity test standard of the kit is determined as follows: the S / P value ranges for sensitive control serum of 1:320, 1:640, 1:1,280 and 1:2,560 are 0.90≤S / P≤1.25, 0.70≤S / P<1.05, 0.45≤S / P<0.75 and 0.30≤S / P<0.55, respectively.

[0319] (4) Specific experimental results showed that the MAP indirect ELISA antibody detection method had no cross-reactivity with bovine serum positive for Mycoplasma bovis, Mycobacterium bovis, Mycobacterium tumefaciens, bovine viral diarrhea virus, Escherichia coli BL21 and recombinant Escherichia coli pET22b-BL21.

[0320] (5) The repeatability test results showed that the intra-assay coefficient of variation of the MAP indirect ELISA antibody detection kit was between 0.73% and 6.38%, and the inter-assay coefficient of variation was between 0.49% and 6.5%.

[0321] (6) The results of the concordance rate experiment showed that the positive concordance rate of the MAP indirect ELISA antibody detection kit and the commercial IDvet paratuberculosis antibody detection kit was 96.7%, the negative concordance rate was 95.1%, and the total concordance rate was 95.2%, and the sensitivity was better than that of the IDvet paratuberculosis antibody detection kit.

[0322] (7) Epidemiological testing results showed that the MAP antibody positivity rates in various provinces using the MAP indirect ELISA antibody detection kit were as follows: Xinjiang 5.4%, Heilongjiang 9.7%, Liaoning 68.2%, Shandong 21.3%, Henan 11.2%, Shanghai 7.6%, and Guangdong 6.7%. Among them, the concordance rate between the MAP antibody-positive cattle samples from Xinjiang, Heilongjiang, and Liaoning and the MAP-IS900 nucleic acid positivity rate was 91.40%.

Claims

1. A bovine paratuberculosis mycobacterium indirect ELISA antibody detection kit, characterized in that, The kit contains a microplate coated with a recombinant protein MAP 625472, which is composed of three proteins MAP1272c, MAP0862 and MAP2154c in tandem. The amino acid sequence of the recombinant protein MAP 625472 is shown in SEQ ID NO.

21.

2. The bovine paratuberculosis mycobacterium indirect ELISA antibody detection kit as described in claim 1, characterized in that, The coating buffer used to prepare the microplates coated with the recombinant protein MAP 625472 was CB solution, the coating concentration of recombinant protein MAP625472 was 10 μg / mL, and the coating conditions were 4℃ for 16-20 h; the blocking solution was PBS solution containing 5% w / w gelatin and 5% w / w trehalose.

3. The bovine paratuberculosis mycobacterium indirect ELISA antibody detection kit as described in claim 1 or 2, characterized in that, The kit also contains a bovine paratuberculosis mycobacterium negative control, a bovine paratuberculosis mycobacterium positive control, a bovine paratuberculosis mycobacterium sample dilution solution, an enzyme-labeled secondary antibody, a 10× concentrated washing buffer, chromogenic solution A, chromogenic solution B, and a stop solution.

4. The indirect ELISA antibody detection kit for Mycobacterium paratuberculosis of bovine strain as described in claim 3, characterized in that, The enzyme-labeled secondary antibody is a goat anti-bovine IgG-HRP enzyme conjugate, the protective solution for the enzyme-labeled secondary antibody is a PB solution containing 5% v / v rabbit serum, and the dilution of the enzyme-labeled secondary antibody is 1:30,000; the sample dilution solution for bovine paratuberculosis mycobacterium is a PBS solution containing 5% v / v horse serum and 0.1% w / w casein; the 10× concentrated washing buffer is a 10× PBST solution.

5. The indirect ELISA antibody detection kit for Mycobacterium bovis paratuberculosis as described in claim 4, characterized in that, When using the kit described above to detect Mycobacterium bovis, the serum dilution is 1:40, the incubation temperature is 25℃, the serum incubation time is 35 min ± 1 min, the enzyme-labeled secondary antibody incubation time is 30 min ± 1 min, and the color development time is 10 min.

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