Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting avian mycobacterium paratuberculosis and application of double-antibody sandwich ELISA method

Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting avian mycobacterium paratuberculosis and application of the double-antibody sandwich ELISA method. The method comprises the following steps: coating an elisa plate by using a 3C2 2A4 monoclonal antibody as a capture antibody; sealing the elisa plate, adding a sample to be detected, and incubating; an enzyme-labeled 2D101D4 monoclonal antibody is added for incubation, and then a reaction is carried out; detecting the elisa plate after color development; the nucleotide sequence of the heavy chain variable region of the 3C2 2A4 is as shown in SEQ ID NO. 3; the nucleotide sequence of the light chain variable region of the 3C2 2A4 is as shown in SEQ ID NO.7; the nucleotide sequence of the heavy chain variable region of the 2D10 < 1 > D4 is as shown in SEQ ID NO. 9; and the nucleotide sequence of the light chain variable region of 2D101D4 is as shown in SEQ ID NO. 11. The method has higher sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a double-antibody sandwich ELISA method for detecting Mycobacterium avium subsp. paratuberculosis and its application. Background Art

[0002] Johne's disease is a bacterial disease of animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). Infected dairy cows show a decrease in milk production, resulting in serious economic losses in the dairy industry. At the same time, it can also cause human infections through the contamination of raw milk. To reduce economic losses, accurate and rapid detection of MAP in raw milk is of great significance. Currently, there are various methods for MAP detection, including bacterial isolation and identification, immunological diagnosis, and molecular biology detection methods.

[0003] In the prior art, the methods for detecting Mycobacterium avium subsp. paratuberculosis mainly include pathogen isolation and culture, PCR detection method, etc., but the detection limits of the above methods are relatively high. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a double-antibody sandwich ELISA method for detecting Mycobacterium avium subsp. paratuberculosis and its application.

[0005] A double-antibody sandwich ELISA method for detecting Mycobacterium avium subsp. paratuberculosis, characterized by comprising the following steps: Coating the reaction wells with the 3C2 2A4 monoclonal antibody as the capture antibody; Blocking the reaction wells, adding the sample to be tested to the reaction wells and incubating; Adding the enzyme-labeled 2D10 1D4 monoclonal antibody, incubating and then reacting; Detecting the enzyme-labeled plate after color development; The nucleotide sequence of the heavy chain variable region of the 3C2 2A4 is as shown in SEQ ID NO.3; The nucleotide sequence of the light chain variable region of the 3C2 2A4 is as shown in SEQ ID NO.7; The nucleotide sequence of the heavy chain variable region of the 2D10 1D4 is as shown in SEQ ID NO.9; The nucleotide sequence of the light chain variable region of the 2D10 1D4 is as shown in SEQ ID NO.11.

[0006] Preferably, the coating concentration of the capture antibody is 0.5 μg / mL to 5 μg / mL.

[0007] Preferably, the dilution concentration of the 2D10 1D4 monoclonal antibody is 1:5000 - 40000.

[0008] Preferably, 1% gelatin by mass fraction is selected as the blocking solution during blocking, and the blocking time is 0.5 h - 2 h.

[0009] Preferably, the incubation time is 0.5 h - 2 h after adding the sample to be tested.

[0010] Preferably, the incubation time is 0.5 h - 2 h after adding the 2D10 1D4 monoclonal antibody.

[0011] Preferably, the color development time is 10 min - 30 min.

[0012] Preferably, the sample to be tested is selected from foods.

[0013] Preferably, the sample to be tested is raw milk.

[0014] Preferably, the method is applied to the qualitative or quantitative detection of Mycobacterium avium subsp. paratuberculosis in samples.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The lowest detection limit of the double - antibody sandwich ELISA method established by the present invention is 1×10 2 CFU / mL, and the detection limit of qPCR is 1×10 0 CFU / mL, which is more sensitive, convenient and accurate in antigen detection.

[0016] The double - antibody sandwich ELISA method established by the present invention has the advantages of time - saving and low cost, and thus is more suitable for large - scale monitoring and quarantine in veterinary clinics. Description of the Drawings

[0017] Figure 1 Showing the titer detection of 3C2 2A4 and 2D10 1D4 cell lines.

[0018] Figure 2 Showing the results of monoclonal antibody purity detection; wherein: M represents protein Marker, 1 represents the purified antibody of 2D10 1D4 cell line, and 2 represents the purified antibody of 3C2 2A4 cell line.

[0019] Figure 3 Showing the results of qPCR identification of the sensitivity evaluation of bacterial liquid; wherein: 1 represents 1×10 7 CFU / mL; 2 represents 1×10 6 CFU / mL; 3 - 8 respectively represent 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×10 0 CFU / mL; 9 - 10 represent negative control and blank control respectively.

[0020] Figure 4 Show partial results of qPCR detection of artificially contaminated raw milk by standard qPCR; among them, 1 represents 1×10 7 CFU / mL; 2 represents 1×10 6 CFU / mL; 3 - 10 respectively represent 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×10 0 CFU / mL, negative control, blank control.

[0021] Figure 5 Show qPCR detection results of clinical samples. Detailed implementation manners

[0022] The following describes the detailed implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the detailed implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0023] Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Escherichia coli, BCG, and Klebsiella pneumoniae of the present invention are all stored in the laboratory of the College of Animal Science and Technology, Shihezi University.

[0024] The sequence information of 3C2 2A4 of the present invention is as follows: 1. Heavy chain information: (1) Leader sequence (bases): ATGGCTGTCTTGGGACTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCC, denoted as SEQ ID NO.1.

[0025] (2) Leader sequence (amino acids): MAVLGLLFCLVTFPSCVLS, denoted as SEQ ID NO.2.

[0026] (3)Base sequence of heavy chain variable region: CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTTATTAAGTAGCTATGGTATACATTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGGGTGATGTGGAGTGGTGGAAGCTCAGACTATAATGCAGCTTTCAAATCCAGACTGAGCATCACCAAGGACAGTTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTAATGACACAGCCATATATTACTGTGCCAGACGTCACTGGGACGTTAGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA, denoted as SEQ ID NO.3.

[0027] (4)Amino acid sequence of heavy chain variable region: QVQLKQSGPGLVQPSQSLSITCTVSGFLLSSYGIHWIRQSPGKGLEWLGVMWSGGSSDYNAAFKSRLSITKDSSKSQVFFKMNSLOANDTAIYYCARRHWDVRYFDVWGAGTTVTVSS, denoted as SEQ ID NO.4.

[0028] 2. Light chain information (1)Leader sequence (base): ATGGAATCACAGACTCTGGTCTTCATATCCATACTGCTCTGGTTATATGGTGCTGATGGG, denoted as SEQ ID NO.5.

[0029] (2)Leader sequence (amino acid): MESQTLVFISILLWLYGADG, denoted as SEQ ID NO.6.

[0030] (3)Base sequence of the light chain variable region: AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTCGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTTCTTATGTATCCTGGTATCAACAGAAACCAGACCAGTCTCCTAAACTGTTGATATACGGGGCATCCATCCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACGGCTATCCGAACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA, denoted as SEQ ID NO.7.

[0031] (4)Amino acid sequence of the light chain variable region: NIVMTQSPKSMSMSVGERVTLSCKASENVGSYVSWYQQKPDOSPKLLIYGASIRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYGYPNTFGGGTKLEIK, denoted as SEQ ID NO.8.

[0032] The sequence information of 2D10 1D4 of the present invention is as follows: 1. Heavy chain information (1)Base sequence of the heavy chain variable region: GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTCATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTCCTGAGGACACAGCCACATATTACTGTGCTCGACGTCCCAATGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA, denoted as SEQ ID NO.9.

[0033] (2) Amino acid sequence of the heavy chain variable region: DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGHSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTPEDTATYYCARRPNVYWGQGTLVTVSA, denoted as SEQ ID NO.10.

[0034] 2. Light chain information (1) Base sequence of the light chain variable region: CAAAATGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTGAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGGGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA, denoted as SEQ ID NO.11.

[0035] (2) Amino acid sequence of the light chain variable region: QNVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGSYPYTFGGGTKLEIK, denoted as SEQ ID NO.12.

[0036] Example 1 Preparation of MAP monoclonal antibody 1. Animal immunization Six 6 - 8 - week - old female BALB / c mice were selected and numbered as No.1, No.2, No.3, No.4, No.5, and No.6 respectively. Inactivated MAP was mixed with Freund's adjuvant for immunization, and the immunization was carried out by subcutaneous injection at 50 μg / mouse. Booster immunization was carried out once every 2 - 3 weeks. Blood was collected from the tail vein for detection, and the titer of the serum antibody produced by the mice was determined by the indirect ELISA method.

[0037] 2. Serum titer detection and screening (1)Dilute the inactivated MAP to 1 μg / mL with 1×ELISA coating buffer, mix well and add 100 μL to each well of a 96-well plate strip, and incubate overnight in a 4°C refrigerator.

[0038] (2)The next day, discard the coating buffer, wash the plate 3 times, add 200 μL of 5% BSA blocking solution to each well, block at 37°C for 2 h, take out the ELISA plate, discard the internal liquid, and wash the plate 3 times.

[0039] (3)Continuously dilute the serum 1:1000-fold with PBST, add 100 μL to each well, and incubate in a 37°C incubator for 1 h.

[0040] (4)Discard the internal liquid, wash the plate 3 times with 1×PBST at 200 μL / well, and then add 100 μL of the diluted enzyme-labeled secondary antibody to each well (enzyme-labeled secondary antibody: goat anti-mouse - HRP, 1:10000. Incubate at 37°C for 1 h).

[0041] (5)Discard the enzyme-labeled secondary antibody, wash the plate 5 times with PBST at 200 μL / well, add 100 μL of the two-component TMB chromogenic solution to each well, and place in the dark at 37°C for 20 min.

[0042] (6)Add 50 μL of the termination solution to each well to terminate the reaction, measure the absorbance of each well at 450 nm with an ELISA reader, record the values, as shown in Table 15. Select mice No. 1 and No. 6 with the best titer for cell fusion.

[0043] Table 15 Titer after the fourth immunization of mice 3. Cell fusion Feeder cell preparation: Under sterile conditions, take the spleens of healthy immunized mice No. 1 and No. 6 Balb / c, and make a single spleen cell suspension with HAT medium containing 20% fetal bovine serum (HAT additive is a mixture of hypoxanthine, aminopterin, and thymidine), and then pre-pave it into a 96-well plate according to the number of plates.

[0044] Mix the myeloma cells and spleen cells so that the ratio of myeloma cells to spleen cells is preferably 1:5 - 1:10. Act on them with 50% PEG1450 for 1 min, dilute and terminate with basic DMEM medium (high glucose, sodium pyruvate, L-glutamine, HEPES, phenol red), after low-speed centrifugation, gently suspend and mix with HAT medium containing 20% fetal bovine serum, and plate them into the pre-prepared feeder cell plate at 2×10 7 / plate, place in 5% CO 2 , culture at 37°C to obtain hybridoma cells.

[0045] 4. Cell line establishment (1)Fusion screening Dilute MAP to 1 μg / mL with 1×ELISA coating buffer and coat overnight. The next day, aspirate 100 μL of hybridoma cell supernatant per well for ELISA detection. Start the detection when the cells in the fusion plate reach medium size, about 10,000 cells or more. After the ELISA quality control is qualified (i.e., negative control < 0.2, positive control > 1.0), select positive wells (generally OD450 ≥ 0.5) for subcloning.

[0046] (2)Subcloning and detection Pick out the wells with high positive detection values in the fusion plate for limiting dilution. Perform subcloning with a count of 60% of the monoclonal well number per plate. Each time, pick the monoclonal wells with higher positive values for limiting dilution. ELISA detection can be carried out 5 - 7 days after each subcloning. After 2 subclonings, finally screen out the monoclonal cell lines that can stably secrete positive antibodies for expansion culture. The monoclonal cell lines that can stably secrete positive antibodies are named 3C2 2A4, 4H1 4G1, 2D10 1D4, 3E4 2G2, 2H6 3E9, 2A5 1B5, and 3D5 2D4.

[0047] (3)Cell line establishment Expand the cell lines that stably secrete positive antibodies screened in the subcloning stage in 24-well plates. After expansion, collect the supernatant for antigen detection. Use ELISA gradient dilution and WB to verify its stability. Collect the cells and expand them in 10-cm culture dishes. Collect the supernatant again and detect the antibody titer in it. Select the cell lines with higher titers and culture them in cell bottles for cryopreservation, and determine the cell subtype of the established cell line.

[0048] 5. Ascites preparation Inject 500 μL of liquid paraffin into the peritoneal cavity of mice 14 days in advance, and inoculate 2×10 6 / mL of hybridoma cells into the peritoneal cavity of mice. After 7 days, collect the ascites for purification.

[0049] 6. Antibody purification Purify the collected ascites by using a Protein G agarose affinity chromatography column after pretreatment.

[0050] The sample obtained by precipitation with 50% SAS was dialyzed overnight in the starting buffer and filtered through a 0.22 μm microporous membrane. The Protein G-agarose affinity chromatography column (HiTrap Protein G 1 mL, Pharmacia Biotech) was equilibrated. 20 mL of the sample to be purified (containing 15 mg of protein per mL of sample) was loaded onto the column at a flow rate of 0.5 mL / min, and then washed successively with 7 mL of the starting buffer, 6 mL of the elution buffer, and 5 mL of the starting buffer at the same flow rate. The eluate was collected in 1 mL fractions. Positive monoclonal antibodies were obtained by ELISA screening.

[0051] Results 1. Detection of the titer of MAP-immunized mice Inactivated MAP was used to immunize mice. Serum samples were collected from the mice at intervals and detected for ELISA titer.

[0052] The results showed that the titer of the mice after the fourth immunization reached 1:64000, meeting the requirements of the cell fusion experiment. Finally, mice No. 1 and No. 6 were selected for the fusion experiment.

[0053] 2. Screening of MAP monoclonal antibodies and identification of antibody subtypes The cell lines 3C2 2A4, 4H1 4G1, 2D10 1D4, 3E4 2G2, 2H6 3E9, 2A5 1B5, and 3D5 2D4, which stably secreted positive antibodies screened at the subcloning stage, were respectively expanded in 24-well plates. After expansion, the supernatants were collected for antigen detection. ELISA gradient dilution and WB were used to verify their stability. The cells were expanded in 10 cm culture dishes, the supernatants were collected again and the antibody titers were detected. The cell lines with higher titers were selected and cultured in cell flasks for cryopreservation, and the subtypes of the established cell lines were determined (Table 1). Table 1 Determination of subtypes of established cell lines 3. Ascites preparation and detection of monoclonal antibody titer By ELISA detection, 3C2 2A4 and 2D10 1D4 with higher titers were selected for antibody purification. The hybridoma cells prepared in this experiment were intraperitoneally injected into mice, ascites were collected, and Protein G-agarose affinity chromatography column was used for ascites purification. Salmonella was diluted to 1 μg / mL and coated in 96-well ELISA plates. The purified MAP 3C2 2A4 and 2D10 1D4 monoclonal antibody cell lines were serially diluted as primary antibodies. The results showed that the antibody had high affinity ( Figure 1 ).

[0054] 4. Detection of monoclonal antibody purity The purified antibody was detected by SDS-PAGE, and the results showed that the concentration and purity of the purified antibody were both high ( Figure 2 ).

[0055] Example 2 Establishment of double antibody sandwich ELISA method 1. Labeling of monoclonal antibody HRP (horseradish peroxidase) Dissolve 5 mg of HRP in 0.5 mL of 0.1 mol / L NaHCO 3 solution; add 0.5 mL of 10 mmol / L NaIO 4 solution, mix well, tighten the bottle cap, and react at room temperature in the dark for 2 hours. Add 0.75 mL of 0.1 mol / L Na 2 CO 3 and mix well. Add 0.75 mL of purified monoclonal antibody and mix well.

[0056] Weigh 0.3 g of Sephadex G25 dry powder and add it into the outer cylinder of a 5 mL syringe with a glass wool pad at the lower end; then transfer the above cross-linked product into the syringe outer sleeve; tighten it and react at room temperature (in the dark) for 3 hours or overnight at 4°C. Wash out all the cross-linked product with PBS, collect the eluate, add 1 / 20 volume of freshly prepared 5 mg / mL NaBH 4 solution, mix well, and react at room temperature for 30 minutes; then add 3 / 20 NaBH 4 solution, mix well, and react at room temperature for 1 h (or overnight at 4°C). Transfer the conjugate into a dialysis bag, place it in 0.15 M PBS (pH = 7.4) for dialysis overnight at 4°C. After adding an equal amount of glycerol, aliquot it in small amounts and store it at -20°C to prevent repeated freezing and thawing.

[0057] 2. Screening of capture antibody and enzyme-labeled antibody Coat the monoclonal antibodies of the prepared 3C2 2A4 and 2D10 1D4 cell lines on the 96-well enzyme-linked immunosorbent assay (ELISA) plate respectively. After adding the antigen, add the HRP-labeled 3C2 2A4 and 2D10 1D4 monoclonal antibodies, and then add the chromogenic solution to measure the absorbance value at OD450nm. The specific method is as follows: (1) Coating: Coat the monoclonal antibody with a concentration of 2 μg / mL on the 96-well ELISA plate using 1×ELISA coating solution and incubate overnight at 4°C.

[0058] (2) Blocking: Discard the coated antibody, wash the plate 3 times with 1×PBST, pat it dry, and then add 200 μL of 1% gelatin to each well for blocking at 37°C for 2 h.

[0059] (3) Incubating antigen: Discard the liquid in the plate, wash the plate 3 times, pat it dry, and then add 100 μL of the antigen to be tested to each well and incubate at 37°C for 1 h.

[0060] (4)Incubation of enzyme-labeled antibody: After taking out the plate, wash the plate by repeating the previous step. Add the self-prepared enzyme-labeled secondary antibody into the wells, 100 μL per well, and incubate at 37 °C for 1 h.

[0061] (5)Add chromogenic solution: After incubation, wash the plate 5 times, 3 min each time. After patting dry, add the prepared two-component TMB chromogenic solution, 100 μL per well, and incubate at 37 °C for 20 min.

[0062] (6)Termination: After color development, add 50 μL of ELISA termination solution to each well to terminate the reaction. Read the absorbance at OD450nm using an enzyme-labeled immunosorbent assay analyzer.

[0063] 3. Determination of the optimal coating concentration of capture antibody and the optimal dilution factor of enzyme-labeled antibody The optimal coating amount of capture antibody, the optimal dilution of enzyme-labeled antibody, the optimal blocking solution and blocking time, the optimal incubation time of antigen and enzyme-labeled antibody, and the optimal color development time were determined by checkerboard titration to determine the optimal reaction conditions for double-antibody sandwich ELISA.

[0064] The capture antibody was serially diluted with ELISA coating buffer to 0.5, 1, 2, 5 μg / mL, 100 μL per well, and coated on a 96-well enzyme-labeled reaction plate at 4 °C overnight; 100 μL of antigen was added per well; the dilution gradients of enzyme-labeled secondary antibody were 1:5000, 1:10000, 1:20000, and 1:40000, and a negative control was set simultaneously. The OD450nm value was measured, the P / N value was calculated, and the optimal reaction concentrations of capture antibody and enzyme-labeled secondary antibody were determined.

[0065] On this basis, 5% BSA, 5% skim milk powder, and 1% gelatin were set as blocking solutions, the blocking times were 0.5 h, 1 h, 1.5 h, 2 h, the reaction times of the serum to be tested were 0.5 h, 1 h, 1.5 h, 2 h, the incubation time of enzyme-labeled secondary antibody was 0.5 h, 1 h, 1.5 h, 2 h, and the color development times were 10 min, 15 min, 20 min, 25 min, 30 min to optimize the ELISA reaction conditions, and the optimal reaction conditions were determined by the maximum P / N ratio.

[0066] 4. Determination of the cut-off value Use the established double-antibody sandwich ELISA method to detect 50 PBS samples, and calculate the average value of OD450nm of PBS samples and the standard deviation s , and the cut-off value is the average value of OD450nm of PBS plus 2 times the standard deviation ( +2 s ). Samples with OD450nm ≥ cut-off value are positive, and those less than the cut-off value are negative.

[0067] Results 1. Determination of the pairing of capture antibody and enzyme-labeled antibody Using 3C2 2A4 and 2D10 1D4 monoclonal antibodies as capture antibodies respectively, and HRP-labeled ones as detection antibodies respectively for pairwise pairing, and reacting with activated MAP. After pairing screening, it was determined that the 3C2 2A4 monoclonal antibody was the capture antibody, and the 2D10 1D4 enzyme-labeled antibody was the detection antibody. The results are shown in Table 2.

[0068] Table 2 Antibody pairing screening 2. Determination of the optimal concentrations of capture antibody and detection antibody The checkerboard titration method was used to determine that the optimal coating concentration of the capture antibody was 0.5 μg / mL, and the optimal dilution concentration of the detection antibody was 1:5000. The results are shown in Table 3.

[0069] Table 3 Determination of the optimal concentrations of capture antibody and enzyme-labeled antibody 3. Selection of the optimal blocking solution and blocking time The blocking solution was selected as 1% gelatin, and the blocking time was selected as 1 h. The results are shown in Table 4-1 and Table 4-2.

[0070] Table 4-1 Selection of blocking time and blocking solution (upper part) Table 4-2 Selection of blocking time and blocking solution (lower part) 4. Determination of the optimal incubation time of antigen The results of incubating antigen at different times of 0.5 h, 1 h, 1.5 h, and 2 h were compared. The maximum P / N value appeared at 1.5 h (Table 5), so 1.5 h was selected as the optimal incubation time of antigen.

[0071] Table 5 Optimal incubation time of antigen 5. Optimal incubation time of detection antibody The effects of 0.5 h, 1 h, 1.5 h, and 2 h on the detection antibody were compared. The results are shown in Table 6. The P / N value was the largest at 1.5 h, so 1.5 h was selected as the optimal incubation time of the detection antibody.

[0072] Table 6 Optimal incubation time of detection antibody 6. Determination of the optimal chromogenic time Detect the color development at 10 min, 15 min, 20 min, 25 min, and 30 min respectively (Table 7). The results show that the P / N value is the largest at 10 min. Therefore, 10 min is selected as the optimal color development time.

[0073] Table 7 Optimal color development time 7. Determination of the critical value In summary, the optimal reaction conditions are as follows: the blocking solution is 1% gelatin, the blocking time is 1 h, the incubation time of the antigen is 1.5 h, the incubation time of the detection antibody is 1.5 h, and the color development time is 10 min.

[0074] Detect 50 samples of PBS, and perform double-antibody sandwich ELISA detection according to the above-screened optimal reaction conditions. The OD450nm is 0.169, and the standard deviation s is 0.019. When the OD450nm of the sample ≥ 0.207, it is determined as positive; when OD450nm ≤ 0.131, it is determined as negative; when 0.131 < X < 0.207, repeated tests are required for re-determination.

[0075] Example 3 Specificity test of the double-antibody sandwich ELISA method Detect activated MAP, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, BCG, and Streptococcus agalactiae according to the optimized double-antibody sandwich ELISA method, and evaluate the specificity of this method.

[0076] Detect MAP, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, BCG, and Streptococcus agalactiae under the optimized ELISA conditions. It is found that only MAP shows positive results (Table 8), proving that this method has good specificity.

[0077] Table 8 Specificity results of the double-antibody sandwich ELISA Example 4 Sensitivity test of the double-antibody sandwich ELISA method Dilute MAP by 10-fold serial dilution. The total number of colonies of the pathogen to be detected is between 10 7 -10 1 CFU / mL. Use the double-antibody sandwich ELISA method established in the present invention for sensitivity detection, and conduct a control experiment with the qPCR method at the same time.

[0078] Dilute the MAP bacterial solution with PBS to 1×10 7-1×10 1 CFU / mL, the detection was carried out by the established optimal double-antibody sandwich ELISA method (Table 9), and at the same time, it was compared with the qPCR method ( Figure 3 ). It was found that the lowest detection limit of the ELISA method was 1×10 2 CFU / mL, and the detection limit of qPCR was 1×10 0 CFU / mL. It was proved that this method had good sensitivity.

[0079] Table 9 Results of the sensitivity of double-antibody sandwich ELISA Example 5 Repeatability test of double-antibody sandwich ELISA method 1. Intra-batch repeatability test Six ELISA plates coated in the same batch were taken, and the MAP bacterial solution was used as the antigen. The detection was carried out according to the established double-antibody sandwich ELISA method, and the coefficient of variation was calculated.

[0080] 2. Inter-batch repeatability test Six ELISA plates coated in different batches were taken, and the MAP bacterial solution was used as the antigen. The detection was carried out according to the established double-antibody sandwich ELISA method, and the coefficient of variation was calculated.

[0081] Results The intra-batch test was to repeat the double-antibody sandwich ELISA method prepared in the same batch multiple times. The results showed (Table 10) that the coefficient of variation within the batch was less than 10%.

[0082] The inter-batch test was to conduct multiple groups of tests on the double-antibody sandwich ELISA methods prepared in multiple batches. The results showed (Table 11) that the coefficient of variation between batches was less than 10%, indicating that the established ELISA method had good inter-batch consistency.

[0083] Table 10 Intra-batch repeat results of double-antibody sandwich ELISA Table 11 Inter-batch repeat results of double-antibody sandwich ELISA Example 6 When the optimized double-antibody sandwich ELISA method and the national standard qPCR method were used to jointly detect 64 artificially contaminated samples, 48 samples were detected as positive by the ELISA method, and 64 samples were detected as positive by the qPCR method (the total number of colonies of MAP bacterial solution was measured using a turbidimeter. Sterile fresh raw milk and MAP bacterial solution were mixed at a ratio of 900 μL:100 μL. 100 μL of the mixed solution from the previous tube was pipetted into the next tube of fresh raw milk. 60 samples of the bacterial solution were diluted to 1×107 CFU / mL - 1×101 CFU / mL, and 4 samples were diluted to 1×100 CFU / mL. Each mixed sample was boiled at 100 °C for 5 min, centrifuged at 12,000 rpm for 5 min, and the middle liquid in the EP tube was pipetted and added to the sample pad for detection). Compared with the national standard qPCR method, the coincidence rate was calculated.

[0084] The negative coincidence rate was 100%, the positive coincidence rate was 75%, and the total coincidence rate was 75.8%. (Tables 12, Table 13, Figure 4 )

[0085] Table 12 Partial Detection Results of Artificially Contaminated Milk Samples Table 13 Coincidence Rate Results Example 7 For the clinical detection results of the double-antibody sandwich ELISA method, the method established in this experiment and the national standard qPCR method were used to detect 168 clinical fresh raw milk samples, and no positive samples were detected (Table 14, Figure 5 )

[0086] Table 14 Partial Clinical Detection Results It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent redundancy, preferred embodiments of the present invention are described.

[0087] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A double antibody sandwich ELISA method for detecting avian Mycobacterium paratuberculosis subspecies, characterized in that: The following steps are involved: The reaction wells are coated with 3C2 2A4 monoclonal antibody as a capture antibody; the reaction wells are sealed, and the sample to be tested is added to the reaction wells for incubation; HRP-labeled 2D10 1D4 monoclonal antibody is added for incubation; and the ELISA plate is detected after color development; The nucleotide sequence of the heavy chain variable region of 3C2 2A4 is shown in SEQ ID NO.3; The nucleotide sequence of the light chain variable region of 3C2 2A4 is shown in SEQ ID NO.7; The nucleotide sequence of the heavy chain variable region of 2D10 1D4 is shown in SEQ ID NO.9; The nucleotide sequence of the light chain variable region of 2D10 1D4 is shown in SEQ ID NO.

11.

2. The double antibody sandwich ELISA method according to claim 1, characterized in that: The coating concentration of capture antibody is 0.5μg / mL~5μg / mL.

3. The double antibody sandwich ELISA method according to claim 1, characterized in that: The dilution concentration of 2D10 1D4 monoclonal antibodies is 1:5000~40000.

4. The double antibody sandwich ELISA method according to claim 1, characterized in that: When blocking, choose 1% gelatin as the blocking solution, and the blocking time is 0.5h~2h.

5. The double antibody sandwich ELISA method according to claim 1, characterized in that: The incubation time after adding the sample to be tested is 0.5h~2h.

6. The double antibody sandwich ELISA method according to claim 1, characterized in that: The incubation time after adding 2D10 1D4 monoclonal antibodies is 0.5h~2h.

7. The double antibody sandwich ELISA method according to claim 1, characterized in that: The color development time is 10min~30min.

8. The double antibody sandwich ELISA method according to claim 1, characterized in that: The sample to be tested is selected from food.

9. The double antibody sandwich ELISA method according to claim 8, characterized in that: The sample to be tested is fresh milk.

10. Use of the method according to any one of claims 1 to 9 in qualitative or quantitative detection of Mycobacterium avium subspecies paratuberculosis in a sample.

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