Colloidal gold immunochromatography test strip as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food safety monitoring and immunoassay detection, and particularly relates to a colloidal gold immunochromatography test strip, a preparation method and application thereof, the colloidal gold immunochromatography test strip comprises a bottom plate, a nitrocellulose membrane, absorbent paper, a conjugate pad and a sample pad; a mycobacterium paratuberculosis monoclonal antibody marked by colloidal gold is adsorbed on the combination pad; a detection line coated with the mycobacterium paratuberculosis monoclonal antibody and a quality control line formed by goat anti-mouse-IgG are arranged on the nitrocellulose membrane; and the bottom plate is a polyethylene chloride bottom plate, so that the MAP in the fresh milk can be rapidly and accurately detected.

Description

Technical Field

[0001] The present invention relates to the technical fields of food safety monitoring and immunoassay detection, and particularly relates to a colloidal gold immunochromatographic test strip, a preparation method thereof, and an application thereof. Background Art

[0002] Monoclonal antibodies (mAbs) are immunoglobulins that bind to a single epitope of an antigen. Due to their ability to specifically recognize antigens, they are widely used in the detection of various pathogens. The colloidal gold immunochromatographic method is one of the commonly used methods for rapid pathogen detection. It is simple to operate, and the visualization effect can directly determine the result. It can qualitatively detect antigens or antibodies without expensive equipment.

[0003] Johne's disease is a bacterial disease of animals caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Dairy cows infected with MAP 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 contaminated raw milk. With the increasing positive rate of antibodies against Mycobacterium paratuberculosis in cattle herds, it is imperative to establish a specific, rapid, and easy-to-operate detection tool. If the existing detection tools do not meet the specificity requirements, false positives are likely to occur. Summary of the Invention

[0004] To solve the above problems, the present invention provides a colloidal gold immunochromatographic test strip, a preparation method thereof, and an application thereof.

[0005] A colloidal gold immunochromatographic test strip includes a base plate, a nitrocellulose membrane, a blotting paper, a conjugate pad, and a sample pad;

[0006] The conjugate pad is adsorbed with a colloidal gold-labeled monoclonal antibody against Mycobacterium paratuberculosis. The nitrocellulose membrane has a detection line coated with a monoclonal antibody against Mycobacterium paratuberculosis and a quality control line composed of goat anti-mouse IgG; the base plate is a polyvinyl chloride base plate;

[0007] The monoclonal antibody against Mycobacterium paratuberculosis subspecies is 3C2 2A4 or 2D10 1D4;

[0008] The nucleotide sequence of the heavy chain variable region of 3C2 2A4 is as shown in SEQ ID NO.3;

[0009] The nucleotide sequence of the light chain variable region of 3C2 2A4 is as shown in SEQ ID NO.7;

[0010] The nucleotide sequence of the heavy chain variable region of 2D10 1D4 is as shown in SEQ ID NO.9;

[0011] The nucleotide sequence of the light chain variable region of 2D10 1D4 is as shown in SEQ ID NO.11.

[0012] A method for preparing the colloidal gold immunochromatographic test strip described above, comprising the following steps:

[0013] Preparing colloidal gold-labeled 3C2 2A4 monoclonal antibody: Combining 3C2 2A4 monoclonal antibody with colloidal gold to obtain colloidal gold-labeled 3C2 2A4 monoclonal antibody;

[0014] Treating the conjugate pad and the nitrocellulose membrane: Spraying the colloidal gold-labeled 3C2 2A4 monoclonal antibody on the conjugate pad; Drawing 2D10 1D4 monoclonal antibody on the nitrocellulose membrane to form the test line; Drawing goat anti-mouse IgG on the nitrocellulose membrane to form the control line;

[0015] Assembling the conjugate pad and the nitrocellulose membrane to form a colloidal gold immunochromatographic test strip.

[0016] Preferably, the concentration of the colloidal gold-labeled 3C2 2A4 monoclonal antibody on the conjugate pad is 4 μg / mL to 20 μg / mL.

[0017] Preferably, the concentration of the 2D10 1D4 monoclonal antibody on the nitrocellulose membrane is 1.5 mg / mL to 2 mg / mL.

[0018] Preferably, the concentration of the goat anti-mouse IgG is 1.5 mg / mL to 2.5 mg / mL.

[0019] Preferably, when the 3C2 2A4 monoclonal antibody binds to colloidal gold, the pH of the colloidal gold is 7.0 to 9.0.

[0020] Application of the colloidal gold chromatographic test strip in detecting Mycobacterium avium subsp. paratuberculosis.

[0021] Preferably, the Mycobacterium avium subsp. paratuberculosis is Mycobacterium paratuberculosis.

[0022] Preferably, the method for detecting Mycobacterium avium subsp. paratuberculosis is: Inserting the sample end into the test sample solution for detection.

[0023] Preferably, if an indicator line appears at the corresponding control region C position on the nitrocellulose membrane and no indicator line appears at the test region T position, the test result is negative, indicating that Mycobacterium avium subsp. paratuberculosis is not present in the test sample;

[0024] If two indicator lines appear at the T and C positions on the nitrocellulose membrane, the result is positive, indicating that Mycobacterium avium subsp. paratuberculosis is present in the test sample;

[0025] When the indication line does not appear in the quality control area, the test strip is judged invalid regardless of whether the indication line appears in the test area T or not.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] Based on the characteristics of the screened monoclonal antibodies and colloidal gold particles, the present invention has established a rapid, simple and accurate immunochromatographic test strip for detecting MAP in raw milk. Using 3C2 2A4 and 2D10 1D4 as monoclonal antibodies against Mycobacterium avium subsp. paratuberculosis, it specifically recognizes MAP, providing an effective means for effectively monitoring the contamination of MAP in raw milk and ensuring human food safety. Description of the Drawings

[0028] Figure 1 It is the detection result of the titers of 3C2 2A4 and 2D10 1D4 cell lines.

[0029] Figure 2 Purity detection of monoclonal antibodies.

[0030] Figure 3 It is the optimal pH value of colloidal gold-labeled 3C2 2A4 monoclonal antibody; Note: 1, pH value not adjusted; 2 - 10, pH = 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5.

[0031] Figure 4 It is the optimal coating amount of colloidal gold-labeled 3C2 2A4 monoclonal antibody; Note: 1, 0 μg / mL; 2 - 8, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL.

[0032] Figure 5 It is the selection of the optimal coating concentration of the monoclonal antibody for T-line detection; Note: 1 - 4, T-line concentration is 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL.

[0033] Figure 6 It is the specificity detection of the colloidal gold immunochromatographic test strip; Note: 1 - 10, MAP, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, BCG, Streptococcus agalactiae.

[0034] Figure 7 It is the sensitivity detection of the colloidal gold immunochromatographic test strip; Note: 1 - 5: 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×102 CFU / mL, 1×10 1 CFU / mL.

[0035] Figure 8 For the sensitivity evaluation of the bacterial suspension identified by the national standard qPCR, 1 - 8, 1×10 7 CFU / mL, 1×10 6 CFU / mL, 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; 9: negative control; 10, blank control.

[0036] Figure 9 For the partial detection results of the artificially contaminated fresh raw latex colloidal gold immunochromatographic test strip; among them, 1: 1×10 0 CFU / mL; 2 - 3: 1×10 1 CFU / mL; 4 - 6: 1×10 0 CFU / mL; 7: 1×10 0 CFU / mL.

[0037] Figure 10 For the partial results of the standard qPCR detection of artificially contaminated fresh raw milk; among them, 1 - 10: 1×10 7 CFU / mL - 1×10 0 CFU / mL.

[0038] Figure 11 For the detection results of some clinical samples of the colloidal gold immunochromatographic test strip.

[0039] Figure 12 For the detection results of clinical samples of the national standard qPCR. Specific Embodiments

[0040] The specific embodiments of the present invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. 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.

[0041] 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.

[0042] The sequence information of the 3C2 2A4 of the present invention is as follows:

[0043] 1. Heavy chain information:

[0044] (1) Leader sequence (base): ATGGCTGTCTTGGGACTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCC, denoted as SEQ ID NO.1.

[0045] (2) Leader sequence (amino acid): MAVLGLLFCLVTFPSCVLS, denoted as SEQ ID NO.2.

[0046] (3) Heavy chain variable region base sequence: CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTTATTAAGTAGCTATGGTATACATTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGGGTGATGTGGAGTGGTGGAAGCTCAGACTATAATGCAGCTTTCAAATCCAGACTGAGCATCACCAAGGACAGTTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTAATGACACAGCCATATATTACTGTGCCAGACGTCACTGGGACGTTAGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA, denoted as SEQ ID NO.3.

[0047] (4) Heavy chain variable region amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFLLSSYGIHWIRQSPGKGLEWLGVMWSGGSSDYNAAFKSRLSITKDSSKSQVFFKMNSLOANDTAIYYCARRHWDVRYFDVWGAGTTVTVSS, denoted as SEQ ID NO.4.

[0048] 2. Light chain information

[0049] (1) Leader sequence (base): ATGGAATCACAGACTCTGGTCTTCATATCCATACTGCTCTGGTTATATGGTGCTGATGGG, denoted as SEQ ID NO.5.

[0050] (2) Leader sequence (amino acids): MESQTLVFISILLWLYGADG, denoted as SEQ ID NO.6.

[0051] (3) Base sequence of the light chain variable region: AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTCGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTTCTTATGTATCCTGGTATCAACAGAAACCAGACCAGTCTCCTAAACTGTTGATATACGGGGCATCCATCCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACGGCTATCCGAACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA, denoted as SEQ ID NO.7.

[0052] (4) Amino acid sequence of the light chain variable region: NIVMTQSPKSMSMSVGERVTLSCKASENVGSYVSWYQQKPDOSPKLLIYGASIRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYGYPNTFGGGTKLEIK, denoted as SEQ IDNO.8.

[0053] The sequence information of 2D10 1D4 of the present invention is as follows:

[0054] 1. Heavy chain information

[0055] (1) Base sequence of the heavy chain variable region: GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTCATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTCCTGAGGACACAGCCACATATTACTGTGCTCGACGTCCCAATGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA, denoted as SEQ ID NO.9.

[0056] (2) Amino acid sequence of the heavy chain variable region: DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGHSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTPEDTATYYCARRPNVYWGQGTLVTVSA, denoted as SEQ ID NO.10.

[0057] 2. Light chain information

[0058] (1) Base sequence of the light chain variable region: CAAAATGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTGAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGGGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA, denoted as SEQ ID NO.11.

[0059] (2) Amino acid sequence of the light chain variable region: QNVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGSYPYTFGGGTKLEIK, denoted as SEQ ID NO.12.

[0060] The goat anti-mouse IgG used in the present invention was purchased from EarthOx, and the product catalog number is E031610.

[0061] Example 1

[0062] Preparation of MAP monoclonal antibody

[0063] 1. Animal immunization

[0064] Use PBS to resuspend the inactivated MAP bacterial solution to a colony count of 1×10 6 CFU / mL, emulsify it 1:1 with Freund's complete adjuvant, and subcutaneously inject it into 6 female BALB / c mice aged 6 - 8 weeks using a disposable syringe, immunizing at 50 μg / mouse. After 14 - 21 days, emulsify the inactivated MAP 1:1 with Freund's incomplete adjuvant and immunize again. After collecting the serum, determine the serum titer by the indirect ELISA method to identify the immunization effect. The 6 BALB / c mice are numbered as No. 1, No. 2, No. 3, No. 4, No. 5, and No. 6 respectively.

[0065] 2. Detection and screening of antiserum titer

[0066] (1) Dilute the inactivated Mycobacterium paratuberculosis to 1 μg / mL with 1×ELISA coating buffer, mix well and add it to the wells of the plate, 100 μL per well, and incubate overnight in a 4℃ refrigerator.

[0067] (2) The next day, discard the coating buffer, wash the plate 3 times, add 200 μL of 5% BSA blocking solution with a mass fraction to each well, block at 37℃ for 2 h, take out the enzyme - labeled plate, discard the internal solution, and wash the plate 3 times.

[0068] (3) Continuously dilute the serum 1:1000 - fold, 100 μL per well, and place it in a 37℃ incubator for 1 h.

[0069] (4) Discard the internal solution, wash the plate 3 times with 1×PBST at 200 μL / well, and add 100 μL of diluted enzyme - labeled secondary antibody (enzyme - labeled secondary antibody: goat anti - mouse - HRP, 1:10000. Incubate at 37℃ for 1 h) to each well.

[0070] (5) Discard the enzyme - labeled secondary antibody, wash the plate 5 times with PBST at 200 μL / well, first add 100 μL of TMB chromogenic solution to each well, and place it in the dark at 37℃ for 20 min.

[0071] (6) Add 50 μL of stop solution to each well to terminate the reaction. Measure the absorbance of each well at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and record the values.

[0072] 3. Cell fusion

[0073] Preparation of feeder layer cells: Under sterile conditions, take the spleen of healthy immunized Balb / c mice and make a single-cell suspension of spleen cells using HAT medium (HAT additive is a mixture of hypoxanthine, aminopterin, and thymidine) containing 20% fetal bovine serum by volume. Then, according to the number of plates to be seeded, pre-seed it into a 96-well plate.

[0074] Mix myeloma cells and spleen cells so that the ratio of myeloma cells to spleen cells is preferably 1:5 - 1:10. Treat with 50% PEG1450 by mass for 1 min, dilute and terminate with basal DMEM medium (high glucose, sodium pyruvate, L-glutamine, HEPES, phenol red), centrifuge at low speed, and then gently suspend and mix with HAT medium containing 20% fetal bovine serum by volume. Seed at 2×10 7 / plate into the pre-prepared feeder layer cell plate, and culture in an incubator with 5% CO2 at 37 °C to obtain hybridoma cells.

[0075] 4. Cell line establishment

[0076] (1) Fusion screening

[0077] Dilute MAP to 1 μg / mL with 1×ELISA coating buffer and coat overnight. The next day, aspirate 100 μL of cell supernatant per well for ELISA detection. Start the detection when the cells in the fusion plate reach medium size, about more than 10,000 cells. 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.

[0078] (2) Subcloning and detection

[0079] Select the wells with high positive detection values in the fusion plate for limiting dilution. Perform subcloning by counting 60% of the monoclonal well numbers per plate. Each time, select the monoclonal wells with higher positive values for limiting dilution. ELISA detection can be performed 5 - 7 days after each subcloning. After 2 subclonings, finally screen out the monoclonal cell line that can stably secrete positive antibodies for expansion culture.

[0080] (3) Cell line establishment

[0081] The cell lines that stably secrete positive antibodies screened in the subcloning stage were expanded and cultured 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 collected and 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, cryopreserved, and the cell subtypes of the established strains were determined.

[0082] 5. Ascites preparation

[0083] 14 days in advance, 500 μL of liquid paraffin was injected into the peritoneal cavity of mice, and 1×10 6 -2×10 6 / mL of hybridoma cells were correctly inoculated into the peritoneal cavity of mice. After 8 days, the ascites were collected for purification.

[0084] 6. Antibody purification

[0085] The collected ascites were pretreated and then purified using a Protein G agarose affinity chromatography column. The specific steps are as follows:

[0086] The sample obtained by precipitation with 50% mass fraction of SAS was dialyzed overnight in the starting buffer and filtered through a 0.22-μm microporous membrane. The Protein G-agarose affinity chromatography column was equilibrated. 20 mL of the sample to be purified, containing 18 mg of protein per 1 mL, was loaded onto the column at a flow rate of 0.5 mL / min. Then, it was washed successively with 8 mL of the starting buffer, 7 mL of the elution buffer, and 5 mL of the starting buffer at the same flow rate, and the eluates were collected in 1-mL tubes each. The MAP monoclonal antibody was obtained by screening and detection using ELISA.

[0087] Among them, the specification of the Protein G-agarose affinity chromatography column is HiTrap Protein G 1 mL, Pharmacia Biotech, and the sample to be purified contains 10.2 - 21.1 mg of protein per mL of the sample.

[0088] Results

[0089] 1. Detection of the titer of MAP-immunized mice

[0090] Inactivated MAP was used to immunize mice. Serum samples were collected from some of the mice at intervals for ELISA titer detection. 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.

[0091] 2. Screening of MAP monoclonal antibody and identification of antibody subtypes

[0092] The cell lines stably secreting positive antibodies screened in the subcloning stage were expanded and cultured 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 collected and expanded in 10-cm culture dishes, and the supernatants were collected again to detect the antibody titer. The cell lines with higher titers were selected and cultured in cell flasks, cryopreserved, and the cell subtypes of the established cell lines were determined. The results are shown in Table 1.

[0093] Table 1 Determination of cell subtypes of established cell lines

[0094] Plant No. 3C2 2A4 4H1 4G1 2D10 1D4 3E4 2G2 2H6 3E9 2A5 1B5 3D5 2D4 Subtype IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a

[0095] 3. Ascites preparation and monoclonal antibody titer detection

[0096] After ELISA detection, 3C2 2A4 and 2D10 1D4 with higher titers were selected for antibody purification.

[0097] The hybridoma cells prepared by the present invention were intraperitoneally injected into mice to collect ascites, and Protein G agarose affinity chromatography column was used for ascites purification. Salmonella was diluted to 1 μg / mL and coated on a 96-well ELISA plate. The purified MAP 3C22A4 and 2D10 1D4 monoclonal antibody cell lines were serially diluted as primary antibodies. The results showed that the antibody had high affinity, as Figure 1 shown.

[0098] 4. Monoclonal antibody purity detection

[0099] The purified antibody was detected by SDS-PAGE. The results showed that the concentration and purity of the purified antibody were both high, as Figure 2 shown.

[0100] 5. Determination of capture antibody and detection antibody

[0101] Pairing of capture antibody and enzyme-labeled antibody

[0102] The monoclonal antibodies of the prepared 3C2 2A4 and 2D10 1D4 cell lines were respectively coated in 96-well ELISA plates. After adding the antigen, the 3C2 2A4 and 2D10 1D4 monoclonal antibodies labeled with HRP were added, and then the TMB two-component chromogenic solution was added to measure the absorbance value at OD450nm. The specific method is as follows:

[0103] (1) Coating: Use 1×ELISA coating buffer to coat the monoclonal antibody with a concentration of 2 μg / mL on a 96-well ELISA plate, and coat overnight at 4°C.

[0104] (2) Blocking: Discard the coated antibody, wash the plate 3 times with 1×PBST, pat dry, and add 200 μL of 1% gelatin by mass fraction to each well for blocking at 37°C for 2 h.

[0105] (3) Incubate the antigen: Discard the liquid in the plate, wash the plate 3 times, pat dry, and add 100 μl of the antigen to be tested to each well. Incubate at 37 °C for 1 h.

[0106] (4) Incubate the enzyme-labeled antibody: After taking out the plate, repeat the previous step of washing the plate. Add the self-prepared enzyme-labeled secondary antibody to the wells, 100 μL per well, and incubate at 37 °C for 1 h.

[0107] (5) Add the chromogenic solution: After incubation, wash the plate 5 times, 3 min each time. Pat dry and add the prepared two-component TMB chromogenic solution, 100 μL per well, and incubate at 37 °C for 20 min.

[0108] (6) Termination: Add 50 μl of ELISA termination solution to each well to terminate the reaction after color development. Read the absorbance at OD450 nm using an enzyme-linked immunosorbent assay reader.

[0109] Use the 3C2 2A4 and 2D10 1D4 monoclonal antibodies as capture antibodies respectively, and use HRP-labeled substances as detection antibodies for pairwise cross-matching, and react with the activated MAP. After pairing and screening, determine the 3C2 2A4 monoclonal antibody as the capture antibody and the 2D10 1D4 enzyme-labeled antibody as the detection antibody.

[0110] Example 2

[0111] Establishment of the MAP colloidal gold immunochromatographic test strip method

[0112] 1. Optimization of the conditions for colloidal gold labeling of monoclonal antibodies

[0113] (1) Exploration of the optimal pH value for colloidal gold labeling of monoclonal antibodies

[0114] Take 10 detachable 96-well plate sample addition wells, add 300 μL of 30-nm colloidal gold solution to each well, and adjust the pH value of the colloidal gold solution in each well to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 respectively with 0.1 mol / L K2CO3. One tube is used as a control group without any treatment. Dilute the 3C2 2A4 monoclonal antibody concentration to 1 mg / mL, add 10 μL of monoclonal antibody to each well, mix well, and incubate at 37 °C for 1 h. Then add 30 μL of 10% (mass fraction) NaCl solution, incubate at 37 °C for 10 min, and react at room temperature for 2 h, and observe the color change of the colloidal gold solution.

[0115] (2) Exploration of the optimal concentration for colloidal gold labeling of monoclonal antibodies

[0116] Take 8 detachable 96-well plate sample wells, add 300 μL of 30 nm colloidal gold solution to each well, adjust to the optimal pH value, and add different volumes of monoclonal antibody to each well so that the concentrations of the monoclonal antibody are 0 mg / mL, 2×10 -3 mg / mL, 4×10 -3 mg / mL, 6×10 -3 mg / mL, 8×10 -3 mg / mL, 10×10 -3 mg / mL, 15×10 -3 mg / mL, 20×10 -3 mg / mL. Mix well, incubate at 37 °C for 1 h, then add 30 μL of 10% (mass fraction) NaCl solution. The best situation for the binding of MAP monoclonal antibody and colloidal gold particles should be that there is no color change in the colloidal gold sol.

[0117] 2. Coupling of colloidal gold and MAP monoclonal antibody

[0118] Take a clean glass bottle, add 30 mL of colloidal gold solution, adjust to the optimal pH value of the colloidal gold with 0.1 mol / L K2CO3, then add the optimal labeling amount (1 mL of 4.8 μg / mL 3C2 2A4 monoclonal antibody) of MAP monoclonal antibody 3C2, react at 37 °C for 1 h, and incubate with 10% (mass fraction) BSA for 0.5 h. Transfer the solution to a new centrifuge tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, resuspend the precipitate with the resuspension solution, and centrifuge. Repeat this process 3 times in total. Resuspend the precipitate with the colloidal gold complex solution and store at 4 °C for later use.

[0119] 3. Assembly of MAP colloidal gold immunochromatographic test strip

[0120] (1) Pretreatment of sample pad

[0121] To improve the binding and release of the sample to be detected and the gold-labeled antibody on the gold-labeled pad. Pretreat with a mixed solution of 0.2% (volume fraction) Tween-20 and 3% (mass fraction) sucrose, place in an incubator at 37 °C, and dry.

[0122] (2) Pretreatment of conjugate pad

[0123] The conjugate pad is an important part of the gold-labeled solution. The quality of the conjugate pad determines the stability and effectiveness of the sample detection result. Pretreating the conjugate pad is very important for the sample detection of the test strip. Pretreat the conjugate pad by soaking it in a 2% (mass fraction) sucrose solution containing surfactant for 20 min, place it in a well-ventilated place to dry naturally, and evenly spray the 4.8 μg / mL colloidal gold-labeled Mycobacterium paratuberculosis monoclonal antibody onto the pretreated conjugate pad, and dry at 37 °C for 12 h.

[0124] 4. Selection of the Optimal Coating Concentration of the T-line Detection Antibody

[0125] Dilute goat anti-mouse IgG with PBS to a concentration of 2 mg / mL and draw it on the nitrocellulose membrane to form a quality control line. Adjust the concentration of the MAP 2D10 1D4 monoclonal antibody to 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 0.5 mg / mL, and draw lines (T-line). Add positive samples to be tested, and determine the optimal antibody concentration for the test line according to the test results.

[0126] 5. Preparation of the Colloidal Gold Immunochromatographic Test Strip

[0127] Place the base plate of the test strip, the PVC (polyvinyl chloride) plate, in a horizontal position, and sequentially paste the NC membrane - absorbent paper - conjugate pad - sample pad. The NC membrane is at the bottom center of the PVC base plate, the absorbent paper is at the upper edge of the PVC plate, and the lower edge of the absorbent paper covers the NC membrane; the upper edge of the conjugate pad covers the NC membrane by 1 mm. The upper edge of the sample pad covers the conjugate pad by 2 mm, and the lower edge is flush with the lower edge of the PVC plate. The NC membrane is the carrier for coating the antigen: the quality control line is goat anti-mouse IgG, and the test line is the 2.0 mg / mL 2D10 1D4 monoclonal antibody, with a distance of 4 mm between the two lines; after assembling the colloidal gold test strip, use a strip cutter to cut it into 4 mm × 6 cm.

[0128] Results

[0129] 1. Optimization of the Conditions for Labeling MAP Monoclonal Antibody with Colloidal Gold

[0130] (1) Optimization of the Optimal pH Value

[0131] Adjust the colloidal gold solution to different pH values using 0.1 M K2CO3, and add the monoclonal antibody of MAP and react for a period of time. Then observe the color of the colloidal gold solution. The results show that when the pH value of the colloidal gold solution is 7.0, the color of the colloidal gold does not change significantly, as Figure 3 shown. Therefore, the optimal pH value for the binding of the 3C2 2A4 monoclonal antibody to colloidal gold is 7.0.

[0132] (2) Exploration of the Optimal Concentration of Labeling the 3C2 2A4 Monoclonal Antibody with Colloidal Gold

[0133] Adjust the colloidal gold solution in 8 wells to the optimal pH = 7.0. Add 0, 0.6, 1.2, 1.8, 2.4, 3.0, 4.5, and 6.0 μL of the 1 mg / mL monoclonal antibody to each well in turn, so that the concentrations are 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL respectively. After reacting for a certain time, observe the color change. The results show that when the labeling amount of the antibody is 4 μg / mL, the color of the colloidal gold does not change significantly, asFigure 4 As shown, the optimal concentration of the monoclonal antibody labeled with colloidal gold is 4.8 μg / mL.

[0134] (3) Selection of the optimal antibody concentration for the test line

[0135] Take 30 mL of colloidal gold solution. After adjusting the optimal pH value, add 48 μL of 1 mg / mL 3C2 2A4 monoclonal antibody. Here, the 3C2 2A4 monoclonal antibody is the antibody that binds to colloidal gold and is the solution for the gold conjugate pad. After reacting for 1 h, add 10% BSA by mass for blocking treatment, and then use the resuspension solution and the reconstitution solution to treat the gold conjugate solution for standby.

[0136] Dilute the 2D10 1D4 monoclonal antibody to 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 0.5 mg / mL for scribing. Here, the 2D10 1D4 monoclonal antibody is the antibody scribed on the nitrocellulose membrane, that is, the test line, and use goat anti-mouse IgG as the C line for the assembly of the test strip. The results show that when the concentration of the T line is 2 mg / mL, the color development result is the best, as Figure 5 shown.

[0137] Example 3

[0138] Specificity test of the MAP colloidal gold immunochromatographic test strip

[0139] Using activated MAP, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, BCG, and Streptococcus agalactiae as samples respectively, perform specificity detection using the colloidal gold immunochromatographic test strip established by the present invention.

[0140] The results show that: the established immunochromatographic test strip has no cross-reaction with other bacterial species and has good specificity, as Figure 6 shown.

[0141] Example 4

[0142] Sensitivity detection of the MAP colloidal gold immunochromatographic test strip

[0143] Perform 10-fold serial dilution on MAP. When the total number of colonies of the pathogen to be detected is between 10 7 -10 1 CFU / mL, perform sensitivity detection using the colloidal gold immunochromatographic test strip established by the present invention, and at the same time conduct a control experiment with the national standard qPCR method.

[0144] After determining the total number of colonies of the activated MAP using a turbidimeter, dilute it to 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×105 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, and it was dropped onto the test strip for observation and compared with the qPCR method.

[0145] The results were as Figure 7 and Figure 8 shown: When the total number of colonies was 1×10 1 CFU / mL, the T line was clearly visible, and the number of colonies detected by the qPCR method was 1×10 0 CFU / mL. Therefore, the sensitivity of the test strip was 1×10 1 CFU / mL.

[0146] Example 5

[0147] Detection Results of MAP in Artificially Contaminated Raw Milk

[0148] When the optimized assembled test strip and the national standard qPCR method were used to jointly detect 64 artificially contaminated samples, 60 samples were detected as positive by the colloidal gold immunochromatographic test strip, and 64 samples were detected as positive by the qPCR method (the total number of colonies of the MAP bacterial solution was measured using a turbidimeter. Sterile raw milk and the MAP bacterial solution were mixed in a ratio of 900 μL:100 μL, and 100 μL of the mixed solution from the previous tube was pipetted into the next tube of raw milk. 60 samples of the bacterial solution were diluted to 1×10 7 CFU / mL - 1×10 1 CFU / mL, and 4 samples were diluted to 1×10 0 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 onto the sample pad for detection and compared with the national standard qPCR method to calculate the coincidence rate.). The positive coincidence rate was 93.75%. Figure 9 、 Figure 10 are partial detection results of artificial contamination.

[0149] Example 6

[0150] Clinical Detection Results of MAP Colloidal Gold Immunochromatographic Test Strip

[0151] After processing clinical raw milk samples, 168 clinical samples were detected. Figure 11 、 Figure 12 It showed that neither the colloidal gold immunochromatographic test strip nor the national standard qPCR method detected clinical positive samples.

[0152] 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 avoid redundancy, the present invention describes preferred embodiments.

[0153] 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.

[0154] 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 changes and modifications.

Claims

1. A colloidal gold immunochromatographic test strip, characterized in that: Including base plate, nitrocellulose membrane, absorbent paper, conjugate pad, sample pad; The conjugate pad is adsorbed with colloidal gold-labeled Mycobacterium paratuberculosis monoclonal antibody, the nitrocellulose membrane is provided with a detection line coated with Mycobacterium paratuberculosis monoclonal antibody and a quality control line composed of goat anti-mouse IgG; the bottom plate is a polyvinyl chloride bottom plate; The Mycobacterium paratuberculosis subspecies monoclonal antibody is 3C22A4 or 2D101D4; The nucleotide sequence of the heavy chain variable region of 3C22A4 is shown in SEQ ID NO.3; The nucleotide sequence of the light chain variable region of 3C22A4 is shown in SEQ ID NO.7; The nucleotide sequence of the heavy chain variable region of 2D101D4 is shown in SEQ ID NO.9; The nucleotide sequence of the light chain variable region of 2D101D4 is shown in SEQ ID NO.

11.

2. A method for preparing the colloidal gold immunochromatographic test strip according to claim 1, characterized in that: The following steps are involved: Preparation of colloidal gold-labeled 3C22A4 monoclonal antibody: combining the 3C22A4 monoclonal antibody with colloidal gold to obtain the colloidal gold-labeled 3C22A4 monoclonal antibody; Treating the conjugate pad and the nitrocellulose membrane: spraying the colloidal gold-labeled 3C22A4 monoclonal antibody on the conjugate pad; streaking the 2D101D4 monoclonal antibody on the nitrocellulose membrane to form the detection line; streaking the goat anti-mouse IgG on the nitrocellulose membrane to form the quality control line; The conjugate pad and the nitrocellulose membrane are assembled to form a colloidal gold immunochromatographic test strip.

3. The preparation method according to claim 2, characterized in that: The concentration of the colloidal gold-labeled 3C22A4 monoclonal antibody on the conjugate pad is 4 μg / mL to 20 μg / mL.

4. The preparation method according to claim 2, characterized in that: The concentration of the 2D101D4 monoclonal antibody on the nitrocellulose membrane is 1.5 mg / mL to 2 mg / mL.

5. The preparation method according to claim 2, characterized in that: The concentration of the goat anti-mouse IgG is 1.5 mg / mL to 2.5 mg / mL.

6. The preparation method according to claim 2, characterized in that: When the 3C22A4 monoclonal antibody is combined with colloidal gold, the pH of the colloidal gold is 7.0-9.

0.

7. Use of the colloidal gold chromatography test strip according to claim 1 in detecting avian Mycobacterium paratuberculosis subspecies.

8. The use according to claim 7, characterized in that: The avian Mycobacterium paratuberculosis subspecies is Mycobacterium paratuberculosis.

9. The use according to claim 7, characterized in that: The method for detecting avian mycobacterium paratuberculosis subspecies is: inserting the sample end into the sample liquid to be tested for detection.

10. The use according to claim 9, characterized in that: If the indicator line is displayed at the corresponding quality control area C position on the nitrocellulose membrane, and the indicator line is not displayed at the detection area T position, it means that the test result is negative, indicating that the test sample does not contain avian Mycobacterium paratuberculosis subspecies; If two indicator lines are displayed at the T and C positions on the nitrocellulose membrane, it means the result is positive, indicating that the sample to be tested contains avian Mycobacterium paratuberculosis subspecies; When the quality control area does not display the indicator line, the test paper is considered invalid regardless of whether the detection area T displays the indicator line or not.

Images