A detection test strip for Leishmania antibody, its preparation method and application

Abstract

The present invention discloses a Leishmania antibody detection test strip and its preparation method and application, belonging to the field of lateral flow chromatography detection. The present invention includes the expression and purification of the recombinant antigen LmSTI1-Nh36 tandem protein of Leishmania, the preparation of a colloidal gold immunochromatographic test strip and its application. The sensitivity of detecting Leishmania antibodies is 1:2560, and the specificity is 100%. The colloidal gold antibody detection test strip prepared with the Leishmania LmSTI1-Nh36 tandem protein as the detection target has the advantages of being convenient, fast, sensitive, specific, etc., can obtain a clear detection result within 15 minutes, is suitable for on-site epidemiological investigation, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of lateral flow chromatography detection, and relates to a Leishmania antibody detection test strip, a preparation method thereof and an application thereof. Background Art

[0002] Leishmaniasis (LSH) is a zoonotic disease caused by Leishmania infection, which is widely distributed in various regions of the world. Dogs are the main intermediate hosts. According to the World Health Organization, cases of Leishmania have been reported in 98 countries around the world. This disease has been endemic in the northwestern regions of China for a long time. In recent years, epidemiological reports on dogs infected with Leishmania have been carried out in many places such as Beijing, Henan, and Hebei. At present, there is no vaccine for canine leishmaniasis. If not treated in time after infection, the mortality rate is extremely high, and it is easy to infect humans. Therefore, a reliable detection method is of great significance for strengthening the screening of Leishmania infection in animals such as dogs and ensuring public health safety.

[0003] The application of immunological methods in the detection of Leishmania has received increasing attention. The most effective immunological detection method is to detect specific antibodies against Leishmania disease using purified natural antigens or recombinant antigens. Common detection methods include indirect hemagglutination assay, enzyme-linked immunosorbent assay, enzyme-linked immunoelectrotransfer blot, gold-labeled immunodot blot, and immunochromatographic strip method. Among them, the immunochromatographic strip method does not require a cold chain system to store reagents, has simple operation, rapid and intuitive results, and low requirements for instruments and equipment and operators, and is very suitable for on-site use. The performance of immunological detection methods highly depends on the type, source and purity of antigens. At present, the recombinant antigen K39 has been reported more. The immunochromatographic strip prepared by the recombinant antigen K39 of this gene fragment by InBios Company in the United States has achieved good results in on-site application. However, although this kit has certain effects in detection, relying only on the K39 protein as the detection target has the risk of missed detection. To meet the urgent market demand for high detection rates, the present invention aims to develop a Leishmania disease detection product and method with high detection rate, high accuracy and strong specificity. Summary of the Invention

[0004] In order to improve the deficiencies of the prior art, the present invention provides a Leishmania antibody detection test strip, a preparation method thereof and an application thereof. The present invention includes the following technical solutions:

[0005] In the first aspect, the present invention provides a preparation method of a Leishmania detection target recombinant antigen. The main preparation method of the target is as follows:

[0006] S1: Synthesis and optimization of LmSTI1 and Nh36 genes: Codon optimization was performed on the LmSTI1 and Nh36 gene sequences. The optimized LmSTI1 has the nucleotide sequence shown in SEQ ID NO:1, and the optimized Nh36 has the nucleotide sequence shown in SEQ ID NO:2. A recombinant plasmid containing the tandem genes of LmSTI1 and Nh36 was constructed.

[0007] S2: Protein expression and purification: The recombinant plasmid was transfected into Expi293 cells, and the culture supernatant of the transfected Expi293 cells was collected. Using protein purification technology, the LmSTI1-Nh36 tandem protein was isolated and purified from the cell culture supernatant, and the purity analysis of the collected protein was performed. The results of protein electrophoresis are shown in Figure 1 .

[0008] In a second aspect, the present invention provides a test strip for detecting Leishmania antibodies. The test strip includes a PVC bottom plate, a blood filtration membrane, a conjugate pad, a nitrocellulose membrane (NC membrane), and a water absorption pad ( Figure 2 ). The preparation method of the Leishmania antibody detection test strip includes the following steps:

[0009] S1: The colloidal gold-labeled LmSTI1-Nh36 tandem protein and DNP antibody were sprayed on the conjugate pad at a spraying amount of 2.5 μL / cm, and placed in an oven at 37 °C for 2 h to dry, obtaining a conjugate pad containing the colloidal gold-labeled LmSTI1-Nh36 tandem protein and DNP antibody;

[0010] S2: Using a membrane scribing instrument, T line and C line were scribed on the NC membrane. The LmSTI1-Nh36 tandem protein (1.25 mg / mL, 0.8 μL / cm) was immobilized in the T line area, and DNP-BSA (0.75 mg / mL, 0.7 μL / cm) was immobilized in the C line area;

[0011] S3: The blood filtration membrane, conjugate pad, NC membrane, and water absorption pad were sequentially adhered to the PVC bottom plate. One end of the bottom plate was the water absorption pad, and the other end was the blood filtration membrane. One end of the T line of the NC membrane was overlapped (overlapping 1-2 mm) and connected to the colloidal gold-labeled conjugate pad, and one end of the C line of the nitrocellulose membrane was overlapped (overlapping 1-2 mm) and connected to the water absorption pad. The test strip was cut into strips with a width of 0.5 cm and a length of about 6 cm using a cutting machine, obtaining the Leishmania antibody detection test strip.

[0012] In a third aspect: The present invention also provides a method for using the test strip for detecting Leishmania antibodies. The method for use includes the following steps:

[0013] S1: Equilibrate the blood sample to be tested (blood sample) to room temperature;

[0014] S2: Centrifuge the blood sample at 3000 rpm for 5 min at room temperature, take the supernatant (serum to be tested) into a microcentrifuge tube for standby. If the sample cannot be tested immediately, it should be stored refrigerated at 2 - 8°C. If it exceeds 24 hours, it should be stored frozen at -20°C.

[0015] S3: Dilute the serum to be tested about 3 times, use a dropper to add 4 drops (about 120 μL) of the serum to be tested onto the blood filter membrane of the test strip, and observe the test result after reacting at room temperature for 10 min.

[0016] S4: The result determination method is as follows: If the C line shows color and the T line is visible to the naked eye, it is judged as positive; if the C line shows color and the T line does not show color, it is judged as negative; if the C line does not show color, it is judged as invalid regardless of whether the T line shows color or not.

[0017] The detection principle of the Leishmania antibody detection test strip provided by the present invention is as follows: After the serum of dogs infected with Leishmania is added dropwise to the blood filter membrane, it moves through the adsorption of the water absorption pad at the rear end. When passing through the conjugate pad, the Leishmania antibody in the serum binds to the colloidal gold-labeled tandem protein fixed on the conjugate pad. During the process of the conjugate moving backward, it binds to the colloidal gold-labeled tandem protein on the T line of the NC membrane, forming an antigen-antibody-antigen "sandwich" and starting to show color. DNP (anti-deoxyribonucleoprotein antibody) does not produce non-specific reactions with interfering substances in the sample. The DNP molecule crosslinked with BSA (bovine serum albumin) and the anti-DNP antibody can form a stable complex. When the target antigen exists in the sample, the anti-DNP antibody will preferentially bind to the target antigen, thus forming a visible mark on the test line. At the same time, the unbound target antigen will bind to the DNP-BSA complex, forming an independent C line.

[0018] The Leishmania antibody detection test strip provided by the present invention, its preparation method and application have the advantages of high accuracy, high sensitivity, strong specificity, simple operation, short detection time, low requirements for operators, and are suitable for the rapid clinical detection of canine leishmaniasis. It is a rapid detection test strip with strong practicability. Brief Description of the Drawings

[0019] Figure 1 Electrophoresis diagram of LmSTI1-Nh36 tandem protein.

[0020] Figure 2 Schematic diagram of the Leishmania antibody detection test strip. Detailed Embodiments

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The test methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0022] Example 1 Preparation of Leishmania Antibody Detection Test Strip

[0023] 1. Preparation of LmSTI1-Nh36 Tandem Protein

[0024] 1.1 Synthesis of LmSTI1-Nh36 Tandem Gene

[0025] S1: Refer to the Nh36 gene sequence and LmSTI1 gene sequence of Leishmania donovani in GenBank, and optimize their codons. The optimized LmSTI1 gene sequence is shown in SEQ ID NO:1, and the optimized Nh36 sequence is shown in SEQ ID NO:2.

[0026] SEQ ID NO:1:

[0027] atggatgcaacggaattgaaaaataagggaaacgaggaatttagcgccggtagatacgtggaggccgtgaactatttcagtaaagccataca

[0028] gctggatgaacagaacagcgtgctgtatagcaataggtctgcatgttttgcagctatgcagaaatataaggacgcactcgacgatgcagacaa

[0029] gtgcatctcaatcaagcccaattgggcgaaaggttatgttcgaagaggggcagcattgcacggaatgaggaggtatgatgacgccatagctg

[0030] cctacgagaaaggactgaaagttgatccctccaactccgggtgtgcgcagggcgtaaaagatgttcaggtggccaaggctagagaggccc

[0031] gggacccaatcgctagagtgttcactcccgaagcgtttagaaagatccaggaaaaccccaagctgagtttgctcatgctccaaccagattacg

[0032] tgaaaatggtggataccgtaatcagagatccaagccagggacgcctctacatggaagatcaaagatttgccctgacactgatgtaccttagcg

[0033] ggatgaaaataccaaatgacggggacggtgaggaggaggaaagaccttccgctaaggcggcggagaccgcaaaacccaaagaggaga

[0034] aacctcttacagacaatgaaaaggaggcccttgctctcaaggaggaaggcaataagctctacctctctaagaagtttgaagaagccctcacta

[0035] agtatcaggaggcacaagtgaaggatcctaacaacaccctgtatattctgaatgtaagtgccgtctatttcgagcaaggggattatgacaaatg

[0036] cattgccgagtgtgaacacggaatcgagcatggccgcgagaatcattgcgattacacaattatcgctaaattgatgacccggaatgccctctgt

[0037] ctccaaaggcagcgaaaatacgaggcagctatagacctgtacaaaagagccctggtggagtggcgcaatccagatacactgaagaagctg

[0038] accgaatgtgagaaagagcaccaaaaggctgttgaagaggcctacatcgatccagaaattgcgaaacaaaagaaggacgaggggaatca

[0039] gtactttaaggaagataagtttccagaagcagtggccgcttacactgaagccatcaagcggaatccagccgaacatacaagctattctaacag

[0040] ggcggcggcttatataaagctgggggcgttcaatgatgctctgaaggacgcggagaaatgcatcgagctgaaacccgattttgtgaagggat

[0041] acgcccgaaaagggcatggatatttttggaccaaacagtataacagagccctgcaggcatatgatgaagggctgaaagtggacccttctaatg

[0042] ctgactgcaaggacggcagatatcgcacaatcatgaaaatccaggaaatggccagtggacaatccgccgacggagatgaagcggctcgga

[0043] gagcaatggatgaccctgaaatcgccgcaatcatgcaagactcatacatgcagctggtgctgaaagaaatgcaaaatgatcccacacggatt

[0044] caggaatacatgaaggatagcggtatttcttctaaaatcaataagctgattagcgcagggatcatcaggttcggataa

[0045] SEQ ID NO:2:

[0046] atgccacgaaaaatcatcctcgactgtgatcctggaatcgatgatgccgttgccatatttttggcccatggaaaccctgaagtcgagctgctggc

[0047] catcactaccgtcgtgggaaatcagactcttgagaaggtgacacggaatgcacggctggtcgctgatgtcgccggtatagtgggagtgccag

[0048] tggcggccgggtgtaccaaacccctggtacgcggggtgagaaacgcatctcagattcatggcgagaccggcatggggaatgtctcctatcc

[0049] cccagagtttaaactcgatgggaggcacgctgttcagttgataattgatctgatcatgtcccacgagccgaagacaatcaccttggtcccaacc

[0050] ggagggctgactaatatcgccatggctgtgagactggagccaagaatcgtagatcgggttaaggaggtggtactgatgggaggaggctatc

[0051] acaccggtaacgctagccccgtggcggagttcaatgtgtttgtggatcccgaggctgctcacattgtgttcaatgagtccaacgtcacaatggt

[0052] cggtctggacctcacgcaccaggctctcgccactccagccgttcagaagcgggtaaaagaagtgggcacaaagcccgccgcatttatgctg

[0053] cagatcctggacttctacacaaaagtatacgagaaggagcggaacacatatgccacctatcacgatccctgcgctgttgcctatgttatcgacc

[0054] ccacggtgatgacaaccgaacaggtgccggtcgacatagagctcaatggtgctctcactacgggtatgactgtcgccgatttccggtacccaa

[0055] ggccaaagcattgtcacacgcaggtcgcagtgaagttggacttcgacttctggtgcctggtgattgatgcccttaaaagaatcggagaccctca

[0056] gtaa

[0057] S2: After concatenating the optimized sequences, submit them to Beijing Tsingke Biotechnology Co., Ltd. for gene synthesis. The synthesized concatenated gene recombinant plasmid is stored at -20°C.

[0058] 1.2 Expression and Purification of LmSTI1-Nh36 Concatenated Protein

[0059] S1: Purchase commercial Expi293 cells, resuscitate and culture them in 25 mL of OPM serum-free medium.

[0060] S2: When the cell density reaches 2.5×10 6 cells / mL, passage the cells into 100 mL of medium.

[0061] S3: When the cell viability reaches over 97% and the density is 1.5 - 2.0×10 6 cells / mL, use CarpTrans transfection reagent to transfect the recombinant plasmid into Expi293 cells. The transfection steps are as follows:

[0062] (a) Preparation of plasmid dilution: Add 100 μg plasmid into 5 mL medium and gently mix;

[0063] (b) Preparation of transfection reagent dilution: Add 400 μL Carp trans transfection reagent into 5 mL medium and gently mix;

[0064] (c) Preparation of plasmid - transfection reagent complex: Add the transfection reagent dilution into the plasmid dilution, gently mix and let it stand at room temperature for 15 min to fully react and form the plasmid - transfection reagent complex;

[0065] (d) Slowly add the incubated plasmid - transfection reagent complex into the cells to be transfected, gently shaking the culture flask while adding;

[0066] (e) Put the transfected cells into a 37°C shaker and continue culturing at 110 rpm.

[0067] S4: After culturing for 24 hours, add 5 mL OPM - 293Pro Feed into the medium and continue culturing in a 37°C shaker at 110 rpm.

[0068] S5: On the sixth day after transfection, collect the culture supernatant of the transfected Expi293 cells.

[0069] S6: Use protein purification technology to isolate and purify the LmSTI1 - Nh36 tandem protein from the cell culture supernatant. The amino acid sequence of the LmSTI1 - Nh36 tandem protein is shown in SEQ ID NO:3.

[0070] SEQ ID NO:3:

[0071] MDATELKNKGNEEFSAGRYVEAVNYFSKAIQLDEQNSVLYSNRSACFAAMQKYKDALDD

[0072] ADKCISIKPNWAKGYVRRGAALHGMRRYDDAIAAYEKGLKVDPSNSGCAQGVKDVQVA

[0073] KAREARDPIARVFTPEAFRKIQENPKLSLLMLQPDYVKMVDTVIRDPSQGRLYMEDQRFA

[0074] LTLMYLSGMKIPNDGDGEEEERPSAKAAETAKPKEEKPLTDNEKEALALKEEGNKLYLSK

[0075] KFEEALTKYQEAQVKDPNNTLYILNVSAVYFEQGDYDKCIAECEHGIEHGRENHCDYTIIA

[0076] KLMTRNALCLQRQRKYEAAIDLYKRALVEWRNPDTLKKLTECEKEHQKAVEEAYIDPEIA

[0077] KQKKDEGNQYFKEDKFPEAVAAYTEAIKRNPAEHTSYSNRAAAYIKLGAFNDALKDAEK

[0078] CIELKPDFVKGYARKGHGYFWTKQYNRALQAYDEGLKVDPSNADCKDGRYRTIMKIQE

[0079] MASGQSADGDEAARRAMDDPEIAAIMQDSYMQLVLKEMQNDPTRIQEYMKDSGISSKINKLISAGIIRFGQ----MPRKIILDCDPGIDDAVAIFLAHGNPEVELLAITTVVGNQTLEKVTRNA RLVADVAGIVGVPVAAGCTKPLVRGVRNASQIHGETGMGNVSYPPEFKLDGRHAVQLIIDLIMSHEPKTITLVPTGGLTNIAMAVRLEPRIVDRVKEVVLMGGGYHTGNASPVAEFNVFVDPEAAHIVFNESNVTMVGLDLTHQALATPAVQKRVKEVGTKPAAFMLQILDFYTKVYEKERNTYATYHDPCAVAYVIDPTVMTTEQVPVDIELNGALTTGMTVADFRYPRPKHCHTQVAVKLDFDFWCLVIDALKRIGDPQ

[0080] The protein purification technology includes affinity chromatography and gel filtration chromatography techniques.

[0081] ① Method for purifying protein by affinity chromatography:

[0082] (a) Preparation of working solutions and samples: Prepare Binding buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0), Elution Buffer (20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0), and filter them through a 0.45 μm filter membrane. Centrifuge the cell culture supernatant at 10,000 rpm for 30 min and filter it through a 0.45 μm filter membrane.

[0083] (b) Turn on the machine to clean the pipeline and install the nickel column: Turn on the AKTA purifier, first rinse the pipeline with pure water and install the nickel column, then continue to rinse with pure water at a flow rate of 3 mL / min for 10 min; then rinse the nickel column with Binding buffer at a flow rate of 3 mL / min for 10 min.

[0084] (c) Loading: Place the A1 pump head into the prepared sample and load at a flow rate of 2 mL / min until all the samples are loaded.

[0085] (d) Washing away impurities: First, wash away most of the impurity proteins with 10% Elution Buffer at a flow rate of 2 mL / min until the baseline is stable, then wash away a small amount of impurity proteins bound to the nickel column with 15% Elution Buffer at a flow rate of 2 mL / min until the baseline is stable.

[0086] (e) Elution: Rinse with 100% Elution Buffer at a flow rate of 2 mL / min and collect the elution peak until the baseline is stable.

[0087] (f) Cleaning the nickel column and pipeline: Rinse with pure water and 20% ethanol respectively at a flow rate of 2 mL / min for 20 min each.

[0088] (g) Remove the chromatography column and turn off the power of the AKTA purifier.

[0089] (h) Detect the size and purity of the target protein in the eluate by SDS-PAGE, aliquot it, and store it at -80 °C for later use.

[0090] ② Method for purifying protein by gel filtration:

[0091] (a) Preparation of working solutions and samples: Prepare PBS (pH 8.0) buffer and filter it through a 0.45 μm filter membrane. Concentrate the collected target protein and filter it through a 0.22 μm filter membrane.

[0092] (b) Turn on the machine to clean the pipeline and install the chromatography column: Turn on the AKTA purifier, first rinse the pipeline with pure water and install Superdex TMIncrease the 75 10 / 300GL chromatography column, then continue to rinse with pure water at a flow rate of 0.5 mL / min for 60 min; then rinse the nickel column with PBS at a flow rate of 0.8 mL / min for 60 min.

[0093] (c) Loading: Pump the concentrated protein sample into the loading loop with a syringe and continue to rinse with PBS at a flow rate of 0.8 mL / min.

[0094] (d) Collecting the target protein: When the 280 absorption peak starts to rise, collect the protein with a peak higher than 5 mAU.

[0095] (e) Cleaning Superdex TM Clean the 75 Increase 10 / 300GL chromatography column and pipeline: Rinse with pure water and 20% ethanol respectively at a flow rate of 0.8 mL / min for 60 min each.

[0096] (f) Remove the chromatography column and turn off the power of the AKTA purifier.

[0097] (g) Detect the size and purity of the target protein in the eluate by SDS-PAGE. After aliquoting, store at -80 °C for later use.

[0098] 2. Colloidal gold labeling of LmSTI1-Nh36 tandem protein

[0099] S1: Adjust the pH of the colloidal gold solution to 7.6 - 8.0 with 0.1 mol / L K 2 CO 3 solution;

[0100] S2: Add 45 - 55 ng / mL of LmSTI1-Nh36 tandem protein to 100 mL of colloidal gold solution and stir for 10 min;

[0101] S3: Slowly add 10% bovine serum albumin to a final concentration of 1%, mix well and let stand at room temperature for 15 min;

[0102] S4: Centrifuge at 4 °C and 10000 rpm for 20 min, aspirate the supernatant, and resuspend the precipitate in 1 mL of 0.01 mol / L pH 9.2 borate buffer containing 5% sucrose, 1% BSA, 0.5% Tween-20, 0.2% PEG20000 and 0.02% sodium azide, and store at 4 °C for later use.

[0103] 3. Preparation of Leishmania antibody detection test strip

[0104] S1: Spray the colloidal gold-labeled LmSTI1-Nh36 tandem protein and DNP antibody onto the conjugate pad respectively, with a spraying volume of 2.5 μL / cm, and place it in an oven at 37 °C for drying for 2 h to obtain a conjugate pad containing the colloidal gold-labeled LmSTI1-Nh36 tandem protein and DNP antibody;

[0105] S2: Use a membrane streaking instrument to streak the NC membrane to set the T line and C line. Fix the LmSTI1-Nh36 tandem protein (1.25 mg / mL, 0.8 μL / cm) in the T line area and fix DNP-BSA (0.75 mg / mL, 0.7 μL / cm) in the C line area; the DNP-BSA is purchased from Shanghai Biotool Biotechnology Co., Ltd.

[0106] S3: Adhere the blood filtration membrane, conjugate pad, NC membrane and absorbent pad to the PVC bottom plate in sequence. One end of the bottom plate is the absorbent pad, and the other end is the blood filtration membrane. One end of the T line of the NC membrane overlaps (overlapping 1 - 2 mm) and connects with the colloidal gold-labeled conjugate pad, and one end of the C line of the NC membrane overlaps (overlapping 1 - 2 mm) and connects with the absorbent pad. Cut it into test strips with a width of 0.5 cm and a length of about 6 cm using a chopping machine to obtain a Leishmania antibody detection test strip.

[0107] Example 2 Method for using the Leishmania antibody detection test strip

[0108] 1. Method for using the test strip

[0109] The method for using the Leishmania antibody detection test strip is as follows:

[0110] S1: Equilibrate the blood sample to be tested to room temperature;

[0111] S2: Centrifuge the whole blood at 3000 rpm for 5 min at room temperature, take the supernatant and place it in a microcentrifuge tube for standby. If the sample cannot be detected immediately, it should be stored refrigerated at 2 - 8 °C. If it exceeds 24 hours, it should be stored frozen at -20 °C;

[0112] S3: After diluting the serum to be tested about 3 times, use a dropper to add 4 drops (about 120 μL) of the serum to be tested to the blood filtration membrane of the test strip. After reacting at room temperature for 10 min, observe the test result.

[0113] 2. Result determination

[0114] Positive: The C line shows color, and the T line is visible to the naked eye, judged as positive;

[0115] Negative: The C line shows color, and the T line does not show color, judged as negative;

[0116] Invalid: The C line does not show color, and it is judged as invalid regardless of whether the T line shows color or not.

[0117] Performance Verification of Leishmania Antibody Detection Test Strip in Example 3

[0118] In this example, clinically positive sera were obtained from dogs infected with Leishmania through epidemiological detection. Double-positive results were obtained through etiological detection (PCR method) and serological IFA detection, which were used as positive quality control products. Clinically negative sera for both the etiology of canine leishmaniasis and serological IFA were used as negative quality control sera.

[0119] Meanwhile, the canine sera required in this example included positive sera for common parasitic diseases in dogs (such as positive sera for toxoplasmosis, trichomonas, coccidia, giardia, and amoeba). The preparation of the quality control products combined with epidemiological investigations to collect clinical sera with clear backgrounds and uniform and stable quality.

[0120] 1. Sensitivity

[0121] The positive quality control products were diluted to eight titers of 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, and 1:10240 with 0.15 mol / L PBS dilution (pH 7.2). Three batches of Leishmania antibody detection test strips and serological IFA detection were used to detect the Leishmania positive quality control products simultaneously, and the detection results were compared.

[0122] The steps of immunofluorescence analysis technique (IFA) are as follows:

[0123] S1: Dilute the serum to be tested;

[0124] S2: Drop the serum onto the standard slide, fix it, drop the fluorescein-labeled Leishmania antigen, incubate at 37 °C for 30 min, and then wash it by shaking in a PBS solution of 0.01 mol / L, pH 7.4;

[0125] S3: Use filter paper to fully absorb the excess water, drop a drop of buffered glycerol, and then cover with a cover slip;

[0126] S4: Result determination: Observe the specific fluorescence intensity of the specimen under a fluorescence microscope. "-" indicates no fluorescence, "±" indicates extremely weak suspicious fluorescence, "+" indicates weak but clearly visible fluorescence, and "++~++++" all indicate bright fluorescence. When the fluorescence intensity reaches "++" or above, it can be determined as positive.

[0127] For the Leishmania antibody detection test strip, "-" indicates a negative result, and "+~+++" all indicate positive results. The number of "+" is positively correlated with the color depth of the T line.

[0128] The results of the Leishmania antibody detection test strip and IFA detection are shown in Table 1. When the antibody titer shown in the IFA detection result is 1:2560, the Leishmania antibody detection test strip can accurately detect positive results.

[0129] Table 1 Sensitivity Test Results

[0130]

[0131] 2. Specificity

[0132] Use Leishmania antibody test strips from 3 batches to detect 5 Leishmania negative control products, 1 positive sample of toxoplasmosis, 1 positive sample of trichomoniasis, 1 positive sample of coccidiosis, 1 positive sample of giardiasis, and 1 positive sample of amoebiasis. The test results are shown in Table 2. The above test results are all negative, without cross-reaction with positive samples of toxoplasmosis, trichomoniasis, coccidiosis, giardiasis, and amoebiasis, and the specificity is 100%.

[0133] Table 2 Specificity Test Results

[0134]

[0135] 3. Clinical Sensitivity Detection

[0136] Collect blood from 30 stray dogs and prepare sera to be tested. Verify whether the serum samples contain Leishmania antibodies by IFA, and use Leishmania antibody test strips from 3 batches to detect clinical samples. The test results are shown in Table 3. Only 1 test result of the test strip is inconsistent with the IFA test result, and the clinical sensitivity of the test strip is 96.7%.

[0137] Table 3 Clinical Sensitivity Test Results

[0138]

[0139] 4. Clinical Specificity Detection

[0140] Collect 5 Leishmania serum samples, 1 positive serum sample of toxoplasmosis, 1 positive serum sample of trichomoniasis, 1 positive serum sample of coccidiosis, 1 positive serum sample of giardiasis, and 1 positive serum sample of amoebiasis. Verify whether the serum contains Leishmania by IFA, and use Leishmania antibody test strips from 3 different batches to detect the collected serum samples. The test results are shown in Table 4, and the clinical specificity of the Leishmania antibody test strip is 100%.

[0141] Table 4 Clinical Specificity Test Results

[0142]

[0143] 5. Repeatability

[0144] Use the Leishmania antibody test strip of the same batch to detect 5 positive Leishmania control products, 5 negative Leishmania control products, and 10 clinical samples, and compare the in-batch test results. Use the Leishmania antibody test strips of 3 batches to detect the above samples and compare the between-batch test results. The test results are shown in Table 5 and Table 6. The test results of the test strip are consistent with the IFA test results, and the in-batch repeatability and between-batch repeatability are good.

[0145] Table 5 Results of in-batch repeatability test

[0146]

[0147] Table 6 Results of between-batch repeatability test

[0148]

[0149] 6. Stability

[0150] Conduct long-term stability tests and accelerated stability tests on each component of the Leishmania antibody test strip and the finished test strip. The long-term test conditions are 2 - 30 °C, and the frequencies are 0 month, 1 month, 2 months, 3 months, 6 months, 12 months, and 15 months. The results show that there is no obvious change in the test results of the test strip within 15 months; the accelerated test conditions are a temperature of 50 °C, a relative humidity of 75% ± 5%, and the frequencies are 0 day, 1 day, 2 days, 3 days, 6 days, 12 days, and 15 days. The results show that there is no obvious change in the test results of the test strip within 15 days; combining the long-term test and the accelerated test, the storage period of the Leishmania antibody test strip is set to be stored at 2 - 30 °C for 12 months.

Claims

1. A test strip for detecting antibodies against Leishmania, characterized in that: The test strip comprises LmSTI1-Nh36 tandem protein, and the LmSTI1-Nh36 tandem protein is prepared by the following steps: S1: referring to the gene sequences of Leishmania donovani LmSTI1 and Nh36 in GeneBank, codon optimization was performed, the optimized LmSTI1 had the nucleotide sequence shown in SEQ ID NO:1, and the optimized Nh36 had the nucleotide sequence shown in SEQ ID NO:2; the optimized nucleotide sequences were used to construct a recombinant plasmid containing the tandem genes of LmSTI1 and Nh36; S2: The recombinant plasmid was transfected into Expi293 cells, the culture supernatant of the transfected Expi293 cells was collected, and the LmSTI1-Nh36 tandem protein was separated and purified from the cell culture supernatant using protein purification technology.

2. A test strip for detecting Leishmania antibodies according to claim 1, characterized in that: The test strip comprises a conjugate pad, on which colloidal gold-labeled LmSTI1-Nh36 tandem protein is fixed. The steps for preparing the colloidal gold-labeled LmSTI1-Nh36 tandem protein are as follows: S1: Use 0.1 mol / L K2CO3 solution to adjust the pH of the colloidal gold solution to 7.6-8.0; S2: Add 45-55 ng / mL of LmSTI1-Nh36 tandem protein to 100 mL of colloidal gold solution and stir for 10 min; S3: Slowly add 10% bovine serum albumin to a final concentration of 1%, mix well and leave at room temperature for 15 min; S4: Centrifuge at 10000 rpm for 20 min at 4°C, aspirate the supernatant, and suspend the precipitate in 1 mL of 0.014 mol / L pH 9.2 borate buffer containing 5% sucrose, 1% BSA, 0.5% Tween-20, 0.2% PEG20000 and 0.02% sodium azide, and store at 4°C for later use.

3. A test strip for detecting antibodies against Leishmania according to claim 2, characterized in that: The colloidal gold-labeled LmSTI1-Nh36 tandem protein was fixed on the conjugate pad by uniform spraying at a spray volume of 2.5 μL / cm , and then placed in a 37° C. oven to dry for 2 h.

4. A test strip for detecting Leishmania antibodies according to claim 1, characterized in that: The test strip comprises a nitrocellulose membrane, on which is a T line for displaying the test result, and on which is coated the LmSTI1-Nh36 tandem protein.

5. A test strip for detecting antibodies against Leishmania according to claim 1, characterized in that: The test strip also includes a control area.

6. A test strip for detecting antibodies against Leishmania according to claim 5, characterized in that: The control area of ​​the test strip is coated with DNP-BSA.

Images