Antibody for detecting tomato New Delhi leaf curl virus, colloidal gold test strip and preparation method of colloidal gold test strip
By preparing specific monoclonal antibodies 3-F11-A8 and 5-H1-C10 and combining them with the design of colloidal gold immunoassay strips, the problems of speed, specificity, and sensitivity in detecting New Delhi leaf curl virus in tomatoes in existing technologies have been solved, enabling efficient field detection with results that can be read visually within 5 minutes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
- Filing Date
- 2026-03-03
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies make it difficult to develop colloidal gold test strips that can detect New Delhi leaf curl virus in tomatoes quickly, specifically, and with high sensitivity. In particular, the high conservation of capsid proteins among members of the Geminiviridae family leads to a high risk of cross-reaction, and conventional PCR and LAMP technologies require specialized equipment and environments, which cannot meet the needs of rapid field detection.
Using specific monoclonal antibodies 3-F11-A8 and 5-H1-C10, combined with a colloidal gold immunochromatographic strip design, rapid detection is achieved through the sandwich immunochromatographic principle. Monoclonal antibodies are prepared using hybridoma cell lines ToLCNDV-Cell line-3-F11-A8 and ToLCNDV-Cell line-5-H1-C10, and test lines and control lines are set on the test strip to achieve specific recognition and sensitive detection.
It enables visual interpretation of results within 5 minutes, accurately, specifically, and sensitively detecting ToLCNDV in field cucurbit samples, avoiding cross-reactions, and is suitable for large-scale sample testing, providing a rapid and convenient diagnostic and monitoring method.
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Figure CN121874133A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant virus biodetection technology, specifically to antibodies, colloidal gold test strips, and their preparation methods for detecting New Delhi leaf curl virus in tomatoes. Background Technology
[0002] Tomato New Delhi Leaf Curl Virus (ToLCNDV) belongs to the genus *Phaseolus Aureobasidium* in the family Geminiviraceae. It is a single-stranded circular DNA virus with a genome consisting of two DNA strands (DNA-A and DNA-B), each encoding 6-8 functional proteins. DNA-A is responsible for viral replication and transcriptional regulation, while DNA-B is involved in viral movement within the plant. First discovered in 1995 on tomatoes in the New Delhi region of India, this virus is primarily transmitted persistently by whiteflies and has since spread widely across Asia, Europe, and Africa, becoming a global disease.
[0003] ToLCNDV infection in tomatoes causes symptoms such as upward curling and yellowing of leaves, stunted growth, and deformed fruits, resulting in yield losses of over 50% in severe cases. This virus has a wide host range, infecting more than 30 other economic crops besides tomatoes, including melons, pumpkins, tomatoes, and peppers. Detection of ToLCNDV mainly includes three methods: symptom observation, serological testing, and molecular biological detection. In field observations, the leaf curling and yellowing symptoms caused by ToLCNDV are easily confused with other geminiviruses (such as TYLCV), making accurate diagnosis difficult based on symptoms alone. In serological testing, ELISA and colloidal gold immunochromatographic assay strips are the most commonly used methods. However, due to the high conservation of the mesocapsid protein (CP) of geminiviruses (amino acid sequence similarity can reach over 85% between different species), there is a risk of cross-reactivity, necessitating the screening of specific monoclonal antibodies to improve detection accuracy. Molecular biological detection is currently the most reliable method. Conventional PCR can specifically amplify the CP or Rep gene fragments of ToLCNDV. Real-time quantitative PCR (qPCR) has high sensitivity and quantitative ability, but requires sophisticated instruments and professional operators. Loop-mediated isothermal amplification (LAMP) technology requires a constant temperature environment and takes 40-60 minutes to determine the results, which cannot meet the needs of rapid detection.
[0004] Colloidal gold immunochromatographic assay strips are particularly suitable for rapid field detection of ToLCNDV due to their ease of use and speed (results can be read visually within 5-10 minutes). However, the development of ToLCNDV-specific colloidal gold assay strips faces significant challenges due to the high conservation of the capsid protein (CP) among members of the Geminiviridae family. Summary of the Invention
[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an antibody, a colloidal gold test strip, and a method for preparing the same for detecting ToLCNDV in tomatoes. This invention develops a specific monoclonal antibody, and the colloidal gold immunoassay strip can rapidly, specifically, and sensitively detect ToLCNDV in cucurbit samples. The test strip allows for direct visual interpretation of results within 5 minutes and is suitable for field testing.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides two hybridoma cell lines, including: (1) a hybridoma cell line ToLCNDV-Cell line-3-F11-A8 that secretes the tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8. The hybridoma cell line 3-F11-A8 was deposited on November 11, 2025 at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: C2025277. (2) The hybridoma cell line ToLCNDV-Cell line-5-H1-C10, which secretes the monoclonal antibody 5-H1-C10 against New Delhi leaf curl virus of tomato, was deposited on November 11, 2025 at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCCNO:C 2025278.
[0007] In a second aspect, the present invention provides two monoclonal antibodies, tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8 and tomato New Delhi leaf curl virus monoclonal antibody 5-H1-C10; wherein, monoclonal antibody 3-F11-A8 is secreted by a hybridoma cell line with accession number CCTCC NO:C 2025277; and monoclonal antibody 5-H1-C10 is secreted by a hybridoma cell line with accession number CCTCC NO:C 2025278.
[0008] In some embodiments, the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8 are shown in SEQ ID NO. 1-3, the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO. 4 and SEQ ID NO. 5, and the amino acid sequence of the light chain variable region CDR2 is LAS.
[0009] In some embodiments, the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the tomato New Delhi leaf curl virus monoclonal antibody 5-H1-C10 are shown in SEQ ID NO. 6-8; the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO. 9 and SEQ ID NO. 10, and the amino acid sequence of CDR2 is FAS.
[0010] In a third aspect, the present invention provides the application of the above-mentioned hybridoma cell line or monoclonal antibody in the preparation of a product for detecting New Delhi leaf curl virus in tomatoes.
[0011] In a fourth aspect, the present invention provides a colloidal gold immunochromatographic assay strip for detecting New Delhi leaf curl virus in tomatoes. The strip includes a base plate, and a sample pad, an immunogold pad, a nitrocellulose membrane, and absorbent filter paper sequentially overlapped and fixed to the base plate. The immunogold pad is a gold-labeled pad conjugated with colloidal gold particles labeled with monoclonal antibody 3-F11-A8. A detection line and a control line are sequentially arranged on the nitrocellulose membrane along the sample flow direction. The detection line is coated with monoclonal antibody 5-H1-C10, and the control line is coated with a secondary antibody. Preferably, the secondary antibody is goat anti-mouse IgG antibody.
[0012] In some embodiments, the detection line and the control line are spaced 5 mm ± 1 mm apart.
[0013] In a fifth aspect, the present invention provides a method for preparing a colloidal gold immunoassay strip, comprising the following steps: (1) Adjust the pH of the colloidal gold solution to 8.00-8.50, add monoclonal antibody 3-F11-A8 to the colloidal gold solution to make the final concentration of monoclonal antibody 3-F11-A8 8-12 μg / ml; mix well, react at room temperature for 35-45 min, add BSA solution to stop the reaction, let stand for 20-40 min, centrifuge at low speed to discard the aggregated gold particles, then centrifuge at high speed, discard the supernatant, and redissolve the precipitate with gold labeling solution to prepare an immunocolloidal gold solution containing colloidal gold particles labeled monoclonal antibody-3-F11-A8; (2) The immunocolloidal gold solution prepared in step (1) is coated onto the gold label pad to obtain the immunocolloidal gold pad; (3) Monoclonal antibody 5-H1-C10 and goat anti-mouse IgG antibody were respectively dotted onto nitrocellulose membrane to form nitrocellulose membrane containing detection line and control line; (4) The sample pad, the colloidal gold immunochromatographic pad, the nitrocellulose membrane and the absorbent filter paper are sequentially stacked on the base plate to prepare the colloidal gold immunochromatographic test strip.
[0014] Preferably, in step (1), the colloidal gold solution is prepared according to the following method: Heat a 10% tetrachloroauric acid solution to boiling, stir for 1 minute to mix thoroughly, and immediately add 2.4 mL of a 1% trisodium citrate solution. Continue heating for 5 minutes, then stop heating and continue stirring for 10 minutes at a speed of 2-3 for 20 minutes. After stopping stirring, allow to stand at room temperature. The prepared colloidal gold solution should be free of significant floating matter or precipitate, and the solution color should be purplish-red.
[0015] Preferably, in step (1), the particle size of the colloidal gold in the colloidal gold solution is 40 nm.
[0016] Preferably, in step (1), the concentration of colloidal gold particles labeled with monoclonal antibody 3-F11-A8 in the immunogold solution is 10 μg / ml.
[0017] Preferably, in step (1), the mass concentration of the BSA solution is 10%.
[0018] Preferably, in step (2), the amount of immunogold solution coated on the gold pad is 50 μl / cm².
[0019] Preferably, in step (3), the monoclonal antibody 5-H1-C10 and the goat anti-mouse IgG antibody are both diluted to 1.5 mg / ml, and the dosage is 1 μl / cm; then they are dotted onto the nitrocellulose membrane to form a detection line and a control line, respectively. Beneficial effects
[0020] (1) This invention is the first to prepare monoclonal antibodies 3-F11-A8 and 5-H1-C10 that can specifically recognize New Delhi leaf curl virus of tomato. The combination of monoclonal antibodies 3-F11-A8 and 5-H1-C10 can effectively distinguish between Tomato yellow leaf curl virus (TYLCV), Squash Leaf Curl China Virus (SLCCV), and Ramie mosaic virus (RaMoV) without cross-reactivity.
[0021] (2) The ToLCNDV colloidal gold immunoassay strip of the present invention can accurately, specifically and sensitively detect ToLCNDV in cucurbits in the field. The test strip can be observed with the naked eye within 5 minutes, which is very suitable for large-scale sample testing in the field. It has great application value in actual production and provides material support for the diagnosis, monitoring and early warning of ToLCNDV. Attached Figure Description
[0023] Figure 1 Immunoblot screening test of 13 cell lines.
[0024] Figure 2 Screening test using dot-ELISA on supernatant of 13 cell lines.
[0025] Figure 3 Standard curve for protein concentration working range.
[0026] Figure 4 : A schematic diagram of the ToLCNDV colloidal gold immunoassay strip structure of the present invention.
[0027] Figure 5 The criteria for determining the results of the colloidal gold immunoassay strip of the present invention.
[0028] Figure 6 Specificity analysis of ToLCNDV colloidal gold immunoassay strip.
[0029] Figure 7 Sensitivity analysis of ToLCNDV CP protein on ToLCNDV colloidal gold immunoassay strip.
[0030] Figure 8 Analysis of field crude extracts of samples tested using ToLCNDV colloidal gold immunochromatographic assay strips. Detailed Implementation
[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Example 1:
[0032] Example 1: Preparation of monoclonal antibodies against New Delhi leaf curl virus in tomatoes 1.1 Immunogen protein information (ToLCNDV-CP, Tomato New Delhi Leaf Curl Virus - Coat protein) Immunogen name: His-ToLCNDV-CP Immunogen sequence (276 AAs, MW=31.67 kDa, pI=10.1): MGSSHHHHHHSSGLVPRGSHMAKRPADIIISTPASKVRRRLNFDSPYGARAVVPIARVTKAKAWTNRPMNRKPRMYRMYRSPDVPRGCEGPCKVQSFESRHDVSHIGKVMCVSDVTRGTGLTHRVGKRFCVKSVYVLG KIWMDENIKTKNHTNSVMFFLVRDRRPTGSPQDFGEVFNMFDNEPSTATVKNMHRDRYQVLRKWHATVTGGTYASKEQALVRKFVRVNNYVVYNQQEAGKYENHTENALMLYMACTHASNPVYATLKIRIYFYDSVTN (SEQ ID NO.11) 1.2 Animal Immunization
[0033] Five mice were immunized with each of the aforementioned target antigens, ToLCNDV-CP protein, for a total of four immunizations over a 22-day period. Using ToLCNDV-CP as both the immunogenic and detection antigens, serum titers were detected by indirect ELISA, and Western blotting was performed on the serum to screen for serum with high titers and specificity. Based on the ELISA and Western blotting results, the most suitable mice were selected for subsequent cell fusion experiments.
[0034] 1.3 Cell fusion, subcloning screening, and line establishment
[0035] Spleens from the most suitable immunized mice were used for cell fusion. Positive hybridoma cells were obtained by screening in HAT medium. The positive hybridoma cells were subcloned using the limiting dilution method to obtain monoclonal cell lines. During each subcloning, 2-3 rounds of indirect ELISA screening were performed. After obtaining the required positive monoclonal cell lines, cell lines were established. The cell supernatant was used to perform Western blotting (WB) and dot-ELISA on virus samples containing ToLCNDV to screen for cell lines with high sensitivity and specificity.
[0036] Results: Thirteen positive monoclonal cell lines that met the requirements were obtained. In Western blotting and dot-ELISA, the supernatant signals of four monoclonal cell lines, namely 1-E4-D8, 3-F11-A8, 5-H1-C10 and 26-E12-H10, were found to be strong and single, and their sensitivity and specificity met the requirements for subsequent experiments.
[0037] 1.4 Production of Monoclonal Antibodies
[0038] The four monoclonal cell lines 1-E4-D8, 3-F11-A8, 5-H1-C10, and 26-E12-H10 were cultured in supernatant; antibodies were purified using the protein G method, and 2 mg of antibody was produced from each cell line.
[0039] Antibody titers were detected using an indirect ELISA method. The antigen PMMoV-CP was coated at a concentration of 5 μg / ml, 100 μl / well, and the coating dilution buffer was CBS buffer (pH=9.6). The secondary antibody was Peroxidase AffiniPure Goat Anti-Mouse IgG (FC). Antibody titer was defined as the highest dilution ratio when the detected value / blank control was ≥3. The antibody titer results are shown in Table 1 below. Table 1. Antibody titer test results
[0040] 1.5 Monoclonal antibody subtype identification
[0041] The subtypes of the above four monoclonal antibodies were identified, and the results are shown in Table 2 below.
[0042] Table 2: Monoclonal Antibody Subtype Identification Results
[0043] Example 2: Screening of monoclonal antibody pairs against New Delhi leaf curl virus in tomato
[0044] 2.1 Selection of Monoclonal Antibody Pairs (1) Biotinylated antibody titer detection Antibody titers were detected using an indirect ELISA method. ToLCNDV-CP and non-specific antigen were used as coating antigens at a concentration of 2 μg / ml (100 μl / well). CBS buffer (pH 9.6) was used as the coating dilution buffer. Streptavidin-HRP was used as the secondary antibody at a dilution of 1:2000. The antibody titer was determined by the highest dilution ratio when the detected value / blank control was ≥3. PBS was used as the blank control. The results are shown in Table 3 below.
[0045] Table 3: Biotinylated Antibody Titer Test
[0046] (2) Antibody pairing Antibody titers were detected using a sandwich ELISA method. Four mouse monoclonal antibodies were used as coating antibodies at a concentration of 2 μg / ml (100 μl / well). The coating dilution buffer was CBS buffer (pH=9.6). The sandwich antigens were PMMoV-CP 100 ng / ml and non-specific antigen 100 ng / ml. The detection antibody was Biotin-labeled mouse monoclonal antibodies at 100 ng / ml. The secondary antibody was Streptavidin-HRP, 1:2000. The combination of high target protein signal values and low non-specific signal values in the sandwich was selected. The detection results are shown in Table 4 below.
[0047] Table 4: Antibody Pairing Screening Results
[0048] In Table 4, Antibody 1 is monoclonal antibody 1-E4-D8; Antibody 3 is monoclonal antibody 3-F11-A8; Antibody 5 is monoclonal antibody 5-H1-C10; Antibody 26 is monoclonal antibody 26-E12-H10; Biotin-1 is monoclonal antibody 1-E4-D8; Biotin-3 is monoclonal antibody 3-F11-A8; Biotin-5 is monoclonal antibody 5-H1-C10; and Biotin-26 is monoclonal antibody 26-E12-H10. (5) Detection of protein working concentration range Antibody titers were detected using a sandwich ELISA method. Monoclonal antibody 3-F11-A8 was used as the coating antibody at a concentration of 2 μg / ml (100 μl / well). The coating dilution buffer was CBS buffer (pH=9.6). The sandwich antigen ToLCNDV-CP was used at concentrations of 100 ng / ml - 1.5625 ng / ml and 50 ng / ml - 0.78125 ng / ml. OD450 and OD620 values were measured separately. The results are shown in Table 5 below. Figure 3 As shown. Test results: The final paired antibody pair was coating antibody 3-F11-A8 and detection antibody 5-H1-C10. The working concentration range of recombinant protein was 0.78125-100 ng / ml, and the calibration curve showed good characteristics.
[0049] Table 5 Protein Working Concentration Range Test 2.2 Detection of 4 antibody pairs in pathogen samples
[0050] Antibody titers were detected using a sandwich ELISA method. Four of the aforementioned mouse monoclonal antibodies were used as coating antibodies at a concentration of 2 μg / ml (100 μl / well). The coating dilution buffer was CBS buffer (pH=9.6). For ToLCNDV-infected leaf lysate, 0.1 g of infected leaf was weighed and placed in a mortar, 1 ml of 0.15 mol / L PBST was added, and the mixture was thoroughly ground. The solution was then transferred to a centrifuge tube and centrifuged at 4℃, 6000 rpm, and 5 min. The supernatant was collected. Lysate from healthy leaves was treated similarly and used as the sandwich antigen. The detection antibody was 100 ng / ml of biotin-labeled mouse monoclonal antibodies from four strains. The secondary antibody was Streptavidin-HRP at a dilution of 1:2000. The wavelength was OD450. The final paired antibody pair was coating antibody 3-F11-A8 and detection antibody 5-H1-C10. This antibody pair could detect the target protein in the viral lysate. No target analyte was detected in the lysate from healthy leaves, indicating good specificity of the antibody pair.
[0051] Table 6. Antibody detection results for pathogen samples
[0052] In Table 6, Antibody 1 is monoclonal antibody 1-E4-D8; Antibody 3 is monoclonal antibody 3-F11-A8; Antibody 5 is monoclonal antibody 5-H1-C10; Antibody 26 is monoclonal antibody 26-E12-H10; Biotin-1 is monoclonal antibody 1-E4-D8; Biotin-3 is monoclonal antibody 3-F11-A8; Biotin-5 is monoclonal antibody 5-H1-C10; and Biotin-26 is monoclonal antibody 26-E12-H10. Finally, monoclonal antibodies 3-F11-A8 and 5-H1-C10 were screened and sequenced, and the relevant sequences are shown in Table 7. The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of antibody 3-F11-A8 are shown in SEQ ID NO. 1-3, the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO. 4 and SEQ ID NO. 5, and the amino acid sequence of the light chain variable region CDR2 is LAS.
[0053] The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of antibody 5-H1-C10 are shown in SEQ ID NO. 6-8; the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO. 9 and SEQ ID NO. 10, and the amino acid sequence of CDR2 is FAS.
[0054] Table 7: Antibody-related sequence information
[0055] Hybridoma cell lines secreting monoclonal antibody 3-F11-A8 and 5-H1-C10 were biopreserved. The biopreservation information is as follows: Preservation materials: ToLCNDV-Cell line-3-F11-A8 hybridoma cell line secreting monoclonal antibody against New Delhi leaf curl virus (NDV), deposited at China Center for Type Culture Collection (Wuhan University, Wuhan, China), deposited on November 11, 2025, accession number CCTCC NO:C2025277; and ToLCNDV-Cell line-5-H1-C10 hybridoma cell line secreting monoclonal antibody against New Delhi leaf curl virus (NDV), deposited at China Center for Type Culture Collection (Wuhan University, Wuhan, China), deposited on November 11, 2025, accession number CCTCC NO:C 2025278.
[0056] Example 3: Preparation and application of ToLCNDV colloidal gold immunochromatographic assay strips
[0057] 3.1 Preparation of ToLCNDV colloidal gold immunochromatographic assay strips: 3.1.1 Preparation of colloidal gold solution: Preparation: Wash the 500mL beaker, 20mL small beaker, rotor, brown bottle, glass rod, etc., and place them in an acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120g:200ml:1000ml) and soak for 24 hours. Remove and rinse 3-4 times with tap water, then rinse 3-4 times with ultrapure water, and dry in a 37℃ oven for later use. Preparation of colloidal gold: Prepare a 10% tetrachloroauric acid solution and measure a 1% trisodium citrate solution, equilibrate at room temperature for 15 minutes. Measure 1L of purified water into a clean glass container, place the container on a magnetic stirrer, set the speed to level 2, and the temperature to 350℃, and heat the solution to boiling. Quickly add 2mL of the 10% tetrachloroauric acid solution at once, stir for 1 minute to mix, and immediately add 2.4mL of the 1% trisodium citrate solution at once. Continue heating for 5 minutes, then stop heating and continue stirring for 10 minutes. Turn off the power and transfer the glass container to an unheated stirrer. Set the speed to 2-3 and continue stirring for 20 minutes. After stopping stirring, let it stand to room temperature. The prepared colloidal gold solution should be free of significant floating matter or precipitate, and the solution color should be purplish-red.
[0058] 3.1.2 Antibody labeling Take the prepared colloidal gold, adjust the pH to 8-8.5 with 0.1M K2CO3, add monoclonal antibody 3-F11-A8, and stir at room temperature to obtain a colloidal gold-antibody conjugate solution. Add 2 ml of 10% BSA to the prepared colloidal gold-antibody conjugate solution, stir at room temperature, centrifuge at 9000-11000 rpm for 40-60 min, discard the supernatant, and dissolve the precipitate in 2 ml of colloidal gold-antibody preservation solution (PBS buffer (pH 7.4, containing 3% BSA, 1% Tween 20), filter through a 0.45 μm filter membrane, and the resulting product is the colloidal gold-antibody conjugate stock solution. Reconstitute the precipitate with gold-labeled solution (PBS buffer (pH 7.4, containing 1% BSA, 1% Tween 20) to prepare an immunochromatographic colloidal gold solution (10 μg / mL) containing colloidal gold particles labeled with monoclonal antibody 3-F11-A, and store at 4℃.
[0059] 3.1.3 Gold spraying An immunogold solution with a final concentration of 10 μg / ml was sprayed onto a gold-labeled pad at a coating volume of 50 μL / cm² to prepare an immunogold pad. The pad was then dried for later use.
[0060] 3.1.4 Membrane Scraping Using a membrane scribing machine, 1.5 mg / ml of monoclonal antibody 5-H1-C10 was applied to the detection (T) line of a nitrocellulose membrane, and 1.5 mg / ml of goat anti-mouse IgG was applied to the control (C) line of the nitrocellulose membrane, with a coating volume of 1 μl / cm for both. The control line was drawn at a distance of 12.1 ± 0.1 cm from the top of the membrane, and the detection line was drawn at a distance of 12.5 cm ± 0.1 cm from the top of the membrane, with a distance of 5 mm ± 1 mm between the control line and the detection line.
[0061] 3.1.5 Assembly Cut the PVC base plate into strips 3mm wide and 60cm long; attach the antibody solid-phase NC membrane to the middle of the PVC base plate, 15cm away from the top; attach the glass fiber membrane probe strip to the bottom of the PVC base plate (i.e., the T-line end near the NC membrane), overlapping it with the solid-phase antibody NC membrane by 0.5cm; then attach a 14.5cm wide sample pad (glass fiber membrane) to the bottom of the PVC base plate, overlapping it with the probe strip by 0.5cm; attach a 17cm wide absorbent paper to the top of the PVC base plate (i.e., the C-line end near the NC membrane), overlapping it with the antibody solid-phase NC membrane by 0.5cm; cut into 3mm wide test strips. Figure 4 This is a schematic diagram of the assembled colloid test strip.
[0062] 3.2 Steps for detecting ToLCNDV using colloidal gold immunochromatographic assay strips 3.2.1. Sample preparation: Grind 0.1g of melon leaves and 1ml of 0.15 mol / L PBST buffer (pH 7.4) evenly in a grinding belt, and then aspirate the grinding liquid.
[0063] 3.2.2. Sample addition: Lay the ToLCNDV colloidal gold immunochromatographic test strip flat and use a pipette to draw 3 drops (about 60 μL) of the homogenate into the sample well.
[0064] 3.2.3. Visual interpretation of results: 5 minutes after sample addition... Figure 5 Interpret the results. Simultaneous color development of the C and T lines indicates a positive result; color development of the C line but no color development of the T line indicates a negative result; no color development of the C line but color development of the T line, or no color development of either the C or T lines, indicates the test strip is invalid.
[0065] The detection principle of the colloidal gold immunochromatographic test strip of the present invention is as follows: The test strip is designed based on the "antibody-antigen-antibody" sandwich immunochromatographic principle. After the sample is added, the sample solution migrates upward under capillary action. When it reaches the immunochromatographic colloidal gold pad, the gold-labeled monoclonal antibody 3-F11-A8 will be dissolved. When the sample contains ToLCNDV, ToLCNDV will bind to the gold-labeled monoclonal antibody 3-F11-A8 and migrate upward together. When it reaches the detection line where the monoclonal antibody 5-H1-C10 is fixed, a gold-labeled antibody 3-F11-A8-ToLCNDV-antibody 5-H1-C10 complex is formed, which eventually forms a red precipitate line. The unfixed gold-labeled antibody 3-F11-A8 continues to ascend to the goat anti-mouse IgG antibody, forming a gold-labeled antibody 3-F11-A8-IgG complex, which forms a red precipitate line, i.e., the control line. When the sample does not contain ToLCNDV, the gold-labeled antibody 3-F11-A8 dissolves in the sample and migrates upward together. When it reaches the detection line where the monoclonal antibody 5-H1-C10 is immobilized, it cannot be immobilized, and no red band appears at the detection line. The unimmobilized gold-labeled antibody continues to ascend to the goat anti-mouse IgG antibody, forming the gold-labeled antibody 3-F11-A8-IgG complex, which forms the red precipitate line, i.e., the control line.
[0066] Example 4: Specificity analysis of ToLCNDV colloidal gold immunochromatographic assay strip Take 60 μL of healthy melon leaf samples and the homogenized samples infected with ToLCNDV, TYLCV, SLCCV, and RaMoV, and add them to the sample wells of colloidal gold immunochromatographic assay strips. After 5 minutes, visually interpret the results. (See also...) Figure 6 The test results showed that 5 minutes after sample addition, the sample infected with ToLCNDV was positive, while the other samples were negative, indicating that the test strip has high specificity.
[0067] Example 5: Sensitivity Analysis of ToLCNDV Colloidal Gold Immunoassay Strips 5.1 Detection of antigen ToLCNDV-CP First, using the antigen standard ToLCNDV-CP protein (1 mg / ml), prepare 1000-fold dilution (1 ug / ml) and 10000-fold dilution (0.1 ug / ml) of antigen, and use PBS as a blank control. Add 60 μL of each (containing approximately 60 ng of ToLCNDV-CP protein) to the sample wells. Visually interpret the results within 15 minutes of sample addition. (See attached image.) Figure 7 The test results showed that 15 minutes after sample addition, a clear positive band was visible in the 1000-fold dilution of the antigen, and a positive band was still visible in the 10000-fold dilution of the antigen, indicating that the test strip has high sensitivity.
[0068] 5.2 Detection of samples infected with ToLCNDV Crude extracts of ToLCNDV-infected plants were prepared in the field and diluted to concentrations of 1:10, 1:50, 1:100, 1:150, and 1:200, with the extracts serving as controls. Three drops (approximately 60 μL) of each solution were added to the sample wells, and the results were visually interpreted 5 minutes after addition. Figure 4 See also Figure 8 The test results showed that 5 minutes after adding the sample, the sample diluted 100 times was positive for ToLCNDV infection, indicating that the test strip has high sensitivity.
[0069] In summary, the ToLCNDV colloidal gold immunoassay strip of this invention can rapidly, specifically, and sensitively detect ToLCNDV in melon samples. The results can be directly interpreted visually within 5 minutes, making it suitable for field testing. It does not react with healthy samples or samples infected with TYLCV, SLCCV, or RaMoV. It can detect 1000-fold dilution of the antigen (containing approximately 60 ng of ToLCNDV-CP protein) and a crude field extract; it can detect 100-fold dilution of ToLCNDV-infected melon leaf samples (containing approximately 60 μg of plant leaves).
[0070] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.
Claims
1. Two hybridoma cell lines, characterized in that, The hybridoma cell lines are (1) ToLCNDV-Cell line-3-F11-A8, which secretes the tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8, and was deposited on November 11, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO:C2025277; and (2) ToLCNDV-Cell line-5-H1-C10, which secretes the tomato New Delhi leaf curl virus monoclonal antibody 5-H1-C10, and was deposited on November 11, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO:C2025278.
2. Two monoclonal antibodies, characterized in that, The monoclonal antibodies are tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8 and tomato New Delhi leaf curl virus monoclonal antibody 5-H1-C10, respectively; wherein, monoclonal antibody 3-F11-A8 is secreted by hybridoma cell line with accession number CCTCC NO:C 2025277; and monoclonal antibody 5-H1-C10 is secreted by hybridoma cell line with accession number CCTCC NO:C 2025278.
3. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the tomato New Delhi leaf curl virus monoclonal antibody 3-F11-A8 are shown in SEQ ID NO.1-3, the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequence of the light chain variable region CDR2 is LAS.
4. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the tomato New Delhi leaf curl virus monoclonal antibody 5-H1-C10 are shown in SEQ ID NO.6-8; the amino acid sequences of the light chain variable regions CDR1 and CDR3 are shown in SEQ ID NO.9 and SEQ ID NO.10, and the amino acid sequence of CDR2 is FAS.
5. The use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to claim 2 in the detection of New Delhi leaf curl virus in tomatoes.
6. A colloidal gold immunoassay strip for detecting New Delhi leaf curl virus in tomatoes, characterized in that, The test strip contains the monoclonal antibody as described in claim 2.
7. The colloidal gold immunochromatographic strip for detecting New Delhi leaf curl virus in tomatoes according to claim 6, characterized in that, The colloidal gold pad on the test strip is a gold-labeled pad with colloidal gold particles-labeled monoclonal antibody 3-F11-A8; the test line is coated with monoclonal antibody 5-H1-C10, and the control line is coated with secondary antibody.
8. The colloidal gold immunochromatographic strip for detecting New Delhi leaf curl virus in tomatoes according to claim 7, characterized in that, The colloidal gold particles have a particle size of 40 nm.
9. The colloidal gold immunochromatographic strip for detecting New Delhi leaf curl virus in tomatoes according to claim 7, characterized in that, The coating amount of the immunogold colloidal solution on the gold-labeled pad was 50 μl / cm².
10. The use of the colloidal gold immunoassay strip according to any one of claims 6-9 in the detection of New Delhi leaf curl virus in tomatoes.