Hybridoma cell strain secreting monoclonal antibody PVY-5, monoclonal antibody and application thereof

Abstract

The application discloses a hybridoma cell strain secreting monoclonal antibody PVY-5, the monoclonal antibody and application, the hybridoma cell strain 4B4D8C3 can secrete anti-potato Y virus monoclonal antibody, the preservation number of the hybridoma cell strain is CCTCC NO: C2022282, the monoclonal antibody PVY-5 has specific immunoreaction with the coat protein of potato Y virus, the heavy chain variable region amino acid sequence of the monoclonal antibody PVY-5 is as shown in SEQ ID NO.6, the light chain variable region amino acid sequence is as shown in SEQ ID NO.7, the sensitivity reaches 1:5120 times dilution by using direct ELISA method detection to the leaf of potato Y virus disease infection, the monoclonal antibody PVY-5 can also be used to prepare ELISA kit, or colloidal gold kit, or nano analog enzyme kit, and the potato Y virus disease can be detected quickly, sensitively, economically and accurately.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen detection technology, and in particular to a hybridoma cell line that secretes monoclonal antibody PVY-5, its monoclonal antibody, and its applications. Background Technology

[0002] my country is the world's largest potato producer, ranking first in both planting area and total output. However, its yield per unit area lags behind, far below that of developed countries in Europe and America. Seed tuber degeneration caused by potato viral diseases (PVX, PVY, PVS, PLRV, etc.) is one of the main reasons for low potato yields. Developed countries in Europe and America, such as the Netherlands, the UK, and the US, began potato seed tuber quality testing more than 70 years ago. Implementing strict seed tuber quality testing and certification and market access systems is a key factor in ensuring seed tuber quality and yield. Currently, the adoption rate of virus-free seed tubers in developed countries in Europe and America exceeds 90%, while in my country it is only 30-50%. With the implementation of the national potato staple food strategy, the strategic importance of potatoes has been further enhanced, and improving the quality and adoption rate of virus-free seed tubers has become an important guarantee for the healthy development of the potato industry. Viruses, as the main harmful organisms affecting potato seed tuber quality, are a key indicator for determining whether seed tuber quality is up to standard. Therefore, there is an urgent need to establish a potato seed tuber quality testing and certification technology system suitable for my country.

[0003] According to current national standards, potato seed potato propagation takes approximately four years from the original seed stock to production seed, during which time it undergoes rigorous field testing, warehouse testing, and laboratory testing to ultimately determine the quality of the seed potatoes. Therefore, unlike the quality testing of other field crop seeds, potato seed potato quality testing personnel need more specialized skills and extensive experience. Consequently, it is necessary to establish and develop rapid, accurate, and sensitive testing technologies and products to assist testing personnel in making scientific, accurate, and timely assessments of seed potato quality.

[0004] PVY is one of the most common and damaging potato viruses, and the second most important disease affecting potatoes, causing severe economic losses. It is a representative member of the Pottyvirus genus, with a wide host range, infecting various plants in the Solanaceae, Leguminosae, and Cucurbitaceae families, and causing significant economic damage. As one of the major pests affecting the quality of potato seed tubers, PVY is a key indicator for determining whether potato seed tubers meet quality standards.

[0005] Therefore, there is an urgent need to develop a simple, rapid, sensitive, economical, and accurate antibody for detecting potato virus disease (PVY) to meet the needs of field quality testing services for potato seed tubers and provide technical support for the comprehensive advancement of potato seed tuber quality testing and certification. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a hybridoma cell line 4B4D8C3 that secretes monoclonal antibody PVY-5; a second objective of the present invention is to provide a monoclonal antibody PVY-5 secreted by the hybridoma cell line 4B4D8C3; a third objective of the present invention is to provide the application of the monoclonal antibody PVY-5 in the detection of potato PVY virus; and a fourth objective of the present invention is to provide a kit containing the monoclonal antibody PVY-5.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A hybridoma cell line 4B4D8C3 that secretes monoclonal antibody PVY-5, wherein the hybridoma cell line can secrete monoclonal antibody against potato virus Y, and the hybridoma cell line is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: C2022282.

[0009] 2. A monoclonal antibody PVY-5 secreted by the hybridoma cell line 4B4D8C3.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody PVY-5 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0011] Preferably, the monoclonal antibody PVY-5 exhibits a specific immune response to the capsid protein of potato virus Y, and the nucleotide sequence of the capsid protein gene of potato virus Y is shown in SEQ ID NO.3; the antibody type and subclass of the monoclonal antibody PVY-5 is IgG1, the chain type is Kappa light chain, and the sensitivity of the direct ELISA method for detecting potato virus Y-infected leaves reaches a 1:5120 dilution.

[0012] 3. Application of the monoclonal antibody PVY-5 in the detection of potato PVY virus.

[0013] 4. A kit containing the monoclonal antibody PVY-5.

[0014] Preferably, the kit is an ELISA kit, a colloidal gold kit, or a nano-enzyme kit.

[0015] Preferably, the nano-mimetic enzyme in the nano-mimetic enzyme kit of the present invention is Fe3O4 magnetic nanoparticles.

[0016] The beneficial effects of this invention are as follows: This invention discloses a hybridoma cell line that secretes monoclonal antibody PVY-5, its monoclonal antibody, and its applications. The hybridoma cell line 4B4D8C3 can secrete monoclonal antibody against potato virus Y. The accession number of the hybridoma cell line is C2022282. The monoclonal antibody PVY-5 has a specific immune response to the 30 kDa coat protein of potato virus Y. The antibody type and subclass is IgG1, and the chain type is Kappa light chain. The sensitivity of detection against potato virus Y-infected leaves using the direct ELISA method reaches 1:5120 dilution. The monoclonal antibody PVY-5 can also be used to prepare ELISA kits, colloidal gold kits, or nano-enzyme kits, which can rapidly, sensitively, economically, and accurately detect potato virus Y disease. Attached Figure Description

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0018] Figure 1 This is the technical approach of the present invention;

[0019] Figure 2 Amplification of the PVY-CP gene fragment;

[0020] Among them, M: 2K plusII marker, 1, 2: PVY-CP gene fragments (807bp);

[0021] Figure 3 Results of PVY-CP prokaryotic expression;

[0022] Where M: Marker, 1: Bacterial precipitate, 2: Bacterial supernatant, 3: Total bacterial protein;

[0023] Figure 4 The results of PVY-CP purification;

[0024] Wherein, A: PVY-CP eluted with different concentrations of imidazole, M: marker, 1: 50mM imidazole elution buffer, 2: 100mM imidazole elution buffer, 3: 300mM imidazole elution buffer; B: PVY-CP purification results, M: marker, 4: concentrated protein);

[0025] Figure 5 For Western blot analysis of PVY monoclonal antibody specificity;

[0026] Wherein, M: protein marker, 1: healthy potato tissue culture seedlings, 2: potato tissue culture seedlings infected with PVY, and 3: potato tissue culture seedlings infected with PVX.

[0027] Figure 6 Sensitivity analysis of PVY monoclonal antibodies;

[0028] Figure 7 For specific detection using PVY nano-mimetic enzyme test strips;

[0029] Wherein, 1: PVA, 2: PVM, 3: PVS, 4: PVX, 5: PLRV, 6: PVY, 7: healthy plant;

[0030] Figure 8 For sensitivity testing of PVY nano-mimetic enzyme test strips;

[0031] Among them, 1:1:10 dilution, 2:1:10 2 Diluted 3 times, 1:10 3 Diluted 4 times, 1:10 4 Diluted 5 times, 1:10 5 6-fold dilution, negative control.

[0032] Biological Preservation:

[0033] Two hybridoma cell lines secreting monoclonal antibodies were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and named 4F1B2G11 and 4B4D8C3, respectively. The deposit date of 4F1B2G11 was September 1, 2022, with accession number CCTCC NO: C2022281, and it was classified as hybridoma cell line 4F1B2G11. The deposit date of 4B4D8C3 was September 1, 2022, with accession number CCTCC NO: C2022282, and it was classified as hybridoma cell line 4B4D8C3. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] Biological materials and reagents required for the examples: Healthy potato seedlings infected with PVX, PVY, PVA, PVS, PVM, and PLRV were all retained in our laboratory; Nanozyme (Fe3O4, 10 mg / mL) was provided by Researcher Duan Demin of the Institute of Biophysics, Chinese Academy of Sciences (preparation method can be found in the literature Duan D, Fan K, Zhang D, et al. Nanozyme-strip for rapid local diagnosis of Ebola); Balb / c mice were purchased from Jiangnan Experimental Animal Base; Tizol, urea, imidazole, penicillin, and streptomycin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Reverse transcription kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; Gel extraction kit was purchased from Beijing TransGen Biotech Co., Ltd.; E. coli BL21 competent cells, recombinant plasmid expressing protein (pET28a), LB medium, Tris buffer, IgG-HRP and SDS-PAGE protein gel were purchased from Aibosen Biotechnology Co., Ltd.; Freund's complete adjuvant, Freund's incomplete adjuvant, PEG1450, HT and HAT were purchased from Sigma-Aldrich; newborn calf serum was purchased from Prairie Green Field Co., Ltd.; DMEM (Glu 4.5 g / L) was purchased from Dalian Meilun Biotechnology Co., Ltd.; dialysis bags, ELISA coating solution, ELISA stop solution, TMB chromogenic solution and DAB chromogenic solution were purchased from Beijing Solarbio Science & Technology Co., Ltd.; antibody subtype detection kit was purchased from Sino Biological; ultrafiltration concentration tubes were purchased from Millipore; affinity chromatography column and Protein A purification column were purchased from Wuhan Huiyan Biotechnology Co., Ltd.; ELISA plates were purchased from Costar; nitrocellulose membrane, absorbent paper, glass cellulose membrane and PVC base plate were purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0036] The technical route of this invention is as follows Figure 1 As shown.

[0037] Example 1: Construction of a prokaryotic expression vector for potato virus Y CP protein

[0038] I. Primer Design and Synthesis

[0039] The PVY gene sequence was retrieved from the National Center for Biotechnology Information (NCBI). Primers for amplifying the full-length CP protein gene were designed based on the full-length sequence of the PVY coat protein (CP) gene (Table 1). The primers were aligned on NCBI and then synthesized by Shanghai Sangon Biotech Co., Ltd. The PVY-CP gene was amplified using primers PVY-CP-F and PVY-CP-R. The amplified product was 807 bp in length, and the nucleotide sequence encoding the PVY-CP gene is shown in SEQ ID NO. 3.

[0040] Table 1. Primer sequences of PVY-CP-F and PVY-CP-R

[0041]

[0042] II. Extraction of viral genome

[0043] (1) Place the sample in a mortar, add liquid nitrogen and grind into powder, transfer to a 1.5 mL centrifuge tube, add 1 mL TRIzol and mix well;

[0044] (2) Centrifuge at 14000g for 5 minutes at 4℃;

[0045] (3) Take the supernatant, add 200 μL of chloroform, shake to mix, and let stand at room temperature for 15 min;

[0046] (4) Centrifuge at 12000g for 15 min at 4℃; transfer the upper aqueous phase to a new 1.5mL centrifuge tube, add 0.5mL isopropanol, mix well, and let stand at room temperature for 10 min.

[0047] (5) Centrifuge at 12000g for 10 min at 4℃;

[0048] (6) Discard the supernatant, keep the precipitate, add 1 mL of 75% ethanol, gently shake the centrifuge tube to suspend the precipitate;

[0049] (7) Centrifuge at 7500g for 5 minutes at 4℃, discard the supernatant, invert the centrifuge tube onto filter paper, and let it air dry.

[0050] (8) Add 25-100 μL of DEPC water to dissolve the precipitate to obtain RNA.

[0051] III. Amplification of the viral CP gene

[0052] (1) cDNA synthesis

[0053] Using total RNA extracted from plant viruses as a template, the procedure was performed according to the kit instructions.

[0054] (2) PCR amplification

[0055] Using the synthesized cDNA as a template, amplification was performed using the primers listed in Table 1. The reaction system consisted of 50 μL of cDNA template, 2 μL each of upstream and downstream viral primers (0.1 μmol·L⁻¹), 25 μL of 2×Taq enzyme, and ddH₂O to a final volume of 50 μL. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, repeated 35 times; and 72℃ extension for 10 min.

[0056] (3) Agarose gel electrophoresis detection:

[0057] Prepare a 1% agarose gel and add GoldView according to the specified ratio. TM Nucleic acid chromogenic dye (add 1 μL of GoldView per 20 ml TAE solution) TM Shake well. Slowly pour into the mold and let it cool until it forms a gel block. Select a suitable location to spot the DNA Maker and PCR product for electrophoresis (140V, 20 minutes). After electrophoresis, observe the target band using a gel imaging system, cut off the target band, and put it into a centrifuge tube for recovery.

[0058] (4) Recovery and purification of PCR products:

[0059] The PCR products were recovered and purified according to the instructions in the full-gold DNA gel recovery kit.

[0060] IV. Ligation of viral CP protein gene with pET28a vector

[0061] The CP protein gene fragment of PVY purified by gel recovery was ligated into the pET28a vector. The ligation system is shown in Table 2. The ligation was carried out at 37℃ for 30 min.

[0062] Table 2. pET28a carrier linkage system

[0063]

[0064] V. Escherichia coli transformation

[0065] Remove BL21 competent cells from the cryogenic freezer and place them on an ice box. After thawing, add 10 μL of the ligation product (PET28a-PVY-CP), mix thoroughly by pipetting, and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then incubate on ice for 2 min. Add 500 μL of antibiotic-free LB liquid medium and incubate at 37℃ with a shaker at 180 rpm for 1.5 h. Centrifuge at 4000g for 5 min at room temperature, discard excess supernatant, mix the cells by pipetting, and evenly spread the concentrated bacterial solution onto LB / Kan... + On solid culture medium, after the bacterial culture has been completely absorbed by the solid medium, seal the plate and invert it in a 37°C incubator overnight. The next day, pick single colony plaques and place them in LB / Kan. + After being shaken, the liquid culture medium was sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0066] VI. Expression of viral CP protein gene in Escherichia coli

[0067] (1) Add the correctly sequenced Escherichia coli culture to 5 mL of LB liquid medium, place it in a 37℃ constant temperature shaking incubator, and incubate overnight at 250 rpm / min for 16-18 h to obtain the seed culture;

[0068] (2) Transfer the culture medium at a ratio of 1:100 to 200 mL of fresh LB medium and incubate at 37°C and 250 rpm / min. When the bacterial culture OD... 600 When the concentration of α=0.6, IPTG inducer was added, and induction culture was continued at 18℃.

[0069] (3) Collect bacterial cells at 4℃, 5000 rpm / min, for 15 min;

[0070] (4) The bacterial cells were resuspended in a lysis buffer (50 mm Tris, 0.5 M NaCl, pH 8.0) and then sonicated. The sonication conditions were: work 3 s, off 2 s, time 15 min, repeated once;

[0071] (5) After sonication, the supernatant and precipitate were collected at 4℃, 5000 rpm / min for 15 min and analyzed for protein by SDS-PAGE gel.

[0072] VII. Purification of viral CP protein

[0073] (1) Take the cultured bacterial cells, add lysis buffer (50mM Tris, 0.5M NaCl, pH 8.0), resuspend, and then sonicate to disrupt the cells. The sonication conditions are: work 3s, off 2s, time 15min, repeat once;

[0074] (2) Centrifuge the ultrasonically disrupted bacterial solution in a low-temperature centrifuge at 4°C, 5000 rpm / min for 15 min, collect the supernatant, add denaturing agent urea to the supernatant to a final concentration of 8M, dissolve, let stand at 4°C for 1 h, centrifuge and collect the supernatant.

[0075] (3) The supernatant obtained above was filtered through a 0.45 μm filter membrane and purified by Ni affinity chromatography column.

[0076] The steps are as follows:

[0077] a) Wash with 5 column volumes of deionized water to remove air and 20% ethanol;

[0078] b) Equilibrate the column with 5 to 10 column volumes of Buffer A (Buffer A: 50 mM Tris, 0.15 M NaCl, 8 M urea, pH 8.0);

[0079] c) Flow the sample through the Ni column at a rate of 0.5 mL / min;

[0080] d) Balance the column with Buffer A;

[0081] e) Elute with 50 mM imidazole, 100 mM, and 300 mM imidazole respectively.

[0082] f) Perform SDS-PAGE gel analysis on the eluted samples to determine if the target protein is present.

[0083] VIII. Conclusion

[0084] (1) Result of PVY-CP gene prokaryotic expression vector construction

[0085] After PCR amplification, the PVY-CP gene was analyzed by agarose gel electrophoresis, and a DNA fragment of 807 bp in length was detected. Figure 2 The PVY-CP gene fragment was obtained by gel extraction, ligated into the pET28a vector, and transformed into BL21 competent cells. After overnight culture at 37°C on inverted plates, single-clone plaques were selected and sent to Sangon Biotech for sequencing. After sequence alignment, bacterial cultures with correct sequencing results were selected for protein expression.

[0086] (2) PVY-CP expression results

[0087] The correctly sequenced bacterial culture was treated with IPTG inducer as recommended, and protein analysis was performed using an SDS-PAGE gel. The recombinant protein PVY-CP was expressed. Figure 3As shown, lane 1 is the bacterial cell precipitate, lane 2 is the bacterial cell supernatant, and lane 3 is the total bacterial protein. This indicates that the recombinant protein PVY-CP exists mainly in the bacterial cell supernatant in a soluble form, and partly in the bacterial cell precipitate in the form of inclusion bodies.

[0088] (3) PVY-CP purification results

[0089] After lysing the cultured bacteria, the mixture was ultrasonically disrupted, centrifuged at low temperature, and the supernatant was collected. Urea was added to denature the supernatant, and the mixture was filtered through a 0.45 μm filter membrane. Elution was performed with 50 mM imidazole, 100 mM, and 300 mM imidazole, respectively. SDS-PAGE analysis was then performed to determine the presence of the target protein. The results are as follows: Figure 4 As shown in Figure A, lane 1 was eluted with 50 mM imidazole, lane 2 with 100 mM imidazole, and lane 3 with 300 mM imidazole. The proteins eluted with 100 mM and 300 mM imidazole were diluted, dialyzed, and concentrated. The purity of the target protein was then determined using an SDS-PAGE gel, and the results are shown below. Figure 4 As shown in Figure B, lane 4 contains the concentrated target protein, indicating high purity.

[0090] Example 2: Preparation of Potato Virus Y Monoclonal Antibody Serum

[0091] I. Immunizing mice

[0092] (1) The recombinant protein PVY-CP prepared in Example 1 after prokaryotic expression and purification was used as an immunogen. Three healthy Balb / c mice were selected. For the first immunization, 50 μg of PVY antigen was used. Each mouse was emulsified with an equal volume of Freund's complete adjuvant and then subcutaneously injected into the abdomen at multiple points.

[0093] (2) After the first immunization, every 14 days, 50 μg of PVY antigen was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously into the abdomen of the mice. The mice were immunized 3 times.

[0094] (3) Starting from the third immunization, blood was collected from the orbital venous plexus (or tail vein) of mice 7 days after each immunization, and the antibody titer in the mouse blood was determined by indirect ELISA.

[0095] (4) After the mice are immunized to the point that the serum titer is qualified, select mice with higher titers and inject 50 μg of PVY antigen into the peritoneum to enhance immunization.

[0096] II. Serum titer detection in immunized mice

[0097] (1) Protein coating: The experimental group was diluted with ELISA coating buffer to 5 μg / mL, while the control group was coated with ELISA coating buffer, 100 μL / well, and coated overnight at 4℃. The protein was washed twice with PBST.

[0098] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0099] (3) Sample addition: Dilute serum to the specified concentration, 100 μL / well, incubate at room temperature for 1 h, and wash twice with PBST;

[0100] (4) Secondary antibody: Rabbit anti-mouse IgG-HRP 1:1000, 100μL / well, incubated at room temperature for 1h, washed 3 times with PBST;

[0101] (5) Color development: TMB color development solution, solution A: solution B = 1:1, 100 μL / well, react at room temperature for 20 min;

[0102] (6) Termination: ELISA stop solution, 50 μL / well;

[0103] (7) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm.

[0104] Mice were immunized with PVY-CP as the immunogen. After the first immunization, booster immunizations were performed every 14 days for a total of 3 immunizations. The serum titer of the mice was measured by indirect ELISA after the fourth immunization (Table 3). Mice No. 3 had the highest immunogenicity. Cell fusion experiments were performed 3–7 days after the booster immunization.

[0105] Table 3. Mouse serum titers

[0106]

[0107] Example 3: Preparation of Viral Monoclonal Hybridoma Cells

[0108] I. Cell Fusion

[0109] (1) In a biosafety cabinet, collect approximately 10 well-grown, morphologically sound myeloma cells (Sp2 / 0). 7 Each sample was resuspended in 50 mL centrifuge tubes in DMEM (Glu 4.5 g / L) medium without serum and preheated at 37 °C.

[0110] (2) Immunized mice 3 to 7 days after booster immunization, under sterile conditions, spleens were taken, ground, sieved and centrifuged to collect spleen cells.

[0111] (3) After centrifuging the spleen cells with Sp2 / 0, chemical fusion was performed using the fusion agent PEG1450, and the reaction was terminated by adding DMEM.

[0112] (4) Collect fused cells by centrifugation, culture and screen them in high-glucose DMEM supplemented with NBS (newborn calf serum) and HAT. After about 8 days, perform initial fusion screening by indirect ELISA, and perform secondary fusion screening in positive cell wells.

[0113] (5) Select monoclonal hybridoma cells that stably express antibodies, expand cell culture, take cells for ascites production, and freeze the cells.

[0114] II. Screening of Fusion Cells

[0115] The BSA competitive ELISA method is used for detection, and the steps are as follows:

[0116] (1) Protein coating: Dilute PVY antigen or Y virus homogenate to the specified concentration with ELISA coating buffer, 100 μL / well, coat overnight at 4℃, and wash twice with PBST.

[0117] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0118] (3) Sample addition: Add the original volume of cell supernatant, 80 μL, incubate at room temperature for 1 h, and wash twice with PBST;

[0119] (4) Secondary antibody: Rabbit anti-mouse IgG-HRP 1:1000, 100μL / well, incubated at room temperature for 1h, washed 3 times with PBST;

[0120] (5) Color development: TMB color development solution A: B = 1:1, 100 μL / well, react at room temperature for 20 min;

[0121] (6) Termination: ELISA stop solution, 50 μL / well;

[0122] (7) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm;

[0123] (8) Select those with good viability and perform the first subcloning.

[0124] Sixteen positive cell lines were obtained from this cell fusion (Table 4), and cell subcloning experiments were carried out.

[0125] Table 4.3 Fusion Screening of Mice

[0126]

[0127] III. Cell Subcloning Screening

[0128] Cell cloning was performed using the limiting dilution method. Positive cells were resuspended and counted, with a target of one cell per 200 μL of culture medium. The positive cells were then diluted according to the count, with 200 μL added to each well of a 96-well plate. Microscopic examination was performed after 7 to 9 days; wells showing single-cell clusters were identified. Positive cells were then detected using an indirect ELISA method, as follows:

[0129] (1) Protein coating: The experimental group was diluted with ELISA coating buffer to 1 μg / mL, while the control group was coated with ELISA coating buffer, 100 μL / well, and coated overnight at 4℃. The protein was washed twice with PBST.

[0130] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0131] (3) Sample addition: Take 80 μL of the original cell supernatant per well, incubate at room temperature for 1 h, and wash twice with PBST;

[0132] (4) Secondary antibody: Rabbit anti-mouse IgG-HRP 1:1000, 100μL / well, incubated at room temperature for 1h, washed 3 times with PBST;

[0133] (5) Color development: TMB color development solution A:B = 1:1, 100μL / well, react at room temperature for 20min;

[0134] (6) Termination: ELISA stop solution, 50 μL / well;

[0135] (7) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm;

[0136] (8) Select monoclonal hybridoma cells that stably express antibodies, expand cell culture, take cells for ascites production, and freeze the cells.

[0137] After two rounds of cell subcloning, only single and binary clones were selected for microscopic examination and tested, resulting in four positive single-clonal hybridoma cell lines (Table 5). These cells were then cultured to expand their cell count, used to produce ascites fluid, and cryopreserved.

[0138] Table 5. Subcloning Results

[0139]

[0140] Example 4: Preparation of potato virus monoclonal antibody

[0141] I. Preparation of Ascites

[0142] Each mouse was injected intraperitoneally with 0.5 mL of liquid paraffin as a sensitizing agent. Seven days later, positive hybridoma cells were injected intraperitoneally, with one cell line injected into each mouse. Each mouse was injected with 10... 5 ~10 6 Cells were collected by centrifugation, resuspended in 1×PBS buffer, and injected. Slight abdominal distension was observed in mice from day 8 onwards. The mice were continued to be fed until the abdomen became rounded and immobile. Ascites fluid was collected multiple times using drainage, centrifuged, and then frozen at -80°C.

[0143] II. Ascites purification

[0144] (1) Collect ascites fluid, dilute with PBS and filter (0.22 μm).

[0145] (2) Take the filtered sample and purify the protein using a Protein G column. The steps are as follows:

[0146] a) Wash with 5 column volumes of deionized water to remove air and 20% ethanol;

[0147] b) Equilibrate the column with 5-10 column volumes of buffer; buffer: PB buffer.

[0148] c) Flow the sample through the Protein G column at a rate of 0.5 mL / min;

[0149] d) Balance the columns using the buffer described above;

[0150] e) Elute with glycine and neutralize with Tris.

[0151] (3) Collect the glycine-eluted sample and dialyze it overnight at 4°C (dialysis buffer: PBS).

[0152] (4) Take the dialysis sample, concentrate it by ultrafiltration (ultrafiltration tube), and detect the purity of the target protein by SDS-PAGE gel.

[0153] (5) Perform performance testing on antibodies that meet the purity requirements.

[0154] III. Monoclonal Antibody Detection

[0155] A. Monoclonal antibody performance testing

[0156] (1) Protein coating: Dilute PVY-CP, Y virus homogenate, X virus homogenate and healthy tissue homogenate with ELISA coating buffer, 100 μL / well, coat overnight at 4℃, and wash twice with PBST;

[0157] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0158] (3) Sample loading: Dilute PVY monoclonal antibody to the specified concentration, 100 μL / well, incubate at room temperature for 1 h, and wash twice with PBST;

[0159] (4) Primary antibody: Rabbit anti-mouse IgG-HRP 1:1000, 100μL / well, incubated at room temperature for 1h, washed 3 times with PBST;

[0160] (5) Color development: TMB color development solution A: B = 1:1, 100 μL / well, react at room temperature for 20 min;

[0161] (6) Termination: ELISA stop solution, 50 μL / well;

[0162] (7) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm.

[0163] B. Monoclonal antibody specificity detection

[0164] (1) Total protein extraction: Take 0.1g of plant tissue, grind it with liquid nitrogen, and add 250μL of total protein extract and 5μL of 50× protease inhibitor;

[0165] (2) Centrifuge at 13000 rpm and 4℃ for 15 min, and collect the supernatant;

[0166] (3) After adding loading buffer to the supernatant, mix well, boil in boiling water for 10 min, cool for 5 min, and centrifuge at 13000 rpm for 10 min at 4℃.

[0167] (4) Prepare a 10% SDS-PAGE separating gel and a 5% stacking gel, add the samples and electrophore at 180V until the loading buffer is reached;

[0168] (5) Before the end of electrophoresis, immerse the PVDF membrane in methanol for 15 seconds;

[0169] (6) After electrophoresis, immerse the gel in transfer buffer for 15 minutes and then transfer the membrane. Transfer at 100V for 1–1.5 hours.

[0170] (7) After the membrane is transferred, wash it once with TBST, stain it with Ponceau S and take pictures. Then wash it several times with TBST and seal it with 5% skim milk powder prepared with TBST at room temperature for 1 hour.

[0171] (8) After blocking, add the primary antibody diluted 1:5000 and incubate overnight at 4°C;

[0172] (9) After the primary antibody reaction is complete, wash the membrane with TBST 4 times, 15 min each time;

[0173] (10) Add the secondary antibody diluted 1:5000 and incubate at room temperature for 1 hour;

[0174] (11) After the secondary antibody reaction is complete, wash the membrane with TBST 4 times, 10 min each time;

[0175] (12) Add ECL colorimetric solution and take a picture.

[0176] Western blotting was used to specifically analyze the PVY-2, PVY-3, PVY-4, and PVY-5 monoclonal antibodies. All antibodies showed specific immune responses to protein extracts from PVY-infected potato tissue culture seedlings, but not to protein extracts from PVX-infected potato tissue culture seedlings or from healthy potato tissue culture seedlings. Figure 5 This indicates that the four PVY monoclonal antibodies have good specificity.

[0177] C. Monoclonal antibody paired detection

[0178] (1) Protein coating: Dilute PVY monoclonal antibody to 1 μg / mL with ELISA coating buffer, 100 μL / well, coat overnight at 4℃, and wash twice with PBST.

[0179] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0180] (3) Sample loading: PVY tissue was diluted 500 times and negative tissue was diluted 500 times. 100 μL / well, incubated at room temperature for 1 h, and washed twice with PBST;

[0181] (4) Primary antibody: Dilute the biotin-labeled PVY antibody to 1 μg / mL, 100 μL / well, incubate at room temperature for 1 h, and wash twice with PBST;

[0182] (5) Secondary antibody: Avidin-HRP 1:10000, 100μL / well, incubate at room temperature for 1h, wash 3 times with PBST;

[0183] (6) Color development: TMB color development solution A: B = 1:1, 100 μL / well, react at room temperature for 20 min;

[0184] (7) Termination: ELISA stop solution, 50 μL / well;

[0185] (8) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm.

[0186] A successful pairing is considered to have an absorbance value that is twice or more greater than the negative control. According to the DAS-ELISA results (Table 6), only one pair of PVY monoclonal antibodies successfully paired; when the coating antibody was PVY-5, the detection antibody was PVY-2, resulting in a successful pairing.

[0187] Table 6. Pairing Results of PVY Monoclonal Antibodies

[0188]

[0189] Note: * indicates successfully paired antibodies.

[0190] D. Monoclonal antibody sensitivity detection

[0191] (1) Protein coating: Dilute potato Y virus tissue culture seedling homogenate (1g / mL) with ELISA coating buffer, and perform serial dilutions of 1:10 to 1:163840. For the negative control group, add ELISA coating buffer to dilute healthy potato tissue extract, 100μL / well, coat overnight at 4℃, and wash twice with PBST.

[0192] (2) Blocking: Prepare 3% skim milk powder, 380μL / well, incubate at room temperature for 1h, and wash twice with PBST;

[0193] (3) Sample loading: Dilute PVY monoclonal antibody to the specified concentration, 100 μL / well, incubate at room temperature for 1 h, and wash twice with PBST;

[0194] (4) Secondary antibody: Rabbit anti-mouse IgG-HRP 1:1000, 100μL / well, incubated at room temperature for 1h, washed 3 times with PBST;

[0195] (5) Color development: TMB color development solution A:B = 1:1, 100μL / well, react at room temperature for 20min;

[0196] (6) Termination: ELISA stop solution, 50 μL / well;

[0197] (7) Reading: Results of the microplate reader at a main wavelength of 450nm and a secondary wavelength of 630nm.

[0198] The results are as follows Figure 6 As shown, when coated with PVY and detected by direct ELISA, the sensitivity of PVY monoclonal antibody can reach 1:5120 dilution.

[0199] Monoclonal antibodies PVY-2 and PVY-5 were sent to Apexon Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the heavy chain variable region of PVY-2 is shown in SEQ ID NO.4, and the nucleotide sequence of the light chain variable region of PVY-2 is shown in SEQ ID NO.5. The nucleotide sequence of the heavy chain variable region of PVY-5 is shown in SEQ ID NO.6, and the nucleotide sequence of the light chain variable region of PVY-5 is shown in SEQ ID NO.7.

[0200] Two hybridoma cell lines secreting monoclonal antibodies PVY-2 and PVY-5 were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and named 4F1B2G11 and 4B4D8C3, respectively. The deposit date of 4F1B2G11 was September 1, 2022, with accession number CCTCC NO: C2022281, and it was classified as hybridoma cell line 4F1B2G11. The deposit date of 4B4D8C3 was September 1, 2022, with accession number CCTCC NO: C2022282, and it was classified as hybridoma cell line 4B4D8C3.

[0201] Example 5: Preparation of PVY nano-mimetic enzyme test strips

[0202] I. Screening using PVY monoclonal antibody nanomimetic enzyme labeling

[0203] (1) Take the required amount of nanozyme solution, add purified water to prepare a concentration of 0.5 mg / mL, and sonicate for 1-2 min (53 kHz);

[0204] (2) Centrifuge at 13000 rpm at room temperature for 5-10 min;

[0205] (3) Remove the supernatant, add purified water to prepare a concentration of 0.5 mg / mL, resuspend, and sonicate for 1-2 min;

[0206] (4) Centrifuge at 13000 rpm at room temperature for 5-10 min, and remove the supernatant;

[0207] (5) Weigh 10 times the mass of the nanozyme, add it to MES solution (50mM, pH 6.0) and mix well to prepare a 10mg / mL NHS solution; weigh 10 times the mass of the nanozyme, add it to MES solution (50mM, pH 6.0) and mix well to prepare a 10mg / mL EDC solution, taking care to avoid light;

[0208] (6) Take equal volumes of the two solutions prepared in step 5 and add them to the nanozyme washed in step 4 to prepare a concentration of 0.5 mg / mL. Resuspend and mix well, sonicate for 30-60 seconds, sonicate 3-5 times in total, and react at room temperature for 30-40 minutes on a mixer (avoid light).

[0209] (7) Centrifuge at 13000 rpm at room temperature for 5-10 min, and remove the supernatant;

[0210] (8) Add MES solution (50mM, pH 6.0) to prepare a concentration of 0.5mg / mL, shake to mix, and then sonicate for 1-2 min; centrifuge at 13000rpm at room temperature for 5-10 min, and remove the supernatant;

[0211] (9) Take 100 μg of PVY monoclonal antibody and add it to MES solution (50 mM, pH 8.0) and mix well to prepare an antibody solution with a concentration of 0.1 mg / mL.

[0212] (10) Take the antibody prepared in step 9 and add it to the nanozyme washed in step 8 to prepare a nanozyme solution with a concentration of 1 mg / mL. Resuspend and mix well, sonicate for 10-20 seconds, sonicate 5-10 times in total, and react on a mixer at 2-8℃ for 14-18 hours.

[0213] (11) Place the reacted solution on a magnetic rack, aspirate the clear liquid into a new centrifuge tube, and use an ultra-micro protein detector to detect the antibody labeling efficiency of the clear liquid. Add Tris-buffer (50mM, pH 7.4) to the centrifuge tube to prepare a 0.5mg / mL nanozyme solution, resuspend and mix, sonicate for 10-20s, 5-10 times, and react at room temperature for 30-40min;

[0214] (12) Place the reacted solution on a magnetic rack, remove the supernatant, add 5% BSA-PBS solution to prepare a 1 mg / mL nanozyme solution, resuspend, and sonicate for 10-20 seconds, 5-10 times, with the ultrasonic cleaner temperature controlled at 2-8℃. After resuspending, place on a mixer at 2-8℃ for 2-4 hours to seal;

[0215] (13) Magnetic adsorption, discard the supernatant, add 1% BSA-PBS treatment solution to prepare a 1 mg / mL nanozyme solution, resuspend, sonicate for 10-20 seconds, 5-10 times, and control the temperature of the ultrasonic cleaner at 2-8℃. Store at 2-8℃ after resuspending and label for later use.

[0216] II. Pretreatment of bonding pads, sample pads, and absorbent pads

[0217] A. Bonding pad pretreatment

[0218] (1) Cut the glass fiber membrane into bonding pads with a width of 7mm using an instrument;

[0219] (2) Prepare the conjugate pad pretreatment solution (1% Triton X-100, 50mM sodium borate, pH 8.0);

[0220] (3) Take an appropriate amount of the cut conjoint pad and place it in the conjoint pad pretreatment box. Use a pipette to draw conjoint pad pretreatment liquid and add it to the conjoint pad to make it completely wet. Soak for 25-35 minutes.

[0221] (4) Use tweezers to clamp the soaked conjugate pads onto the drying net, arrange them neatly, and dry them in an oven at 40℃ for 2 hours until completely dry;

[0222] (5) Place the dried bonding pad into a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier cabinet for later use.

[0223] B. Sample pad pretreatment

[0224] (1) Cut the glass fiber membrane into sample pads with a width of 11 mm using an instrument;

[0225] (2) Prepare sample pad pretreatment solution (10mM PBS, 1% Tween 20, 0.1g / L PVP K30, pH 7.4);

[0226] (3) Take an appropriate amount of the cut sample pad and place it in the sample pad pretreatment box. Use a pipette to draw sample pad pretreatment liquid and add it to the sample pad to completely wet it. Soak for 25-35 minutes.

[0227] (4) Use tweezers to clamp the soaked sample pads onto the drying net, arrange them neatly, and dry them in an oven at 40℃ for 2.5 hours until completely dry;

[0228] (5) Place the dried sample pad into a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier cabinet for later use.

[0229] C. Absorbent Pad Treatment

[0230] (1) Cut the absorbent paper into absorbent pads with a width of 22mm using an instrument;

[0231] (2) Place the cut absorbent pads on the drying net, arrange them neatly, and dry them in an oven at 40℃ for 2.5 hours;

[0232] (3) Place the dried absorbent pad into a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier cabinet for later use.

[0233] D. NC film marking operation

[0234] (1) Take a certain amount of PVY antibody, add coating buffer solution, and dilute to a coating antibody solution with a concentration of 1.5 mg / mL;

[0235] (2) Take a certain amount of sheep anti-mouse IgG, add coating buffer solution, and dilute to a sheep anti-mouse IgG solution with a concentration of 1 mg / mL;

[0236] (3) Turn on the scrubbing sprayer and execute the cleaning program. After cleaning, put the tubing of pump 1 into the diluted PVY (detection line) antibody solution and the tubing of pump 3 into the diluted goat anti-mouse IgG (control line) solution.

[0237] (4) Place the PVC backing board with the NC film attached in the correct position on the scribing spraying machine and execute the scribing program at a scribing speed of 1μL / cm.

[0238] (5) After the line marking is completed, perform the cleaning procedure and turn off the machine. Mark the PVC backing board and put it into the oven to dry for 1 hour at a temperature of 37°C.

[0239] E. Combined with gold plating operation

[0240] (1) Take the nanozyme labeled with PVY antibody, put it into a centrifuge tube, adsorb it with a magnetic rack, discard the solution, add a certain amount of nanozyme labeled antibody dilution buffer (50mM Tris, 10% trehalose, 5% BSA, 1% Triton X-100, 1% Tween20, 0.05% proclin, 1% PVP K30, pH 8.5), and dilute to a nanozyme concentration of 1mg / mL;

[0241] (2) Place the diluted nanozyme-labeled antibody solution in an ultrasonic instrument and sonicate for 5 to 10 times, each time for 10 to 20 seconds.

[0242] (3) Turn on the scrubbing sprayer and execute the cleaning program. After cleaning, put the cannula of pump 2 into the ultrasonicated nanozyme-labeled antibody solution.

[0243] (4) Place the pretreated bonding pad in the correct position on the scribing sprayer and execute the pad spraying program at a spraying speed of 5 μL / cm.

[0244] (5) After the spray pad is finished, perform the cleaning procedure and turn off the machine. Mark the bonding pad and put it in the oven to dry for 1 hour at 37°C.

[0245] III. Assembly of PVY Nanomimetic Enzyme Test Strips

[0246] (1) Turn on the plate applicator, press the PVC board to press the plate applicator work plate, press the start suction to fix the PVC board, and place the PVC board in the middle position as much as possible to avoid it being in a moving state when the release paper is lifted.

[0247] (2) Assemble the test strips according to the selected combination;

[0248] (3) Press the start button and attach the dried NC membrane, conjugate pad, absorbent pad, and pretreated sample pad to the backing plate according to the setting that the absorbent pad presses the NC membrane by 2mm, the conjugate pad presses the NC membrane by 2mm, and the sample pad presses the conjugate pad by 2mm.

[0249] (4) After the plating process is complete, turn off the plating machine. Store the large test strips in a dark environment with humidity ≤30% for later use;

[0250] (5) Place the pasted PVC board on the chopping machine, turn on the power and switch, lift the flipping frame, adjust the position of the placing board, hold the finished large board in place, align the left end of the finished large board with the blade, and lower the flipping frame.

[0251] (6) Set the test strip cutting width to 0.4cm and click Start to cut;

[0252] (7) Insert the cut test strips into the reagent cartridges; turn on the power of the automatic casing machine, place the casing-filled reagent strips on the casing machine conveyor belt, click the "Run" button, the machine runs until all reagent cartridges are casing-filled, and then turn off the casing machine.

[0253] (8) Place the test strip into a heat-sealing bag containing a desiccant and heat-seal it;

[0254] (9) After heat sealing, mark the test strips and store them in a dehumidifier with a humidity of no more than 30%.

[0255] IV. Performance Testing of PVY Nanoscale Enzyme Test Strips

[0256] A. Screening can be performed using PVY nano-mimetic enzyme test strips.

[0257] (1) Grind the PVY-infected tissue culture seedlings into powder with liquid nitrogen, add extraction buffer, vortex to mix, and centrifuge at room temperature for 4000g for 2min.

[0258] (2) Add positive control and blank control to the assembled test strips respectively;

[0259] (3) After the sample is dropped into the sample well, time for 15 minutes at room temperature;

[0260] (4) Observe whether false positives occur in the test strips and record the numbers of test strips that do not show false positives.

[0261] PVY nano-mimetic enzyme test strips were prepared, and antibodies were selected based on the DAS-ELISA pairing results.

[0262] Only one pair of PVY monoclonal antibodies successfully paired, namely PVY-2 and PVY-5. PVY-2 and PVY-5 were labeled and coated, respectively. Using PVY-2 as the labeling antibody and PVY-5 as the coating antibody, a PVY nano-enzyme test strip was prepared. When a positive sample was added, both the C line (control line) and T line (detection line) appeared simultaneously, indicating a positive result. However, after adding a negative control, both the C line and T line also appeared simultaneously, indicating a false positive. Conversely, using PVY-5 as the labeling antibody and PVY-2 as the coating antibody, when a positive sample was added, both the C line and T line appeared simultaneously, indicating a positive result. After adding a negative control, only the C line appeared, indicating a negative result.

[0263] Ultimately, PVY nano-enzyme test strips were prepared using PVY-5 as the labeling antibody and PVY-2 as the coating antibody.

[0264] B. Specificity detection using PVY nano-mimetic enzyme test strips

[0265] (1) Perform specific testing on test strips that do not show false positives.

[0266] (2) Plants infected with PVA, PVM, PVS, PVY, PVX and PLRV and healthy plants were ground into powder with liquid nitrogen, and the extraction buffer was added and vortexed to mix.

[0267] (3) Centrifuge at room temperature for 4000g for 2min and collect the supernatant;

[0268] (4) After adding the sample to the sample well, time for 15 minutes at room temperature, observe and record the experimental results.

[0269] The results are as follows Figure 7 As shown, when sap from PVA, PVM, PVS, PVX, PLRV, and healthy plants was added to the test strip, only the C line appeared, indicating a negative result. This means the test strip does not react with PVA, PVM, PVS, PVX, PLRV, or healthy plants. When sap from PVY plants was added to the test strip, both the C and T lines appeared, indicating a positive result. This means the test strip reacts with PVY and has good specificity.

[0270] C. Sensitivity detection of PVY nano-mimetic enzyme test strips

[0271] (1) Grind the PVY-infected tissue culture seedlings into powder with liquid nitrogen, add 1 ml of extraction buffer to 0.1 g of plant tissue, and vortex to mix.

[0272] (2) Centrifuge at room temperature for 4000g for 2min;

[0273] (3) Mix the supernatant at a ratio of 1:10 and 1:10. 2 1:10 3 1:10 4 1:10 5 Dilute using a gradient;

[0274] (4) After vortexing the diluted sample, add it dropwise into the sample well, time for 15 minutes at room temperature, and observe and record the experimental results.

[0275] The results are as follows Figure 8 As shown, PVY nano-mimetic enzyme test strips can achieve ratios of 1:10 and 1:10. 2 1:10 3 After dilution, both C and T lines appear simultaneously, indicating a positive test result and detection of PVY. This is true when the dilution ratio is 1:10. 4 1:10 5 After dilution, only the C line appeared, and the T line did not appear, resulting in a negative result and PVY could not be detected.

[0276] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A hybridoma cell line 4B4D8C3 secreting monoclonal antibody PVY-5, characterized in that, The hybridoma cell strain can secrete the anti-potato virus Y monoclonal antibody, and the hybridoma cell strain is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2022282.

2. The monoclonal antibody PVY-5 produced by the hybridoma cell strain 4B4D8C3 of claim 1, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody PVY-5 is shown in SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The mAb PVY-5 according to claim 2, characterized in that, The monoclonal antibody PVY-5 is specifically immunoreactive with the coat protein of the potato virus Y, the nucleotide sequence of the coat protein gene of the potato virus Y is shown in SEQ ID NO. 3; the antibody type and subclass of the monoclonal antibody PVY-5 are IgG1, the chain type is Kappa light chain, and the sensitivity of the direct ELISA method for detecting the potato virus Y infected leaf reaches 1:5120-fold dilution.

4. The monoclonal antibody PVY-5 of claim 2 or 3 is used for detecting the potato PVY virus.

5. A kit containing the monoclonal antibody PVY-5 of claim 2 or 3.

6. The kit of claim 5, wherein the PVY-5 monoclonal antibody is contained in a composition comprising a pharmaceutically acceptable carrier. The kit is an ELISA kit, a colloidal gold kit or a nano-mimetic enzyme kit.

7. The kit containing mAb PVY-5 according to claim 6, characterized in that, The nano-mimetic enzyme in the nano-mimetic enzyme kit is Fe3O4 magnetic nanoparticles.

Images