Hybridoma cell strain secreting anti-polystyrene monoclonal antibody and its application

Abstract

This invention discloses a hybridoma cell line that secretes anti-polystyrene monoclonal antibodies and its applications. The hybridoma cell line includes PS-8, classified as Hybridoma cell line PS-8. The accession number for PS-8 is CCTCC NO: C2024144, the accession date is May 10, 2024, and the depositary institution is the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The hybridoma cell line provided by this invention can stably secrete anti-polystyrene monoclonal antibodies with high titer, good specificity, and high sensitivity, making it suitable for detection in reagent kits and possessing potential application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a hybridoma cell strain secreting anti-polystyrene monoclonal antibodies and application thereof. BACKGROUND

[0002] Microplastics are of various types, great harm, heavy pollution and wide range. The microplastics that pollute the environment mainly include polystyrene (PS), polypropylene (PP), polyethylene (PE) and the like. Microplastics have sublethal toxicity, cytotoxicity and other hazards, which seriously threaten human life and health.

[0003] Existing microplastic detection mostly relies on microscopic spectral methods, Raman spectrum and other instrument analysis methods, which is difficult to meet the needs of rapid and accurate detection. The microplastic pollution environment in China is complex, the geographical environment difference is large, and the pollution links are many, involving the fields of environment, food, textiles and the like. In the production process of synthetic fibers (such as polyester fibers, nylon and the like), some plastic particles or fibers smaller than 5 mm may be generated. For example, the friction of production equipment, incomplete polymerization of raw materials and the like may cause the generation of microplastics. In addition, in the process of textile, such as spinning, weaving, printing and dyeing and the like, the operation of machines, the wear of parts and the stress action in the processing process may cause the microplastic fibers on the surface of textiles to fall off. Especially when some high-strength processing technology is used or poor-quality raw materials are used, the possibility of microplastic generation will increase. When people wear textiles, due to the movement and friction of the body, the fibers on the surface of textiles will gradually wear and fall off, and microplastic fibers will be generated. For example, some parts that are frequently rubbed, such as cuffs, collars and cuffs and the like, are more likely to have fiber falling off. At the same time, in the washing process of a washing machine, the friction between clothes, the impact of water flow and the action of detergent will cause a large amount of fibers to fall off from the clothes, forming microplastic fibers. It is estimated that in a typical household washing activity, a piece of clothing can release millions of fibers. Moreover, some synthetic fiber materials, such as nylon and polyester, are more likely to generate microplastic fibers in the washing process due to their fiber structure and characteristics. When the discarded textiles are landfilled, the textiles will gradually degrade and break down in the natural environment over time, generating microplastics. These microplastics may enter the soil, water and other environments through rainwater erosion and the like, causing pollution. Although incineration can reduce the amount of solid waste of textiles, some smoke and ash containing microplastics may be generated in the incineration process. If these smoke and ash are not properly treated, they will also be released into the environment, causing microplastic pollution.

[0004] The current microplastics detection methods mainly include visual method, microscope method, electron microscope, Raman spectrum analysis method, infrared spectrum method, thermal analysis method, thermal cracking-gas chromatography-mass spectrometer, laser infrared imaging, optical light-thermal infrared technology, but these detection technologies can only be qualitatively determined, some have low detection sensitivity, some rely on laboratory large instrument detection, and it is difficult to monitor the pollution distribution and environmental effect of microplastics in the environment in real time, therefore, it is urgent to study a high-sensitivity and rapid detection method of microplastics in complex matrix. The key of the rapid technology for quantitative analysis of microplastics is to develop a specific antibody of microplastics. At present, there is a preparation of polystyrene (PS) polyclonal antibody, but there is no preparation of microplastics (PS and other types of microplastics) monoclonal antibody. Microplastics are inert small particles, and there are great challenges in the preparation of haptens and the determination of affinity parameters.

[0005] Therefore, a hybridoma cell strain secreting anti-polystyrene monoclonal antibody and its application are urgently needed. SUMMARY

[0006] In order to solve the defects in the prior art, the application provides a hybridoma cell strain secreting anti-polystyrene monoclonal antibody and its application.

[0007] In order to solve the above technical problems, the application provides the following technical solutions:

[0008] The first object of the application provides a hybridoma cell strain secreting anti-polystyrene monoclonal antibody, which comprises a hybridoma cell strain PS-8, which is preserved in the China Center for Type Culture Collection, and the preservation number of the hybridoma cell strain PS-8 is CCTCC NO: C2024144.

[0009] The second object of the application provides a hybridoma cell strain secreting anti-polystyrene monoclonal antibody, which comprises a hybridoma cell strain PS-17, which is preserved in the China Center for Type Culture Collection, and the preservation number of the hybridoma cell strain PS-17 is CCTCC NO: C2024145.

[0010] The third object of the application provides a monoclonal antibody, which is secreted by the hybridoma cell strain PS-8 or the hybridoma cell strain PS-17, and the monoclonal antibody can specifically recognize polystyrene.

[0011] The fourth object of the application provides a kit, which is characterized in that the kit contains a monoclonal antibody secreted by the hybridoma cell strain PS-8 or the hybridoma cell strain PS-17.

[0012] The fifth object of the application provides an application of a monoclonal antibody, which is used for preparing a reagent for detecting polystyrene.

[0013] Compared with the prior art, the present application has the following beneficial effects: the passage cell strain of the hybridoma cell provided by the present application can stably secrete the anti-polystyrene protein monoclonal antibody, the antibody has high secretion titer, good specificity and high sensitivity, and can be applied to kit detection, thereby significantly improving the sensitivity of kit detection.

[0014] Cell preservation:

[0015] The present application provides a hybridoma cell strain secreting an anti-micro-nano plastic polystyrene monoclonal antibody, including hybridoma cell strain PS-8 and hybridoma cell strain PS-17, wherein the hybridoma cell strain PS-8 is screened by the inventors of the present application, the preservation number of the hybridoma cell strain PS-8 is CCTCC NO: C2024144, the preservation number of the hybridoma cell strain PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, the preservation unit is China Center for Type Culture Collection, and the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a SDS-PAGE electrophoresis schematic diagram of PS-BSA and PS-KLH in embodiment 1 of the present application;

[0017] Figure 2 A is a reduced SDS-PAGE electrophoresis schematic diagram of each antibody in embodiment 4 of the present application;

[0018] Figure 2 B is a non-reduced SDS-PAGE electrophoresis schematic diagram of each antibody in embodiment 4 of the present application;

[0019] wherein, lanes 1-12 correspond to antibody names PS-1, PS-3, PS-4, PS-7, PS-8, PS-9, PS-13, PS-15, PS-17, PS-18, PS-22, and PS-23;

[0020] Figure 3 is a control experiment result of each antibody on five different microplastics including PS, PVC, PET, PE and PP in embodiment 4 of the present application;

[0021] Figure 4 is a flowchart for obtaining the variable region of the antibody in embodiment 5 of the present application;

[0022] Figure 5 is a standard curve diagram in embodiment 6 of the present application. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present application will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0024] Example 1: Coupling of microplastics (PS-CHOOH) to conjugated proteins (BSA / KLH).

[0025] 1. Cool down microplastics, polylink coupling buffer, polylink EDAC, polylink wash / storage buffer to room temperature.

[0026] 2. Take 12.5 mg microplastics to 1.5 mL centrifuge tube, centrifuge at 500-1000 g for 5-10 min, discard supernatant; add 0.4 mL coupling buffer solution to resuspend, centrifuge again, discard supernatant; add 0.17 mL coupling buffer solution to resuspend again.

[0027] 3. Dissolve 10 mg polylink EDAC in 50 μL coupling buffer solution to prepare a 200 mg / mL EDAC solution, and note that it should be prepared immediately before use.

[0028] 4. Add 20 μL EDAC solution to the 0.17 mL coupling buffer solution suspension, and mix on a rotator at room temperature for 15 min for activation.

[0029] 5. Add 200-500 μg conjugated protein (BSA / KLH), and mix on a rotator at room temperature for 30-60 min (dissolve the protein in coupling buffer solution to 1-5 mg / mL).

[0030] 6. Centrifuge at 500-1000 g for 10 min, and aspirate the supernatant to detect the amount of conjugated protein (BSA / KLH).

[0031] 7. Add 0.4 mL polylink wash / storage buffer to resuspend.

[0032] 8. Repeat steps 6 and 7, and the precipitate at the bottom of the tube is PS-BSA or PS-KLH, which is finally stored at 4°C.

[0033] 9. Perform SDS-PAGE analysis (4% concentrated gel and 7.5% separation gel). The results of the reduced SDS-PAGE detection are shown in Figure 1 , which proves that the coupling of PS-KLH and PS-BSA is successful.

[0034] Example 2: Preparation of monoclonal antibodies.

[0035] The prepared PS-BSA artificial antigen is used for immunization injection of 6-8 week old female Balb / c mice. The adult Balb / c mice are given primary immunization by subcutaneous administration (in the area between the shoulders) and are given booster immunization every 2 weeks. Blood samples are collected at 0 weeks (before immunization), 8 weeks (2 weeks after the fourth booster injection), 10 weeks (before the third booster injection), 12 weeks and 14 weeks, and then serum separation is performed to obtain B lymphocytes, extract RNA and transcribe into a cDNA library. Specific gene fragments are obtained by re-amplification of the cDNA, and are recombined on phages to construct a nanobody gene library. Then specific antibody screening is performed to obtain the required antibody, and the PS-BSA artificial antigen is fixed on a carrier and then allowed to interact with the phage library. Then the carrier surface is washed to wash away the unbound or non-specifically bound phages, and then strong acid is used to destroy the binding, thereby obtaining a positive phage solution. After 2-3 rounds of repetition, the required monoclonal antibody can be obtained.

[0036] The PS-BSA artificial antigen is injected to observe the specific response of the immune system of the Balb / c mice, and the mechanism of activation of the specific immune response of the antibody is explored.

[0037] Table 1: Immunization mouse scheme.

[0038]

[0039] ELLSA tail blood titer detection

[0040]

[0041] Example 3: Monoclonal antibody subtype analysis and titer detection.

[0042] Monoclonal antibody subtype analysis: The antibodies produced by the 2 hybridoma cells secreting specific monoclonal antibodies, PS-8 and PS-17, were identified for antibody subtype, and the results showed that PS-8 was IgG2b and PS-17 was IgG1.

[0043] Monoclonal antibody titer detection: PS-KLH 1 μg / ml was used to coat the plate, and each antibody was gradiently diluted (1:100, 1:500, 1:2500, 1:12500, 1:32500, 1:612500), and goat anti-mouse IgG-HRP 1 w-fold dilution was added to determine the titer of the purified monoclonal antibody (the results are shown in Table 2).

[0044] Table 2: PS-BSA mouse five-immune tail blood titer detection.

[0045]

[0046] Example 4: Screening and identification of monoclonal antibodies.

[0047] The PS-BSA immunized mice were subjected to cell fusion, and 12 positive hybridoma cell lines were obtained after four rounds of subcloning. The monoclonal antibodies were prepared according to the foregoing method, and each antibody was subjected to reduced and non-reduced SDS-PAGE electrophoresis detection (results shown in Figure 2 At the same time, control experiments were performed on each antibody, and PS, PVC, PET, PE, and PP were coated as five different microplastics (results shown in Figure 3 It was confirmed that the PS monoclonal antibody did not cross-react with other types of micro-nano plastics.

[0048] Example 5: Sequencing of monoclonal antibodies (according to the flowchart to obtain the sequence of the variable region of the antibody). Figure 4

[0049] (1) The monoclonal antibody secreted by the hybridoma cell line PS-8 contains a heavy chain variable region and a light chain variable region, both of which are composed of a complementarity-determining region and a framework region; the complementarity-determining region of the heavy chain variable region and the light chain variable region is composed of CDR1, CDR2, and CDR3;

[0050] The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID No. 1 at positions 50-54;

[0051] The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID No. 1 at positions 69-85;

[0052] The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID No. 1 at positions 118-127;

[0053] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID No. 2 at positions 44-54;

[0054] The amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID No. 2 at positions 70-76;

[0055] The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID No. 2 at positions 109-117.

[0056] The amino acid sequence of the heavy chain variable region of the PS-8 antibody is shown in SEQ ID No. 1:

[0057] ​MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFT SYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCASSYYYGSSYGYWGQGTTLTVSS.

[0058] The amino acid sequence of the light chain variable region of the PS-8 antibody is shown in SEQ ID No. 2:

[0059] MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDV STAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAED LAVYYCQQHYSTPLTFGAGTKLELK.

[0060] (2) The monoclonal antibody secreted by the hybridoma cell strain PS-17 contains a heavy chain variable region and a light chain variable region, both of which are composed of a complementarity determining region and a framework region; the complementarity determining region of the heavy chain variable region and the light chain variable region is composed of CDR1, CDR2 and CDR3;

[0061] The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID No. 3, positions 50-54;

[0062] The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID No. 3, positions 69-85;

[0063] The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID No. 3, positions 118-128;

[0064] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID No. 4, positions 44-54;

[0065] The amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID No. 4, positions 70-76;

[0066] The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID No. 4, positions 109-117.

[0067] The amino acid sequence of the heavy chain variable region of the PS-17 antibody is shown in SEQ ID No. 3:

[0068] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTD YYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQ LSSLTSEDSAVYFCARSEIYGIYYFDYWGQGTTLTVSS.

[0069] The amino acid sequence of the heavy chain variable region of the PS-17 antibody is shown in SEQ ID No. 3:

[0070] METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQD VGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQS EDLADYFCQQYSSYPYTFGGGTKLEIK.

[0071] Example 6: Application of monoclonal antibodies.

[0072] Water samples, soil samples were collected from the sea, rivers and lakes. Textile wastewater samples were collected from textile enterprises. Food samples were collected from the field or the table. The collected samples were pretreated and extracted, and then quantitatively detected by the kit.

[0073] The pretreatment was carried out according to the following steps:

[0074] (1) For liquid samples such as water samples, each sample was centrifuged at 17000xg for 3 minutes, and then the supernatant was digested and extracted. The digestion extract was diluted with sample diluent and used for immunochromatographic quantitative detection.

[0075] (2) For non-liquid samples, the samples were digested, microplastic extracted, diluted with diluent, and then subjected to immunochromatographic quantitative detection.

[0076] Taking the soil sample (spiked detection) as an example:

[0077] Deionized water was added to the soil sample (1:1 w / v), and then vortexed for 10 s. The slurry mixture was centrifuged at 17000xg for 3 minutes, and the supernatant was used as a specific matrix extract for spiked recovery analysis. The sample detection was carried out according to the following steps:

[0078] 1. Add 50 μL of sample pretreatment extract solution to a sterile glass tube (set up parallel replicates for sample testing), and at the same time, add different volumes of microplastic working solution to the same batch of tubes. The final concentration of the standard working solution added is 0, 1.57, 3.125, 6.25, 12.5, 25, 50 ug, respectively, thereby establishing the standard curve for sample testing.

[0079] 2. Then add 950 μL of PBST washing buffer to each test tube.

[0080] 3. Place the test tube on a rotator and rotate at a speed of 20 revolutions per minute for 3 minutes.

[0081] 4. Centrifuge the test tube at a speed of 5500 rpm for 3 minutes to precipitate the microplastic particles. Carefully remove and discard the supernatant.

[0082] 5. Add 1,000 μL of antibody diluent (32500-fold dilution) to each test tube. Vortex the test tube briefly or move it up and down several times with a pipette to ensure that the microplastics are completely mixed with the antibody solution.

[0083] 6. Rotate the test tube at room temperature at a speed of 20 revolutions per minute for 1 hour.

[0084] 7. Washing step. Centrifuge the test tube at a speed of 5500 rpm for 3 minutes to precipitate the microplastic particles. Carefully remove and discard the supernatant. Add 1,000 μL of washing buffer to each test tube and vortex briefly. Place the test tube on a rotator and rotate at a speed of 20 rpm for 3 minutes.

[0085] 8. Repeat step 7 three times.

[0086] 9. After completing the last washing step, centrifuge the test tube at 5500 rpm for 3 minutes to precipitate the microplastic particles. Carefully remove and discard the supernatant.

[0087] 10. Add 1,000 μL of goat anti-rabbit antibody labeled with horseradish peroxidase to each test tube, which has been diluted 2,000 times with PBST solution. Vortex the test tube for 1 min to ensure that the microplastics are completely mixed with the secondary antibody solution.

[0088] 11. Place the test tube on a rotator and rotate at a speed of 20 rpm at room temperature for 1 hour.

[0089] 12. Wash the microplastics four times with washing buffer (repeat steps 8-10).

[0090] 13. Add 100 μL of TMB substrate and move it up and down several times with a pipette, then immediately transfer the suspension to the microwells of a microplate.

[0091] 14. Place the microplate in a slow speed shaker at room temperature for 30 minutes.

[0092] 15. Add 100 μL of 1 M H2SO4 to each well to stop the reaction.

[0093] 16. Measure the OD value at 450 nm using a microplate reader; plot the standard curve using Origin with the standard concentrations as the x-axis and B / B0 as the y-axis. Figure 5

[0094] 17. Enter the OD value of the sample into the standard curve to calculate the amount of microplastics in the sample.

[0095] Finally, it should be noted that the above only for the preferred embodiments of the present application, and is not intended to limit the present application, although the foregoing detailed description of the application has been made, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement of the technical solutions described in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included within the scope of the present application.​

Claims

1. A hybridoma cell line secreting an anti-polystyrene monoclonal antibody, characterized in that, The application relates to a hybridoma cell line PS-8, the classification name of the hybridoma cell line PS-8 is Hybridoma cell line PS-8, the preservation number of the hybridoma cell line PS-8 is CCTCC NO: C2024144, the preservation date is May 10, 2024, the preservation unit is China Center for Type Culture Collection, and the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A monoclonal antibody, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line PS-8 in claim 1 and can specifically recognize polystyrene.

3. A kit characterized in that, The kit contains the monoclonal antibody secreted by the hybridoma cell line PS-8 in claim 2.

4. The use of a monoclonal antibody according to claim 2, characterized in that, The monoclonal antibody is used for preparing a reagent for detecting polystyrene.

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