Hybridoma cell strain capable of secreting anti-polystyrene monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain secreting an anti-polystyrene monoclonal antibody and application of the hybridoma cell strain, the hybridoma cell strain comprises a hybridoma cell strain PS-8 and a hybridoma cell strain PS-17, the hybridoma cell strain PS-8 and the hybridoma cell strain PS-17 are preserved in the China Center for Type Culture Collection, and the preservation numbers of the hybridoma cell strain PS-8 and the hybridoma cell strain PS-17 are CCTCC NO: C2024144 and CCTCC NO: C2024154. The passage cell strain of the hybridoma cell provided by the invention can stably secrete the anti-polystyrene protein monoclonal antibody, the secreted antibody is high in titer, good in specificity and high in sensitivity, and the passage cell strain can be applied to kit detection and has potential application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a hybridoma cell line secreting anti-polystyrene monoclonal antibody and its application. Background Art

[0002] Microplastics are diverse in type, harmful, heavily polluting, and widespread in scope. The microplastics polluting the environment mainly include polystyrene (PS), polypropylene (PP), polyethylene (PE), etc. Microplastics have hazards such as sublethal toxicity and cytotoxicity, seriously threatening human life and health.

[0003] Existing microplastic detection mostly relies on instrumental analysis methods such as microscopic spectroscopy and Raman spectroscopy, which are difficult to meet the needs of rapid and accurate detection. The microplastic pollution environment in China is complex, with large geographical environment differences and many pollution links, covering fields such as environment, food, and textiles. During the production process of synthetic fibers (such as polyester fibers, nylon, etc.), some plastic particles or fibers smaller than 5 millimeters may be generated. For example, the friction of production equipment and the incomplete polymerization of raw materials may both lead to the generation of microplastics. In addition, during the textile process, such as spinning, weaving, and dyeing, the operation of machines, the wear of components, and the stress during processing may cause microplastic fibers to fall off the surface of textiles. Especially in some high-intensity processing processes or when using poor-quality raw materials, the possibility of generating microplastics will increase. When people wear textiles, due to body movements, friction, etc., the fibers on the surface of textiles will gradually wear and fall off, generating microplastic fibers. For example, in some frequently rubbed parts, such as cuffs, collars, and trouser legs, fiber shedding is more likely to occur. At the same time, during the washing process in a washing machine, the friction between clothes, the impact of water flow, and the action of detergents will cause a large amount of fibers to fall off the clothes, forming microplastic fibers. It is estimated that in a typical household washing activity, one piece of clothing can release millions of fibers. Moreover, some synthetic fiber clothing materials, such as nylon and polyester, are more likely to generate microplastic fibers during the washing process due to their fiber structure and characteristics. When waste textiles are landfilled, over time, the textiles will gradually degrade and break in the natural environment, generating microplastics. These microplastics may enter the soil, water bodies and other environments through rainwater scouring and other ways, causing pollution. Although incineration can reduce the amount of solid waste of textiles, some soot and ash containing microplastics may be generated during the incineration process. If these soot and ash are not properly treated, they will also be released into the environment, causing microplastic pollution.

[0004] Currently, the detection methods of microplastics mainly include: visual method, microscopy, electron microscopy, Raman spectroscopy analysis, infrared spectroscopy, thermal analysis, pyrolysis-gas chromatography / mass spectrometry, laser infrared imaging, and photothermal infrared technology. However, some of these detection techniques can only make qualitative judgments, some have low detection sensitivity, and some rely on large laboratory instruments for detection, making it difficult to monitor the pollution distribution and environmental effects of microplastics in the environment in real time. Therefore, there is an urgent need to study a highly sensitive and rapid detection method for microplastics in complex matrices. The key to the rapid technology for quantitative analysis of microplastics is to develop specific antibodies for microplastics. Currently, polyclonal antibodies against microplastics (PS) have been developed, but monoclonal antibodies against microplastics (PS and other types of microplastics) have not been prepared. Microplastics are inert small particles, and there are great challenges in preparing haptens and measuring parameters such as affinity.

[0005] Therefore, there is an urgent need to propose a hybridoma cell line secreting anti-polystyrene monoclonal antibodies and its application. Summary of the Invention

[0006] To solve the defects of the existing technology, the present invention provides a hybridoma cell line secreting anti-polystyrene monoclonal antibodies and its application.

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] The first object of the present invention is to provide a hybridoma cell line secreting anti-polystyrene monoclonal antibodies, including the hybridoma cell line PS-8, which is deposited in the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line PS-8 is CCTCC NO: C2024144.

[0009] The second object of the present invention is to provide a hybridoma cell line secreting anti-polystyrene monoclonal antibodies, including the hybridoma cell line PS-17, which is deposited in the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line PS-17 is CCTCC NO: C2024145.

[0010] The third object of the present invention is to provide a monoclonal antibody, which is secreted by the hybridoma cell line PS-8 or the hybridoma cell line PS-17, and the monoclonal antibody can specifically recognize polystyrene.

[0011] The fourth object of the present invention is to provide a kit, characterized in that the kit contains a monoclonal antibody secreted by the hybridoma cell line PS-8 or the hybridoma cell line PS-17.

[0012] The fifth object of the present invention is to provide an application of a monoclonal antibody, using the monoclonal antibody to prepare a reagent for detecting polystyrene.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The subculture cell line of hybridoma cells provided by the present invention can stably secrete monoclonal antibodies against polystyrene protein, with high antibody titer, good specificity, and high sensitivity. It can be applied to kit detection, significantly improving the sensitivity of kit detection.

[0014] Cell preservation:

[0015] A hybridoma cell line secreting monoclonal antibodies against micro-nano plastic polystyrene provided by the present invention includes hybridoma cell line PS-8 and hybridoma cell line PS-17. The hybridoma cell line PS-8 was self-screened by the inventors of the present invention. The preservation number of the hybridoma cell line PS-8 is CCTCC NO: C2024144, and the preservation number of the hybridoma cell line PS-17 is CCTCC NO: C2024145. The preservation date is May 10, 2024, and the preservation unit is the China Center for Type Culture Collection, with the address at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Brief description of the drawings

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

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

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

[0019] Among them, the antibody names corresponding to lanes 1-12 are PS-1, PS-3, PS-4, PS-7, PS-8, PS-9, PS-13, PS-15, PS-17, PS-18, PS-22, PS-23;

[0020] Figure 3 It is the control experiment results of each antibody when coating 5 different microplastics, namely PS, PVC, PET, PE, and PP, in Example 4 of the present invention;

[0021] Figure 4 It is the acquisition process of the variable region of the antibody in Example 5 of the present invention;

[0022] Figure 5 It is the standard curve graph in Example 6 of the present invention. Detailed implementation manners

[0023] The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

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

[0025] 1. Cool the microplastics, coupling buffer solution (polylink coupling buffer), coupling carbodiimide (polylink EDAC), and coupling washing / storage solution (polylink wash / storage buffer) to room temperature.

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

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

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

[0029] 5. Add 200 - 500 μg of conjugated protein (BSA / KLH), fix it on a rotator, and mix well 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, aspirate the supernatant to detect the amount of conjugated protein (BSA / KLH).

[0031] 7. Add 0.4 mL of coupling washing / storage solution (polylink wash / storage buffer) to resuspend.

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

[0033] 9. Perform SDS-PAGE analysis (4% stacking gel and 7.5% separating gel). The detection results of reducing SDS-PAGE are as Figure 1 shown, proving the successful coupling of PS-KLH and PS-BSA.

[0034] Example 2: Preparation of monoclonal antibodies.

[0035] Female Balb / c mice aged 6 - 8 weeks were selected, and the prepared PS-BSA artificial antigen was used for immunization injection. Adult Balb / c mice received primary immunization by subcutaneous administration (in the area between the shoulders), and booster immunization was carried out every 2 weeks. Blood samples were collected at week 0 (before immunization), week 8 (2 weeks after the fourth booster injection), week 10 (before the third booster injection), week 12, and week 14, and then serum was separated to obtain B lymphocytes. RNA was extracted and transcribed into a cDNA library. Through cDNA re-amplification, specific gene fragments were obtained and recombined on phages to construct a nanobody gene library. Then, specific antibodies were screened to obtain antibodies that met the requirements. The PS-BSA artificial antigen was immobilized on a carrier, and then allowed to interact with the phage library. Then the surface of the carrier was washed to remove unbound or non-specifically bound phages, and then the binding was disrupted with strong acid to obtain a positive phage solution. After repeating 2 - 3 rounds, monoclonal antibodies that met the requirements could be obtained.

[0036] By injecting the PS-BSA artificial antigen, the immunization protocol for mice is shown in Table 1, observing what specific reactions it produces on the immune system of Balb / c mice, and exploring the activation mechanism of antibody specific immune responses.

[0037] Table 1: Immunization protocol for mice.

[0038]

[0039] ELLSA tail blood titer detection

[0040]

[0041] Example 3: Analysis of monoclonal antibody subtypes and titer detection.

[0042] Analysis of monoclonal antibody subtypes: The antibodies produced by 2 hybridoma cells secreting specific monoclonal antibodies were identified for antibody subtypes as PS-8 and PS-17 respectively. The results showed that PS-8 was IgG2b and PS-17 was IgG1.

[0043] Detection of monoclonal antibody titer: The plate was coated with 1 μg / ml of PS-KLH, and each antibody was serially diluted (1:100, 1:500, 1:2500, 1:12500, 1:32500, 1:612500), and 10,000-fold diluted goat anti-mouse IgG-HRP was added to determine the titer of the purified monoclonal antibody (the results are shown in Table 2).

[0044] Table 2: Detection of the titer of the tail blood of PS-BSA mice after five immunizations.

[0045]

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

[0047] Mice were immunized with PS-BSA for cell fusion, and 12 positive hybridoma cells were obtained after four rounds of subcloning. Monoclonal antibodies were prepared from these 12 positive hybridoma cells according to the aforementioned method, and each antibody was detected by reducing and non-reducing SDS-PAGE electrophoresis (the results are as Figure 2 shown). At the same time, a control experiment was conducted on each antibody by coating five different microplastics, namely PS, PVC, PET, PE, and PP (the results are as Figure 3 shown), to confirm that there is no cross-reaction between the PS monoclonal antibody and other types of micro- and nano-plastics.

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

[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, and both the heavy chain variable region and the light chain variable region are composed of complementarity-determining regions and framework regions; the complementarity-determining regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2, and CDR3;

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

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

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

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

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

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

[0056] The amino acid sequence of the heavy chain variable region of the PS-8 antibody is as 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 line PS-17 contains a heavy chain variable region and a light chain variable region, and both the heavy chain variable region and the light chain variable region are composed of a complementarity determining region and a framework region; the complementarity determining regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3;

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

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

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

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

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

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

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

[0068] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTDYYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSEIYGIYYFDYWGQGTTLTVSS。

[0069] The amino acid sequence of the light chain variable region of the PS-17 antibody is shown in SEQ ID No. 4:

[0070] METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQD VGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQS EDLADYFCQQYSSYPYTFGGGTKLEIK。

[0071] Example 6: Application of monoclonal antibody.

[0072] Water samples and soil samples are collected from marine, river, lake and reservoir waters. Textile wastewater samples are collected from textile enterprises. Food samples are collected from fields or dining tables. The collected samples are quantitatively detected by a kit after pretreatment and extraction.

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

[0074] (1) For liquid samples such as water samples, each sample is centrifuged at 17000×g for 3 minutes, and then the supernatant is digested and extracted. The digested and extracted solution is diluted with a sample diluent and used for quantitative immunochromatographic detection with a test strip.

[0075] (2) For non-liquid samples, the samples need to be digested, microplastics extracted, and diluted with a diluent before quantitative immunochromatographic detection.

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

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

[0078] 1. Add 50 μL of sample pretreatment extraction solution into a sterile glass tube (set parallel repeated samples during sample testing), and at the same time add different volumes of microplastic working solution into the same batch of tubes. The final concentrations of the added standard working solutions are: 0, 1.57, 3.125, 6.25, 12.5, 25, 50 μg, thereby establishing a calibration curve for sample detection.

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

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

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

[0082] 5. Add 1,000 μL of antibody dilution solution (diluted 32,500 times) to each test tube. Briefly vortex the test tubes or move the pipette up and down several times to ensure complete mixing of the microplastics with the antibody solution.

[0083] 6. Rotate the test tubes at 20 revolutions per minute at room temperature for 1 hour.

[0084] 7. Washing step. Centrifuge the test tubes at 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 briefly vortex. Place the test tubes on a vortexer and rotate at 20 rpm for 3 minutes.

[0085] 8. Repeat step 7 three times.

[0086] 9. After completing the last washing step, centrifuge the test tubes 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. This antibody has been diluted with PBST solution (diluted 2,000 times). Vortex the test tubes for 1 min to ensure complete mixing of the microplastics with the secondary antibody solution.

[0088] 11. Place the test tubes on a vortexer and rotate at 20 rpm at room temperature for 1 hour.

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

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

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

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

[0093] 16. Measure the OD value using an ELISA reader at 450 nm; use Origin to plot a standard curve ( Figure 5 ), with the concentration of each standard as the abscissa and B / B0 as the ordinate to plot the standard curve.

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

[0095] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting anti - polystyrene monoclonal antibody, characterized in that, It includes the hybridoma cell line PS-8, which is deposited in the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line PS-8 is CCTCC NO: C2024144.

2. A hybridoma cell line secreting anti-polystyrene monoclonal antibody, characterized in that, It includes the hybridoma cell line PS-17, which is deposited in the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line PS-17 is CCTCC NO: C2024145.

3. A monoclonal antibody, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line PS-8 described in claim 1 or by the hybridoma cell line PS-17 described in claim 2, and the monoclonal antibody can specifically recognize polystyrene.

4. A kit, characterized in that, The kit contains the monoclonal antibody secreted by the hybridoma cell line PS-8 or the hybridoma cell line PS-17 as described in claim 3.

5. Use of a monoclonal antibody as described in claim 3, characterized in that, Use the monoclonal antibody to prepare a reagent for detecting polystyrene.

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