Hybridoma cell strain capable of secreting anti-micro-nano plastic broad-spectrum monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain secreting an anti-micro-nano plastic monoclonal antibody and application thereof.The hybridoma cell strain comprises a hybridoma cell strain MP-4 and a hybridoma cell strain MP-9, the hybridoma cell strain MP-4 and the hybridoma cell strain MP-9 are obtained through self screening of the inventor, the preservation number of the hybridoma cell strain MP-4 is CCTCC NO: C2024262, the preservation number of the hybridoma cell strain MP-9 is CCTCC NO: C2024262, and the preservation number of the hybridoma cell strain MP-9 is CCTCC NO: C2024262. The preservation number of the hybridoma cell strain MP-9 is CCTCC NO: C2024261. The passage cell strain of the hybridoma cell provided by the invention can stably secrete an anti-broad-spectrum micro-nano plastic protein monoclonal antibody, the secreted antibody has high titer and good sensitivity, can simultaneously recognize PS, PVC, PP, PE and PET, has good broad-spectrum recognizability, provides a core raw material for establishing a broad-spectrum immunodetection method of micro-plastics, and has good application prospects. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a hybridoma cell line secreting monoclonal antibodies against micro / nano plastics and its applications. Background Art

[0002] Microplastics have a wide variety, great harm, heavy pollution, and a wide range. 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 detections of microplastics mostly rely 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 differences and many pollution links, covering fields such as the environment, food, and textiles. During the production process of synthetic fibers (such as polyester fibers and nylon), 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. Additionally, 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 technologies 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 the 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 number 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 materials, such as nylon and polyester, are more likely to generate microplastic fibers during the washing process due to their fiber structures 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 ashes containing microplastics may be generated during the incineration process. If these soot and ashes are not properly treated, they will also be released into the environment, causing microplastic pollution.

[0004] Currently, the detection methods for microplastics mainly include: visual method, microscopy, electron microscopy, Raman spectroscopy analysis, infrared spectroscopy, thermal analysis, pyrolysis-gas chromatography / mass spectrometry, laser infrared imaging, and photoacoustic 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. It is 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. There are a wide variety of microplastics with different properties, and it is challenging to develop a broad-spectrum antibody that can recognize multiple different types of microplastics. Therefore, a hybridoma cell line secreting monoclonal antibodies against micro / nano plastics and its application are urgently needed to be proposed. Summary of the Invention

[0005] To solve the defects of the existing technology, the present invention provides a hybridoma cell line secreting monoclonal antibodies against micro / nano plastics and its application.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against micro / nano plastics, including the hybridoma cell line MP-4, which is deposited in the China Center for Type Culture Collection. The deposit number of the hybridoma cell line MP-4 is CCTCC NO: C2024262.

[0008] The second object of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against micro / nano plastics, including the hybridoma cell line MP-9, which is deposited in the China Center for Type Culture Collection. The deposit number of the hybridoma cell line MP-9 is CCTCC NO: C2024261.

[0009] The third object of the present invention is to provide a monoclonal antibody, which is secreted by the hybridoma cell line MP-4 or the hybridoma cell line MP-9, and the monoclonal antibody can recognize micro / nano plastics in a broad spectrum.

[0010] Preferably, the micro / nano plastics include at least one of polystyrene, polyvinyl chloride, PET, PE, and PP.

[0011] The fourth object of the present invention is to provide a kit, which contains the monoclonal antibody secreted by the hybridoma cell line MP-4 or the hybridoma cell line MP-9.

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

[0013] Compared with the existing technology, the present invention has the following beneficial effects:

[0014] The subculture cell line of the hybridoma cells provided by the present invention can stably secrete monoclonal antibodies against broad-spectrum micro-nano plastic proteins, with high antibody titer and good specificity, and can be applied to kit detection, significantly improving the detection sensitivity of the kit.

[0015] The present invention can more accurately detect trace amounts of microplastics in the environment. Whether in water, soil or air, even when the concentration of microplastics is very low, the broad-spectrum antibody can effectively recognize and bind to microplastics, providing more accurate detection results for researchers.

[0016] The present invention can detect various different types of microplastics. Due to the wide variety of microplastics, with different shapes, sizes and chemical compositions, the broad-spectrum antibody can recognize microplastics made of different materials, such as polyethylene, polypropylene, etc., greatly expanding the detection coverage.

[0017] Cell preservation:

[0018] The present invention provides a hybridoma cell line secreting monoclonal antibodies against micro-nano plastics, including hybridoma cell line MP-4 and hybridoma cell line MP-9. The hybridoma cell line MP-4 and hybridoma cell line MP-9 were self-screened by the inventors of the present invention. The preservation number of the hybridoma cell line MP-4 is CCTCC NO: C2024262, and the preservation number of the hybridoma cell line MP-9 is CCTCC NO: C2024261. The preservation date is August 14, 2024, and the preservation unit is the China Center for Type Culture Collection, and the address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Brief description of the drawings

[0019] Figure 1 It is a SDS-PAGE electrophoresis schematic diagram of PS-BSA and PS-KLH in Example 1 of the present invention;

[0020] Figure 2 It is the acquisition process of the antibody variable region in Example 5 of the present invention;

[0021] Figure 3 It is the detection result in Example 6 of the present invention. Detailed implementation manners

[0022] The following describes the preferred embodiments of the present invention with reference to 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.

[0023] Example 1: Coupling of microplastics (PS-CHOOH) with conjugated proteins (BSA / KLH).

[0024] 1. Microplastics, coupling buffer (polylink coupling buffer), coupling carbodiimide (polylink EDAC), and coupling wash / storage solution (polylink wash / storage buffer) are cooled to room temperature.

[0025] 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 to resuspend, centrifuge again, and discard the supernatant; add 0.17 mL of coupling buffer again to resuspend.

[0026] 3. Dissolve 10 mg of polylink EDAC in 50 μL of coupling buffer to prepare a 200 mg / mL EDAC solution, noting that it should be prepared freshly before use.

[0027] 4. Add 20 μL of the EDAC solution to the 0.17 mL coupling buffer suspension, fix it on a rotator, and mix evenly at room temperature for 15 min for activation.

[0028] 5. Add 200 - 500 μg of coupling protein (BSA / KLH), fix it on a rotator, and mix evenly at room temperature for 30 - 60 min (dissolve the protein in coupling buffer to 1 - 5 mg / ml).

[0029] 6. Centrifuge at 500 - 1000 g for 10 min, aspirate the supernatant to detect the amount of coupling protein (BSA / KLH).

[0030] 7. Add 0.4 mL of coupling wash / storage solution (polylink wash / storage buffer) to resuspend.

[0031] 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.

[0032] 9. Perform SDS - PAGE analysis (4% stacking gel and 7.5% separating gel). The results of reduced SDS - PAGE detection are as Figure 1 shown, proving the successful coupling of PS - KLH and PS - BSA.

[0033] Example 2: Preparation of monoclonal antibodies.

[0034] 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. The cDNA was amplified again to obtain specific gene fragments, which were 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 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.

[0035] 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.

[0036] Table 1: Immunization protocol for mice.

[0037]

[0038]

[0039] Example 3: Subtype analysis and titer detection of monoclonal antibody MP-4.

[0040] Subtype analysis of monoclonal antibody: For the antibody produced by 1 strain of hybridoma cells secreting specific monoclonal antibody, the MP-4 antibody subtype was identified, and the result showed that MP-4 was IgG2b.

[0041] Titer detection of monoclonal antibody: 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 MP-4 monoclonal antibody (the results are shown in Table 2).

[0042] Table 2: Titer detection of the tail blood of PS-BSA immunized mice for five times.

[0043]

[0044] Coat the plate with 1 μg / ml of PS-KLH, serially dilute each antibody (1:100, 1:500, 1:2500, 1:12500, 1:32500, 1:612500), add 10,000-fold diluted goat anti-rabbit IgG-HRP, and determine the titer of the purified MP-4 monoclonal antibody (the results are shown in Table 3).

[0045] Table 3: Detection of the titer of PS rabbit fifth-immunized serum

[0046]

[0047]

[0048] Example 4: Subtype analysis and titer detection of monoclonal antibody MP-9

[0049] Subtype analysis of monoclonal antibody: Identify the antibody subtype of MP-9, which is the antibody produced by 1 hybridoma cell secreting specific monoclonal antibody. The result shows that MP-9 is IgG2b

[0050] Titer detection of monoclonal antibody: Coat the plate with 1 μg / ml of PS-KLH, serially dilute each antibody (1:100, 1:500, 1:2500, 1:12500, 1:32500, 1:612500), add 10,000-fold diluted goat anti-mouse IgG-HRP, and determine the titer of the purified MP-9 monoclonal antibody (the results are shown in Table 4).

[0051] Table 4: Detection of the titer of PS-BSA mouse fifth-immunized tail blood

[0052]

[0053] Coat the plate with 1 μg / ml of PS-KLH, serially dilute each antibody (1:100, 1:500, 1:2500, 1:12500, 1:32500, 1:612500), add 10,000-fold diluted goat anti-rabbit IgG-HRP, and determine the titer of the purified MP-9 monoclonal antibody (the results are shown in Table 5).

[0054] Table 5: Detection of the titer of PS rabbit fifth-immunized serum

[0055]

[0056] Example 5: Sequencing of monoclonal antibodies MP-4 and MP-9 (obtained according to the Figure 2 procedure for obtaining the variable region of the antibody).

[0057] (1) The monoclonal antibody secreted by the hybridoma cell line MP-4 contains a heavy chain variable region and a light chain variable region. 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.

[0058] The amino acid sequence of CDR1 of the heavy chain variable region is shown as positions 50-54 of SEQ ID No.1.

[0059] The amino acid sequence of CDR2 of the heavy chain variable region is shown as positions 69-85 of SEQ ID No.1.

[0060] The amino acid sequence of CDR3 of the heavy chain variable region is shown as positions 118-125 of SEQ ID No.1.

[0061] The amino acid sequence of CDR1 of the light chain variable region is shown as positions 46-55 of SEQ ID No.2.

[0062] The amino acid sequence of CDR2 of the light chain variable region is shown as positions 71-77 of SEQ ID No.2.

[0063] The amino acid sequence of CDR3 of the light chain variable region is shown as positions 110-118 of SEQ ID No.2.

[0064] The amino acid sequence of the heavy chain variable region of the MP-4 antibody is as shown in SEQ ID No.1:

[0065] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTDYYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQLSSL TSEDSAVYFCAREDYGTPDYWGQGTTLTVSS.

[0066] The amino acid sequence of the light chain variable region of the MP-4 antibody is as shown in SEQ ID No.2:

[0067] MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRTFGGGTKLEIK.

[0068] (2) The monoclonal antibody secreted by the hybridoma cell line MP-9 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;

[0069] The amino acid sequence of CDR1 of the heavy chain variable region is shown as positions 50-54 of SEQ ID No.3;

[0070] The amino acid sequence of CDR2 of the heavy chain variable region is shown as positions 69-84 of SEQ ID No.3;

[0071] The amino acid sequence of CDR3 of the heavy chain variable region is shown as positions 117-128 of SEQ ID No.3;

[0072] The amino acid sequence of CDR1 of the light chain variable region is shown as positions 44-54 of SEQ ID No.4;

[0073] The amino acid sequence of CDR2 of the light chain variable region is shown as positions 70-76 of SEQ ID No.4;

[0074] The amino acid sequence of CDR3 of the light chain variable region is shown as positions 109-117 of SEQ ID No.4.

[0075] The amino acid sequence of the heavy chain variable region of the MP-9 antibody is shown as SEQ ID No.3:

[0076] MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTSY GVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNS LQANDTAIYYCASYYYGSSWYAMDYWGQGTSVTVSS.

[0077] The amino acid sequence of the light chain variable region of the MP-9 antibody is shown as SEQ ID No.4:

[0078] MNMLTQLLGLLLLWFAGGKCDIQMTQSPASQSASLGESVTITCLASQTIG TWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDF VSYYCQQLYSTPLTFGAGTKLELK.

[0079] Example 6: Monoclonal antibodies MP-4 and MP-9 are used for NC membrane detection.

[0080] 1. Take 30 μL of PS-OVA, PVC-OVA, PP-OVA, PE-OVA, and PET-OVA, and use a scribing instrument to scribe lines on the NC membrane at a spraying volume of 0.08 μL / cm. Take 30 μL of 1 mg / mL p16 protein as a control and scribe lines on another NC membrane. Among them, the P16 protein is a cyclin-dependent kinase inhibitor. Place the NC membrane in a humidity below 15% and dry it overnight at 50 °C.

[0081] 2. Dilute the monoclonal antibodies MP-4 and MP-9 to 1 mg / mL with 20% NBS-PBS respectively, and take 2 mL of the antibody solution in a 5 mL centrifuge tube; dilute the control monoclonal antibody p16-25 to 1 mg / mL with 20% NBS-PBS, and take 2 mL of the antibody solution in a 5 mL centrifuge tube.

[0082] 3. Cut the NC membranes coated with PS-OVA, PVC-OVA, PP-OVA, PE-OVA, and PET-OVA into small segments of the same width and place them into the MP-4 antibody solution and MP-9 antibody solution respectively; cut the NC membrane coated with p16 into small segments of the same width and place them in the p16-25 antibody solution, and cut another small segment and place it in the PS-1 antibody solution as a negative control.

[0083] 4. After incubating at 37 °C for 1 h, rinse the NC membrane once with PBST.

[0084] 5. Dilute the goat anti-mouse IgG-HRP 10-fold with 20% NBS-PBS, take 10 mL in a centrifuge tube, and place the NC membrane in the centrifuge tube as well. Incubate at 37 °C for 30 min.

[0085] 6. Wash the NC membrane 4 - 5 times, blot dry on a paper towel, immerse the entire NC membrane in precipitating TMB for 30 s and then take it out, blot dry on a paper towel, and let it develop color at room temperature. Take pictures of the results the next day ( Figure 3 ).

[0086] Example 7: Application of monoclonal antibodies.

[0087] Collect water samples and soil samples from marine, river, and lake waters. Collect textile wastewater samples from textile enterprises. The collected samples are quantitatively detected by a kit after pretreatment and extraction.

[0088] Perform pretreatment according to the following steps:

[0089] (1) For liquid samples such as water samples, centrifuge each sample at 17000 × g for 3 minutes, then digest and extract the supernatant, and dilute the digestion and extraction solution with the sample diluent for quantitative immunochromatographic detection using a test strip.

[0090] (2) For non-liquid samples, the samples need to be digested, microplastics extracted, and diluted with diluent before immunochromatographic quantitative detection.

[0091] Taking soil samples (spiked detection) as an example:

[0092] Add deionized water to the soil sample (1:1 w / v), and then vortex for 10 s. Centrifuge the slurry mixture at 17000×g for 3 minutes, and use the supernatant as the specific matrix extract for spiked recovery analysis. The sample detection is carried out according to the following steps:

[0093] 1. Add 50 μL of sample pretreatment extract (set parallel replicate samples during sample testing) to a sterile glass tube. At the same time, add different volumes of microplastic working solution to 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, thus establishing a calibration curve for sample detection.

[0094] 2. Then add 950 μL of PBST washing buffer to each test tube.

[0095] 3. Place the test tubes on a rotator and rotate at a speed of 20 revolutions per minute for 3 minutes.

[0096] 4. Centrifuge the test tubes at 5500 rpm for 3 minutes to precipitate the microplastic particles. Carefully remove and discard the supernatant.

[0097] 5. Add 1,000 μL of antibody diluent (diluted 32500 times) sample to each test tube. Briefly vortex the test tubes or move them up and down with a pipette several times to ensure complete mixing of the microplastics and the antibody solution.

[0098] 6. Rotate the test tubes at a speed of 20 revolutions per minute at room temperature for 1 hour.

[0099] 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 a speed of 20 rpm for 3 minutes.

[0100] 8. Repeat step 7 three times.

[0101] 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.

[0102] 10. Add 1,000 μL of goat anti-rabbit antibody labeled with horseradish peroxidase to each test tube. This antibody has been diluted 2,000 times with PBST solution. Vortex the test tube for 1 min to ensure complete mixing of the microplastics with the secondary antibody solution.

[0103] 11. Place the test tube on a vortexer and rotate it at 20 rpm for 1 hour at room temperature.

[0104] 12. Wash the microplastics four times with the washing buffer (repeat steps 8 - 10).

[0105] 13. Add 100 μL of TMB substrate, pipette up and down several times, and then immediately transfer the suspension to the wells of a microplate.

[0106] 14. Place the microplate on a slow shaker at room temperature for 30 minutes.

[0107] 15. Add 100 μL of 1 M H2SO4 to each well to terminate the reaction.

[0108] 16. Measure the OD value using an ELISA reader at 450 nm; use Origin to plot a standard curve, with the concentration of each standard as the abscissa and B / B0 as the ordinate to plot the standard curve.

[0109] 17. Substitute the OD value of the test sample into the standard curve to calculate the amount of microplastics in the sample.

[0110] 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 a broad-spectrum monoclonal antibody against micro- and nano-plastics, characterized in that, It includes the hybridoma cell line MP-4, which is deposited with the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line MP-4 is CCTCC NO: C2024262.

2. A hybridoma cell line secreting a broad-spectrum monoclonal antibody against micro- and nano-plastics, characterized in that, It includes the hybridoma cell line MP-9, which is deposited with the China Center for Type Culture Collection, and the deposit number of the hybridoma cell line MP-9 is CCTCC NO: C2024261.

3. A monoclonal antibody, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line MP-4 described in claim 1 or the hybridoma cell line MP-9 described in claim 2, and the monoclonal antibody can recognize micro-nano plastics in a broad spectrum.

4. The monoclonal antibody according to claim 3, wherein The micro-nano plastics include at least one of polystyrene, polyvinyl chloride, PET, PE, and PP.

5. A kit, characterized in that, The kit contains the monoclonal antibody secreted by the hybridoma cell line MP-4 or the hybridoma cell line MP-9 described in claim 3.

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

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