[0068] Example 3 Preparation and identification of monoclonal antibodies
[0069] 1. Immunization of Balb/c mice with a derivative of procalcitonin B cell epitope peptide
[0070] The procalcitonin B cell epitope peptide antigen obtained in Example 2 was taken out from the refrigerator at -80°C as an antigen, dissolved and filtered.
[0071] 6-week-old female Balb/c mice weighing about 20g were selected for immunization. Antigen emulsification uses the double syringe mutual push method. For the first immunization, the procalcitonin B cell epitope peptide antigen shown in SEQ ID NO: 4 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain an antigen mixture. Each mouse was skinned in an amount of 100 μg. Add intraperitoneal injection at more points. The second and third immunizations were performed on the 14th and 28th days. The adjuvant was changed to incomplete Freund's adjuvant. The antigen amount, injection volume and route were unchanged. After the third immunization, the titer was determined by indirect ELISA. Three days before fusion, a booster immunization was performed. Each mouse was intraperitoneally injected with 100 μg PCT without adjuvant, and the cells were fused three days later.
[0072] 2. Determination of serum titer of immunized Balb/c mice
[0073] 10 days after the third immunization, blood was taken from the tail vein of the mouse to detect the serum antibody titer. Soak the newly purchased ELISA plate in double distilled water overnight, dry it for later use; use the coating solution (0.05mol/L sodium carbonate buffer: 0.16g Na 2 CO 3 , 0.293g NaHCO 3 , 0.02g NaN 3 , Add deionized water to dissolve 100ml) Dilute the PCT recombinant protein antigen obtained in Example 1 to the best working concentration of 5μg/ml, add 100μl of antigen diluent to each well, incubate at 37°C for 1 hour, seal with tape, Overnight at 4℃, pour out the liquid in the wells, absorb the remaining reaction liquid in the wells, fill up the washing liquid once, then fill the washing liquid slowly and shake for 2min, decanted, repeat five times, and finally put the reaction plate upside down to absorb water On the paper, make the washing liquid drain out of the holes. After natural drying, it is sealed with tape. This is an ELISA plate coated with PCT recombinant protein antigen, add 300μl blocking solution, incubate at 37°C for 1.5 hours, wash 5 times; blood collection and diluted serum: pinch the tail of the rat and disinfect with 75% alcohol Cut a gap in the tail vein with scissors, take 20μl of blood, centrifuge at 2000rpm for 30min, take 1μl of the supernatant and add 999μl of antibody diluent to mix, and perform volume-fold dilution from 1:100 to 1:3200, and the diluted test Add 100μl of serum to each well, and at the same time take mouse pre-immune serum diluted 1:100 as negative control, and antibody dilution as blank control. Incubate at 37°C for 1 to 1.5 hours, wash 5 times; Dilute horseradish peroxidase goat anti-mouse IgG (Shanghai Yixin Biotechnology Co., Ltd.) to 1:10000, add 100μl to each well, incubate at 37°C for 1.5 hours, Wash 5 times; add 100μl of o-phenylenediamine solution per well, 15 minutes in the dark at room temperature, add 100μl of stop solution to each well. Observe the result. The product after OPD oxidation is orange-red. Use an enzyme-linked immunoassay to record the 492nm reading. After the control hole is adjusted to zero, the OD value of each hole is measured to be greater than 2.1 times of the negative control OD value, that is, it is positive. Serum titer reaches 1:3200 and can be used for cell fusion.
[0074] 3. Preparation of mouse spleen cell suspension and SP2/0 cell suspension
[0075] Take the immunized Balb/c mice, remove the eyeballs of the mice and let them be put to death. After centrifugation of the eye blood, the serum is collected as a positive control for ELISA. The spleen is taken out aseptically and placed in a glass dish containing 10ml of incomplete medium, and washed , Carefully peel off the surrounding connective tissue and adipose tissue, change a glass dish, remove the spleen, place it in a 200-mesh stainless steel net, grind it with the inner core of a syringe, and rinse with incomplete medium from time to time to allow the spleen cells to pass through When the mesh enters the solution, the spleen cells are transferred to a 10ml glass centrifuge tube, centrifuged horizontally at 800rpm for 10min, and the supernatant is removed. In the same way, wash the cells once with 10ml of incomplete medium, collect the precipitated cells by centrifugation, resuspend the cells in 10ml of incomplete medium and mix, the cell count is about 1×10 8 Cells.
[0076] Remove the SP2/0 cells from the liquid nitrogen, quickly put them in a 37°C water bath, and shake them until the cell solution is completely dissolved. Transfer the cells to a 10ml centrifuge tube and centrifuge horizontally at 800rpm for 10min. Discard the supernatant and 10ml complete culture medium. Resuspend the pellet, transfer the cell suspension to a 50ml culture flask and place it in a 37°C, 5% CO2 incubator. After the cells have grown well, select the cells with 8-AG-containing selective medium for a week; 2 days before fusion, transfer 1 flask of cells to 4 flasks, then the cells are in the logarithmic growth phase on the day of fusion, the viability is just right, and the cell size is uniform. Round and translucent, on the day of fusion, use an elbow dropper to gently blow down the SP2/0 cells from the tube wall, collect them in a centrifuge tube, centrifuge, discard the supernatant, and wash the pellet with incomplete medium, 10ml is incomplete Resuspend the medium and count the cells, about 5×10 7.
[0077] Four preparation of trophoblast
[0078] Take an unimmunized Balb/c mouse, remove the eyeballs and let them bloodlett, put them to death, soak in 75% ethanol for 5 minutes, cut the skin of the mouse, lift the peritoneum with tweezers, cut a small mouth with scissors, and take the pre-cooled dropper with an elbow dropper. Flush the abdominal cavity with complete medium, and aspirate the lotion into a 50ml centrifuge tube. In the same way, the abdominal cavity was washed with incomplete medium for 3 times, the washing liquid was collected, and the supernatant was removed by horizontal centrifugation at 1000 rpm at room temperature for 10 minutes. The cells were resuspended in 10 ml of incomplete medium and counted.
[0079] Five fusion of myeloma cells and spleen B lymphocytes
[0080] Before fusion, place PEG1500 in a 37℃ incubator to pre-warm, and draw 1×10 7 Myeloma cell suspension and 1×10 8 Put a spleen B lymphocyte suspension (cell number 1:10) into a 50ml centrifuge tube, add 30ml incomplete medium, mix well, centrifuge at 1000rpm for 10min, discard the supernatant, flick the bottom of the tube to loosen the cell mass Make a paste, put the centrifuge tube in a 37°C water bath, use a dropper to draw 0.8ml of pre-warmed 50% PEG1500 solution, slowly add cells to the cells along the tube wall about 2cm from the bottom of the tube, and rotate the centrifuge tube while adding it for 1 min After adding left and right, then let stand for 90s, add 30ml of pre-warmed incomplete medium at 37℃ drop by drop to stop the fusion, add it within 3min, the speed will be slow first and then fast, the action will be gentle, put the centrifuge tube in the 37℃ incubator Set aside for 5 minutes, take out the centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 10ml HAT medium to resuspend the cells, gently pipette, mix, inoculate the fused cells into a 96-well cell culture plate with trophoblasts, press 100μl/well, each culture plate leaves 6 wells to inoculate SP2/0 cells as a negative control for HAT selection, set at 37℃, 5% CO 2 Cultivate in an incubator.
[0081] 6. Selective cultivation of fusion cells and screening of hybridoma cells
[0082] On the 5th day after the fusion, the growth of the cells can be observed under an inverted microscope and supplemented with 100μl of HAT medium, and cultured with HT medium on the 14th day. 10 to 14 days after fusion, when the cells grow to the bottom of 1/2 of the full culture well, the culture supernatant is detected by indirect ELISA to screen positive clones; the purified PCT recombinant protein prepared in Example 1 is coated with an enzyme label Plate (0.5μg/well), overnight at 4℃, wash 5 times with washing buffer, 5min each time, pat dry the liquid, add 300μl blocking solution to each well, incubate at 37℃ for 2h, add 100μl cell culture supernatant, select a small positive control Mouse immune serum, select SP2/0 culture supernatant for negative control, wash solution for blank control, incubate at 37°C for 2h; wash the plate: add 100μl 1:10000 diluted HRP-labeled goat anti-mouse IG antibody to each well, Incubate at 37°C for 2h; wash, pat dry the liquid, add 100μl of freshly prepared o-phenylenediamine solution per well, react in the dark at room temperature for 10-15 minutes, add 100μl of stop solution per well to stop the reaction, and detect the absorbance at 450nm with a microplate reader. The result was that BALB/C mice were immunized with PCT epitope peptides as antigens. After successful fusion, cloned and ELISA screened, hybridoma cell lines secreting PCT monoclonal antibodies were obtained, and the cell culture supernatant titer reached 1:6400. This hybridoma cell has been cryopreserved several times and can still secrete antibodies stably after subculturing in vitro for more than 3 months.
[0083]
[0084] Seven cloning of positive hybridoma cells
[0085] After screening the positive clones, the positive hybridoma cells were cloned and cultured immediately by the limiting dilution method, feeder cells were prepared, resuspended in 10ml incomplete medium, and positive clones were collected and counted, and the positive clones were counted with incomplete medium Dilute to 100 cells/20ml, take a 96-well cell culture plate pre-filled with trophoblasts, add 200μl cell suspension, transfer the remaining positive cloned cells to a 24-well plate for expansion, collect the cells and cryopreserve in liquid nitrogen , And put the culture plate at 37℃, 5% CO 2 Incubate in an incubator, observe the cell growth under the microscope after 3 days, detect the titer by ELISA after 10 days, and clone the strongest positive clone again until the positive rate of the cells reaches 100%, then the strain can be determined; After the titer of the culture supernatant of the hybridoma cell line of the strain, the hybridoma cell of the fixed strain is expanded and cultured and sent to the China Type Culture Collection (Wuhan University Collection Center) for preservation. The preservation number is CCTCC NO: C201137. Stable secretion of the monoclonal antibody.
[0086] 8. Preparation of mouse ascites, purification of antibody and determination of titer
[0087] Choose 10 healthy female Balb/c mice, intraperitoneally inject 0.5ml of sterilized paraffin oil/mouse, 1-2 weeks later, each mouse intraperitoneally inject 0.5×10 6 ~1×10 6 Hybridoma cells were injected with 0.25 ml of a mixture of liquid paraffin and incomplete Freund’s adjuvant at the same time. After the mice had obvious ascites, they were killed by pulling their necks. The ascites were taken out from the abdominal cavity with a straw, centrifuged at 4°C for 15 minutes, and the clear ascites fluid in the middle section was separated and collected. Use HiTrap rProtein A HP column to access AKTA Explorer purified antibody, see figure 1 , The purity is greater than 95% by SDS-PAGE. The titer of the purified antibody detected by the indirect ELISA method reached 1:256000, which preliminarily shows that the obtained monoclonal antibody has a high binding ability to the PCT molecule, and it is packed and cryopreserved for use in Example 5.
[0088] Nine Anti-PCT monoclonal antibody affinity determination
[0089] In order to test the binding ability of anti-PCT monoclonal antibodies to the PCT antigen, the affinity constant (Kd) detection method of monoclonal antibodies based on the principle of antigen/antibody competitive binding was used to determine the affinity of the obtained monoclonal antibodies. Dissolve the purified PCT antigen in 0.05mol/l carbonic acid buffer (pH9.5), adjust the final concentration of PCT to 1μg/ml, add 100μl to each well of the ELISA plate, and seal the strip with tape at 4℃ overnight . The liquid in the wells was patted dry the next day, and each well was sealed with 1% BSA in PBS solution for 2 hours, washed and dried, and stored at 4°C for later use. The antigen-antibody reaction system was established according to the measurement principle and method. The initial reaction concentration of anti-PCT monoclonal antibody was diluted to 40ng/ml, and the initial concentration of PCT antigen was diluted to 360mg/ml. The antigen concentration is diluted by 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.235 (unit is 10 -12 mol/l) to calculate the affinity constant of PCT monoclonal antibody. The results show that it has high affinity, Kd=5.3×10 -8 mol/L.
[0090] For the experimental procedures of this embodiment, please refer to "Modern Immunological Experimental Technology" (edited by Shen Yan and Zhou Rulin).
