Monoclonal antibodies

Transcription

1 Monoclonal antibodies <PREPARATION for FUSION> 1. Boost the best-responded mouse 3-5 days before fusion. 2. Build up frozen stock of myeloma cells (NS-1 or P 3 U 1 etc.) in MEM + 10% FCS containing 0.1 mm 8-azaguanine (which will kill aminopterin resistant mutants). Forty ml at 3x105 cells/ml should be set up a day before fusion so that cells will be in the log phase. 3. Prewarm all media and reagents in a 37 C water bath before starting fusion. <FUSION> 1. Anaesthetise the mouse with chloroform or ether and take blood from the heart for reference serum. 2. Remove spleen aseptically using sterile dissecting instruments. Pin out mouse and spray the abdomen with 70% ethanol. Make an incision into the left side of the abdomen and cut skin away. Care must be taken not to cut or rupture the spleen. 3. Carefully remove and place the spleen into a sterile petri dish containing 8 ml of MEM+g+g. 4. In a laminar flow hood, add 8 ml of MEM+g+g into each of 3 petri dishes and wash the spleen by transferring dish to dish and swirling gently. 5. Using a sterile needle (18 G or thicker) bent at an angle of 90 C and forceps, carefully tease out spleen cells. Holding one side of the spleen with forceps, make a few little scratch on the other side and start teasing out gently like stroking the surface. Syringe Needle

2 6. Pipette the cell suspension into a 50 ml sterile conical tube. Rinse the petri dish with 3-4 ml of MEM+g+g and transfer to the 50 ml tube. Pipette the cell suspension up and down a few times and allow large fragments to settle down. Suck up the suspension above the sedimented fragments and transfer into a 15 ml sterile conical tube. 7. Centrifuge at 1,000 rpm for 10 minutes at room temperature. 8. Remove the supernatant and resuspend the cells in sterile 2.5 ml of 0.85% NH 4 Cl to rupture RBC. Pipette the cells up and down a few times and leave for two minutes. Add 10 ml of MEM+g+g. 9. Centrifuge at 1,000 rpm for 10 minutes at room temperature to wash off NH 4 Cl. 10. During the centrifugation, combine myeloma cells in a 50 ml conical tube from culture bottles (if there are several) and count using haemocytometer by mixing 0.2 ml of myeloma cell suspension and equal volume of 0.4% aqueous trypan blue in a microcentrifuge tube. Dead myeloma cells take up dye and are stained blue. 11. Discard supernatant, resuspend spleen cells in 10 ml of MEM+10% FCS and count. There should be 108 spleen cells in 10 ml approximately. 12. Combine spleen and myeloma cells in a ratio of 10 spleen cells : 1 myeloma cell in a sterile 50 ml conical tube. 13. Centrifuge at 1,000 rpm for 10 minutes at room temperature. 14. Remove supernatant and resuspend in 10 ml of MEM+g+g. 15. Centrifuge at 1,000 rpm for 10 minutes at room temperature. 16. Discard the supernatant and tap the base of the conical tube to resuspend the cells in a small amount of remaining medium. 17. With a 1 ml pipette, add 0.3 ml of 50% PEG (M.W. 1450, Sigma) drop by drop, constantly stirring the cells with the tip of the pipette as each drop is added. When all the PEG has been added, pipette the cells up and down a few times. Make sure that these all have to be done within 60 seconds since PEG is toxic to the cells.

3 18. Gradually dilute the cells in 15 ml of MEM+g+g over a period of 90 seconds. Pipette the cells up and down in the first 5 ml of the medium and repeat when all the medium has been added. 19. Incubate the cell suspension for 10 minutes at room temperature to allow proper fusion to take place. 20. Centrifuge at 1,000 rpm for 10 minutes at room temperature. 21. Discard supernatant and resuspend the cell pellet in about ml of OPI-HAT medium per 108 spleen cells (the volume used should be aimed at getting one fused cell per one microtitre well). Using a multiple-channel pipette, distribute the cells into 96-well flatbottomed plates, 100 µl per well. Incubate in a CO 2 incubator. 22. Twenty four hours later, add 100 µl of OPI-HAT medium to each well and return the plates to the incubator. 23. After 6 days of undisturbed incubation, check clones, pull and feed with OPI-HAT. 24. At day 10, check clones and assay for monoclonal antibodies. The clones ready for assay should show good growth covering about 1/20 or more of the total base of a well. Always ensure that the medium in the well does not remain yellow for too long. If the medium turns yellowish, sample the supernatant for assay and add fresh OPI-HAT medium back into the well. 25. For assay, aseptically remove 100 µl of supernatant. Do not forget to add fresh OPI-HAT medium into the wells from which samples have been removed for assay. (Adapted from: Lane, R. D., J. Immunol. Methods, 81, 223.) <PROPAGATION> 1. After a positive well has been identified, the cells are transferred from the culture in the 96-well plate to 1 ml of medium supplemented with OPI-HT in a 24-well plate.

4 2. After the culture becomes dense, make a good cell suspension and use it as inoculum into 3 new wells supplemented with 1 ml of OPI-HT. Remember to add 1 ml of fresh medium into the original well. 3. When good growth is observed, transfer the cell culture from a single well to 5 ml of OPI-HT in a culture bottle. The cell suspensions of 2 wells can be combined in a bottle but not more than 2 wells. 4. When the culture becomes dense, freeze down the cells and carry out isotyping and further screening using the supernatant. NOTE: Often the transfer of hybridomas from one size of culture dish to the next is a difficult step to maintain cell viability. Presumably this is caused by the dilution of the growth factors in the medium and may be caused in part by overgrowth in the previous stage. If these problems exist, try using feeder cultures at these stages. <STORAGE> 1. Five ml of hybridoma culture medium from a culture bottle was centrifuged at 1,000 rpm for 10 minutes at room temperature. 2. Remove and keep the supernatant for further assays. Resuspend the cells in 0.5 ml of MEM+20% FCS (the total number of the cells should be approximately 5x106 to 5x107). 3. Add 0.5 ml of MEM+20% FCS+20% DMSO drop by drop and make a good suspension by pipetting the cells up and down a few times. Transfer it in a sterile cryotube. 4. Hold the suspension on ice for minutes and replace in a thick walled polystyrene box overnight at -70 C (frozen slowly at 1-2 C per minutes). Frozen cells were stored at -70 C or in liquid nitrogen. NOTE: Prolonged exposure to DMSO at these concentrations is toxic to cells, and trying to handle more than about vials at one time will lead to extended exposure to DMSO prior to freezing.

5 <RECOVERING CELLS from STORAGE> 1. Thaw the cells in warm water keeping the lip of the cryotube above the suface of the water to lessen the chances of contamination. Occasionally swirl the contents to hasten the thaw. 2. When the cells are almost thawed, transfer the contents to a 15 ml centrifuge tube containing 10 ml of OPI-HT. 3. Spin the cell suspension at 1,000 rpm for 10 minutes at room temperature. 4. Discard the supernatant and resuspend the cells with 5 ml of medium. Transfer the cells to a tissue culture bottle, and then to a CO 2 incubator. With good recoveries, subculture the cells within 24 hours of plating. <CLONING by LIMITING DILUTION> NOTE: When a hybridoma is assayed and found to be secreting the specific antibody, it is necessary to obtain pure clones to ensure that the secretors are selected and propagated. 1. Count the total number of viable cells with a haemocytometer. 2. Make dilutions in OPI-HT to obtain suspensions containing 1 cell/well (10 cells/ml), 1 cell/2 wells (5 cells/ml) and 1 cell/4 wells (2.5 cells/ml). Ten ml of each dilution is necessary. 3. Add 100 µl of each dilution into every well of a 96-well culture plate and incubate. 4. Twenty four hours later, add 100 µl of OPI-HT medium to each well and return the plates to the incubator. 5. Clones should be visible by microscopy after a few days and normally will be ready to screen after 7-10 days. Assay only wells with one clone and disregard wells containing multiple colonies. 6. Select the best wells and either grow up or repeat the cloning procedure with a single cell pick.

6 <COLLECTING ASCITES> 1. Prime adult Balb/c female mice (at least 6 weeks old) by injecting 0.5 ml of pristane (2,6,10,14-tetramethyldecanoic acid) or Freund's incomplete adjuvant into the peritoneum. 2. After 7-14 days, inject 5x105 to 5x106 hybridoma cells intraperitoneally. Prior to injection, the cells should be growing rapidly so start cell culture well before the inoculation. Centrifuge the cells and wash once in PBS. Inject the cells in no more than 0.5 ml of PBS. 3. Ascitic fluid may begin to build up within 1-2 weeks following the injection of the cells. Collect the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. Carefully tap as much fluid as possible with 18-gauge needle NOT attached to a syringe. Insert a needle into the lower part of belly and collect ascites flowing out of the needle in a centrifuge tube. 4. Return the mouse to its cage. Many mice will produce a second or third batch of ascites within 2-3 days. 5. Incubate the fluid in a 37 C water bath for 1 hour and transfer to 4 C overnight. 6. Spin the fluid at 3,000 g for 10 minutes. If there is an oil layer, remove this first and discard. Carefully remove the supernatant from the cell pellet. Spin again if necessary. NOTE: A single mouse may yield as much as 10 ml of ascitic fluid per batch. Antibody concentrations will typically be between 1 and 10 mg/ml. NOTE: Between 2 and 10% of the antibodies from ascites will be from the mouse's current antibody repertoire and not from the hybridoma. <ISOTYPING of IMMUNOGLOBULIN> Use commercial kit such as "Mouse Monoclonal Antibody Isotyping Kit (product code: MMT RC1)" from Serotec.