l a b o r a t o r y   r e s e a r c h   i n   i m m u n o l o g y   ::   v i r o l o g y   ::   h e m a t o l o g y
virus
bar
v i r a l   i m m u n i t y   &   p a t h o g e n e s i s   g r o u p

::  p r o t o c o l s  ::  m o n o c l o n a l  a n t i b o d y  p r o d u c t i o n.  f u s i o n  ::


ƒ




OUTLINE
Fusion is an important procedure to produce monoclonal antibodies (MoAb).

PROTOCOL
  1. prepare the culture of myeloma cells and maintain the concentration of the cells at the level of less than 10^6 cells/ml (IMDM + 10% FCS/FBS). Just before the fusion wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ƒC. Count cells.
  2. sacrifice the mouse and dissect the spleen.
  3. tease the spleen in ice-cold IMDM (serum free!); pass through the nylon  filter; wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ƒC. Count cells.
  4. mix 10^8 spleen cells (in 25 ml of IMDM [serum free]) with 2x10^7 myeloma cells (in 25 ml of IMDM [serum free]) in Falcon-50 ml conical tube.
  5. centrifuge at 1200 rpm, 5 min, 37ƒC.
  6. aspirate the supernatant (SN) with a Pasteur pipette (all till the last drop!!!)
  7. gently break the pellet by tapping of the bottom of the tube.
  8. place the tube on 37ƒC water bath; add slowly, dop by drop 1 ml of pre-warmed (37ƒC) 50% PEG 1500 (polyethylene glycol, Roche, Germany) continously stirring the cells with the pipette tip (this procedure lasts for a period over 1 min)
  9. add 1 ml of pre-warmed to 37ƒC IMDM continously stirring the mixture (this procedure lasts for a period over 1 min).
  10. add 3 ml of pre-warmed to 37ƒC IMDM continously stirring the mixture (this procedure lasts for a period over 3 min).
  11. add 10 ml of pre-warmed to 37ƒC IMDM continously stirring the mixture (this procedure lasts for a period over 1-2 min).
  12. let to stay for 5 min on the 37ƒC water bath.
  13. centrifuge at 1200 rpm, 5 min, 37ƒC; aspirate the SN.
  14. resuspend the cells in IMDM-FCS(10%v/v) - HTx100 (1%v/v) - AJ(5%v/v) - IL-6, 0.5x10^6 cells/ml (calculation figured out from the number of spleen cells the fusion was started from); let to stay in incubator, 37ƒC, pCO2 7% for 2 hours in a big flasc.
  15. add aminopterin; distribute on 96-wells cell culture plates (Nunc) (200µl/well); place in incubator (37ƒC, pCO2 7%) enveloped in aluminium paper.
  16. check the cultures 1-2-7 days after the fusion.
  17. on day 7-10 change the culture medium.
  18. test clones when thay occupy 25% of the bottom surface of the well.
SOLUTIONS
  1. IMDM (Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F
  2. IMDM-FCS(10%v/v)-HTx100(1%v/v)-AJ(5%v/v)-IL-6 (100 U/ml)
  3. IMDM - FCS(10%v/v) - HTx100(1%v/v) - AJ(5%v/v) - IL 6 (100 U/ml) - aminopterine x100 (1% v/v)
  4. HT (hypoxantine-thymidine)x100, 0.22 (or 0.45) µm-filtered, store at -20ƒC = hypoxantine solution, 50 ml + thymidine solution, 50 ml + ddH2O up to 250 ml
  5. hypoxantine solution = hypoxantine, 340 mg + ddH2O up to 50 ml + NaOH 1 N, 3 ml -> heat to 37ƒC
  6. thymidine solution = thymidine (2'-desoxy-thymidine), 96 mg + ddH2O up to 50 ml
  7. AJ (from "ajouter" , to add, local slang), 3 L, 0.22 (or 0.45) µm-filtered, store at -20ƒC =  Asp-Arg x100, 1 L + Gluta x 50, 2 L + mercaptoethanol, 350 µl
  8. Asp-Arg x100, 1 L, pH 6.0 = L-Asparagine, 3.6 g (0.24 mM) + L-Arginine (0.55 mM), 11.6 g + HCl, 0.5N, 100 ml -> heat to 37ƒC, add ddH2O up to 1 L
  9. Gluta x50, 1 L, pH 6.0 = glutamine, 10.8 g (1.48 mM) + HCl, 0.5N, 100 ml -> heat to 37ƒC, add ddH2O up to 1 L
  10. aminopterine x100, 0.22-0.45 µm-filtered, store at -20ƒC = aminopterine, 4.4 mg (3.8µM)+ ddH2O, 50 ml + NaOH, 1N, 100 µl-> heat to 37ƒC, add ddH2O up to 250 ml
ADDITIONAL INFO
[  read more  ] about fusion and PEG 1500

SCHEME
fusion
[  enlarge  ]

REFERENCES

Lane, R.D., Cristmann, R.S. and Lachmann, M.F. (1984) J.Immunol. Methods 72: 71-76
created by Andrei Musaji          ::          updated : 2004