Note: We highly recommend that the following protocol be followed carefully. Several laboratories have experienced difficulty in developing hybrids with the 240E1 fusion partner, if the following protocol is not carefully followed.
Preparation of the fusion partner 240E-1e The rabbit plasmacytoma cell line is grown in an enriched RPMI 1640. We use RPMI Medium powder: one bag is for one liter; it contains L-glutamine but no bicarbonate.
We prepare the medium with various additions in the following way:
|RPMI 1640 GIBCO 31800||
powder for 1 L
L-glutamine GIBCO 25030, 200 mM ( 100x)
|We also add gentamicin, penicillin, streptomycin and fungizone, all purchased from GIBCO.
Now we fill up to 1L and filter sterilize. At this stage the medium can be kept frozen, but, after thawing it has to be mixed well. Fetal Calf Serum, 15%, is added when we use the medium. A final pH adjustment, if it is necessary, can be done with 1N HCl or 1N NaOH. (If the pH is not on the dot it is better to use medium that is a little more acidic. Do not use medium that is too alkaline).
We culture the cells at a concentration of 0.2 - 0.6 x 106/ml. The doubling time is approximately 36-48 h. For 4-6 days before a fusion the cells are fed daily, either by adding fresh medium or by dilution. It is important that cells are in log phase growth at the time of fusion. When cells that were frozen in liquid nitrogen are put back into culture we grow them in the presence of 8-azaguanine (20µg/ml) for several days. Three days before fusion the cells are pelleted and resuspended in medium without 8-azaguanine. In general we do not use cells for fusion that are in culture longer than 4 weeks.
We have used only New Zealand White rabbits, we have no experience with other strains. For the primary immunization, when we plan to use spleen cells for a fusion, rabbits are injected with a total of 2 mg of protein or
2 x 107 cells in complete Freund's adjuvant subcutaneously, intramascularly, and intraperitoneal. The animals are boosted once or twice at 2 - 3 week intervals in the same manner with incomplete Freund's adjuvant. The final boost is given intraperitoneally and intravenously with saline 4 days before the fusion. We find that we can increase the fusion efficiency if we incubate the isolated lymphocytes for 24 - 48 h on plastic dishes or in flasks with the antigen (about 50 µg/ml) for in vitro boosting. It is possible to increase the fusion efficiency by the addition of CD40L-transfected cells ( x-irradiated) during this step of preincubation. We use 1/10 of the number of spleen cells.
A word of caution: When spleen cells are used for the fusion we frequently encounter excessive growth of adherent cells in many wells which prevent the hybridomas from establishing themselves. The growth of these adherent cells can be minimized if the lymphocytes are very gently teased out of the spleen. We have been most successful if we "balloon" the spleen by injecting medium or HBSS through a fine needle and then squeeze the cells out carefully. Do not rub the tissue between slides and also do not force the spleen tissue through a sieve. Another way to avoid or at least to minimize this problem, is to incubate the spleen cells for 24-48 h on plastic dishes to remove adherent cells before fusion. If still adherent cells grow and interfere with the growth of the hybridomas it is necessary to transfer the hybridomas into another well as soon as possible.
We have also been successful in using, instead of spleen cells, lymphocytes from the popliteal lymph nodes for the fusion. We found much less adherent cells to grow out of these tissues. In this case, rabbits were immunized by subcutaneous injection in several places of the leg below the popliteal nodes. The first injection was given with complete Freund's adjuvant; three or four subsequent injections were given at 5 day intervals with the antigen dissolved in saline and again the fusion was performed four days after the last boost. Another possibility is to boost the rabbits once or twice at 2 - 3 week intervals (with antigen dissolved in saline) and again the fusion was performed four days after the last boost.
Note: Some investigators have successfully used frozen spleen cells of an immunized rabbit for developing Hybridomas
The fusions were performed using conventional methodology (Michell and Shigi, 1980 Selected Methods in Cellular Immunology. W. F. Freeman & Co. p354-367): 1.5-3 x 108 lymphocytes from an immunized rabbit and the fusion partner were fused at a ratio of 2:1 with 50% PEG 4000 (EM Science, Cherry Hill, N.J. 08304) at 370C in serum-free medium. The cells were plated in 48-well microtiter plates at 2 x 105 lymphocytes or less per well in 0.5 ml medium with 15% FCS. (The aim is that no more than 25% of the wells are positive. This assures us that the upcoming hybridomas are mostly clonal.) After 3 - 5 days HAT (i.e., 0.5 ml of 2x concentrated HAT in medium) was added. Medium was changed every 4-5 days. (About 50% of the medium is aspirated and replaced with fresh medium containing 1x HAT.) Clones usually were observed after 2-5 weeks. Supernatants were tested for the presence of antibody specific for the immunogen. In a typical fusion we obtain 100 - 300 positive wells of 1000 wells plated. Typically 5 - 15 % of these clones are specific for the antigen. The hybridomas are cloned by limited dilution in 48-well microtiter plates. For feeder cells we used the fusion partner 240E-1 at 5 x 104 cells per well. These feeder cells are killed 5-6 days later by the addition of HAT.