Notes on Making Rat x Y3 Monoclonal Antibody Producing Hybridomas

This note is not intended as a full protocol for making monoclonal antibodies, but is to give a few hints and tips for the use of Y3/Ag1.2.3. Generally speaking the methodology is similar to making murine (eg. with NS0) monoclonal antibodies, but there are a few critical differences which are commented on below:
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1) Why we like to make monoclonal antibodies with Y3/Ag1.2.3
Although Y3/Ag1.2.3 has its own myeloma light chain, their are numerous characteristics that make this a better parent line for making hybridomas than the 'Y0' chain loss variants or using NS0 to make rat x mouse hybrids. These are:

a) Hybridomas are very stable - even in long term large scale cultures (bioreactors).
b) High levels of monoclonal antibody secretion in culture - often 100 micrograms per ml of ordinary tissue culture, without recloning, and up to 5 milligrams per ml in hollow fibre bioreactors.
c) Y3/Ag1.2.3 and hybridoma offspring are relatively resistant to nutrient starvation, which combined with their high stability makes tissue culture much less intensive, so screening can be at your leisure (or you can do complex screening on uncloned cultures without fear of them going negative on you).

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2) Notes on immunization.
a) We prefer to use strains that have a different light chain allotype to the kappa 1a of Y3 (eg. DA which is kappa 1b) to allow later screening of myeloma light chain loss variants (if required).
b) The final boost should be by the intravenous route, four days before the fusion, to ensure maximal activity in the spleen.

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3) The fusion.
a) Prepare Y3/Ag1.2.3 by growing it in a roller bottle, feeding 24 hours before the fusion, and harvesting the cells on the day of the fusion (remember Y3/Ag1.2.3 is loosely adherent to both the plastic and each other - a happy culture will have an almost confluent layer on the plastic, and small balls of cells in suspension - the roller should rotate quite slowly - only 1-2 revs/min).
b) Use half of one rat spleen (approx. 1undefined8_cells~B_together_with_6_x_1undefined7_Y3~HAg1.2.3_per_fusion_~Athe_other_half_of_the_spleen_cells_can_be_frozen_as_a_backup_in_90~7_FCS_~D10~7_DMS0~B.~Kbr_~H~M~2~1~0c~B_Try_and_keep_all_cells~E_PEG_and_medium_components_at_37_deg._C_throughout_the_procedure.~Kbr_~H~M~2~1~0d~B_Plate_the_entire_fusion_out_in_just_120_x_1ml_cultures_in_6_x_~9Linbro~9_24_well_plates~E_using_DMEM_~D_20~7_FCS_~Acontaining_penniclin_~D_streptomicin~E_Na_pyruvate_and_glutamine~B.~Kbr_~H~M~2~1~0e~B_DO_NOT_use_Isoves_medium_~AIMDM~B_as_this_will_encourage_overgrowth_by_primary_fibroblasts_from_the_spleen.~Kbr_~H~M~2~1~0f~B_Select_with_HAT_as_normal.~Kbr_~H~M~2~1~0g~B_Split_cultures_into_duplicate_plates_once_confluent_~Aaround_10_days~B~E_and_if_there_are_no_infection_problems_you_can_wait_for_most_of_the_wells_to_become_confluent_by_feeding_the_others_~Aand_collecting_supernatants_for_testing~B_~F_again_remember_that_most_hybridomas_will_be_ADHERENT~E_so_will_need_vigorous_pipetting_to_get_them_into_suspension_for_splitting.~Kbr_~H~M~2~1~0h~B_Split_wells_into_25ml_flasks_to_make_2_x_2_freezes_and_collect_larger_volumes_of_supernatants.~Kbr_~H~M~2~1~0i~B_Screening_can_continue_throughout_or_even_after_this_culture~Hfreezing_process_until_you_know_what_hybridomas_you_want_to_keep.~Kbr_~H~M~2~1~0j~B_After_2_weeks_the_interesting_wells~Hcultures_can_be_weaned_from_HAT_via_HT_and_into_IMDM_5~7_FCS~E_and_cloned_by_limiting_dilution_in_96_well_plates._~K~Hfont~M~K~Hp~M~2~1~Kp~M~2~1~0~Kfont~M~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~K~Hfont~M~K~Hp~M~2~1~Kp~M~2~1~0~Kfont~M4~B_Chain_loss_and_isotype_switch_variants~Kbr_~H~M~2~1~0a~B_If_you_want_to_make_light_chain_loss_~Aor_isotype_switch~B_variants~E_this_can_be_confounded_by_the_very_stability_of_these_hybridomas._You_can_sometimes_make_them_less_stable_by_growing_them_for_a_few_weeks_in_Azaguanine_or_Thioguanine_selection_~Aie._against_HPRT_or_for_HAT_sensitivity_~F_don~9t_ask_me_why_this_works~3~B_before_cloning_out_for_variants.~Kbr_~H~M~2~1~0b~B_You_can_screen_by_ELISA_or_with_mAb_coupled_RBC_using_mouse_monoclonal_antibodies_against_the_Y3_derived_Kappa_1a_~ARG11~H15.5~B_and_the_specific_DA_Kappa_1b_~AG9~H1.3~B~E_or_the_appropriate_heavy_chain_~Aeg_NORIG_7.16_anti~Frat_IgG2b~B._~K~Hfont~M~K~Hp~M~2~1~Kp~M~2~1~0~Kfont~M~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~F~K~Hfont~M~K~Hp~M~2~1~Kp~M~2~1~0~Kfont~M5~B_For_full_details_see~I~Kbr_~H~M~2~1~0Clark~E_M.R.~E_~8amp~J_Waldmann~E_H._Production_of_murine_monoclonal_antibodies_in_Methods_in_Hematology_13~I1~F20_~A1986~B._~K~Hfont~M~K~Hp~M~2~1~8lt~Jcenter~8gt~J~2~1~0~8lt~Jp~8gt~J~2~1~0~0 </p> </center>
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