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:

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).

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.

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. 10**8 cells) together with 6 x 10**7 Y3/Ag1.2.3 per fusion (the other half of the spleen cells can be frozen as a backup in 90% FCS +10% DMS0).
• c) Try and keep all cells, PEG and medium components at 37 deg. C throughout the procedure.
• d) Plate the entire fusion out in just 120 x 1ml cultures in 6 x 'Linbro' 24 well plates, using DMEM + 20% FCS (containing penniclin + streptomicin, Na pyruvate and glutamine).
• e) DO NOT use Isoves medium (IMDM) as this will encourage overgrowth by primary fibroblasts from the spleen.
• f) Select with HAT as normal.
• g) Split cultures into duplicate plates once confluent (around 10 days), and if there are no infection problems you can wait for most of the wells to become confluent by feeding the others (and collecting supernatants for testing) - again remember that most hybridomas will be ADHERENT, so will need vigorous pipetting to get them into suspension for splitting.
• h) Split wells into 25ml flasks to make 2 x 2 freezes and collect larger volumes of supernatants.
• i) Screening can continue throughout or even after this culture/freezing process until you know what hybridomas you want to keep.
• j) After 2 weeks the interesting wells/cultures can be weaned from HAT via HT and into IMDM 5% FCS, and cloned by limiting dilution in 96 well plates.

4) Chain loss and isotype switch variants

• a) If you want to make light chain loss (or isotype switch) variants, 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 (ie. against HPRT or for HAT sensitivity - don't ask me why this works!) before cloning out for variants.
• b) You can screen by ELISA or with mAb coupled RBC using mouse monoclonal antibodies against the Y3 derived Kappa 1a (RG11/15.5) and the specific DA Kappa 1b (G9/1.3), or the appropriate heavy chain (eg NORIG 7.16 anti-rat IgG2b).

5) For full details see: