1. It is preferable to use NS-0 as a fusion partner, it does not secrete a constitutive antibody but has the ability to produce monoclonal antibodies once fused to an immune spleen cell. NS-1 has a constitutive antibody, which may cause interference in screening assays for recombinant hybridoma cells.

2. Cell lines are available that have other defective pathways, aminop-terin cannot be used to select for their growth, and an alternative agent must be used.

3. Antigens must be representative but do not need to be identical to the target substance. Synthetic peptides modelling single epitopes and fusion proteins containing homologous regions to the target can be used very effectively as antigens.

4. It is not always possible to use exactly the same format for screening as will be used in the final assay but it is advisable to attempt to ensure that the "position" that the monoclonal antibodies (MAb) will occupy in the final format is the position that is used for screening. For example MAbs which will eventually be conjugated to enzymes and used in Double Antibody Sandwich (DAS) ELISA should be screened for using TAS ELISA. This ensures that the epitope-bind-ing portion of the antibody is in the same position as it will be in the final test format with the analyte bound to the coating antibody.

5. Balb-c mice are normally used for MAb production, partly because they are the strain of mouse that the NS-0 cell was derived from and also because they are easily handled and the females can be communally housed.

6. Batches of FBS vary enormously in their ability to support cell growth and it is important to establish that the batch you are buying will be suitable. Most suppliers will allow you to reserve quantities from a batch and will provide small samples for testing. Usually a few batches are tested at the same time and then the best performer is selected. Testing is carried out by cloning an established hybridoma line by limiting dilution in medium containing the test FBS and then observing numbers of resulting colonies.

7. Polyethylene glycol 4000 MW is normally used for cell fusions. Batches may vary in their ability to produce viable hybridomas and it is wise to test a few batches from different sources prior to undertaking hybridoma project work. A number of biochemical suppliers now produce ready-to-use PEG/media solutions in sterile ampoules, which workers may find is a more practical source.

8. If the antigen to be used is derived from fungal mycelium, bacterial cell walls or is known to be highly glycosylated then repeated immunization is of no value. Antigens of this type are known to be anamnestic and do not produce "memory" B-lymphocytes. The animal sees each immunization as a primary challenge and the likelihood of producing an IgG response is remote.

9. Frozen spleen cells work well for cell fusions and this approach allows a greater degree of flexibility in performing fusions than using splenocytes directly ex vivo. The vial of spleen cells should be rapidly thawed in a 37°C water bath or between the palms of the hands. One milliliter of PBS is then added to the vial and the contents aspirated and added to 5 mL of PBS. The cells are washed once by centrifugation at 400g and resuspended in 5 mL of cold PBS; they should then be stored on ice until required. Spleen cells that have been frozen may have a tendency to clump and the pellet will look very pale. This does not affect their ability to produce viable hybridomas.

10. Disposable cell strainers (Falcon) that fit into the top of universal containers may be used to remove large fragments of spleen tissue.

11. The volumes of media used during cell fusion and subsequent plating in to tissue culture wells do not need to be accurately measured. Disposable Pasteur pipets can be used to approximate volumes and most manufacturers publish specifications including drop volume for dispensing cells and medium.

Was this article helpful?

0 0

Post a comment