Protein Protocols Protein Protocols

An Efficient Method for MAb Production 1137
5. The myeloma cells should look uniform and healthy by this time with no sign of cell debris.
6. We normally perform all steps up to this point in a separate sterile hood. The mouse is
nonsterile externally, and particular care is taken to avoid hairs; the outer skin is torn,
rather than cut, for this reason. Mice should not be introduced into the hood used for
cell culture.
7. Some protocols remove red blood cells by differential lysis at this point, but we have not
found it necessary.
8. The final concentration of PEG during fusion is thought to be critical for the yield of
colonies. If initial yields are low, try adding increasing (but small) amounts of DMEM/
25 mM HEPES to the pellet before adding PEG.
9. Never use unplugged micropipet tips for adding to culture plates; sterile tips can be used
for removing culture supernatant only.
10. Bacterial and yeast contaminations rarely occur and are usually the result of a major failure
in sterile technique (e.g., inadequate sterilization of culture medium or glassware).
Sources of any sporadic fungal contamination should be tracked down and eliminated.
11. Some protocols recommend freezing down uncloned or partly cloned cells as a precautionary
measure. Following our rapid cloning protocol, however, the first round of cloning
and screening is often complete before the original colony is sufficiently expanded
for freezing. It is certainly advisable to keep the original culture alive, however, by adding
0.1 mL of cloning medium back to the fusion well and by feeding the 24-well culture, if
necessary. Cloning should not be regarded as an ordeal suitable only for healthy cells, but
rather as a means of invigorating a failing culture. We always clone twice for this reason,
even if the line is evidently clonal after the first round. As a general maxim: if in doubt or
trouble, clone immediately.
12. There are rare exceptions to the general principle that cloning should be continued until
all colonies are positive in the screens. We were once performing an initial screen by
ELISA and then testing ELISA-positive wells by immunofluorescence microscopy (IMF)
on muscle sections. After cloning one well that was positive in both assays, we found that
only half the clones were ELISA-positive and very few of these were also IMF-positive.
We recloned wells that were positive in both assays again with the same result. We thought
we had come across our first “unstable” hybridoma, but by chance we tested ELISAnegative
wells in IMF and found they were all IMF-positive. Only then did we realize that
the original fusion well had contained two different hybridomas, one ELISA-positive and
one IMF-positive. The purpose of cloning is to separate such lines, but by selecting wells
that were positive in both assays (and hence still had two clones), we had been systematically
defeating this objective.
13. When very few cells survive after being kept in liquid nitrogen, “cloning” by limiting
dilution at about 100 total cells/well may be the only way to recover the cell line.
14. Cells may also be collected by centrifugation and resuspended in 5% fetal calf serum at
this stage, if a culture supernatant with low levels of nonmouse Ig is required.
15. Most MAb are stable for long periods in sterile culture medium, but there are undoubtedly
some that lose activity rapidly even at 4°C. These are perhaps best avoided, but, if required,
bulk culture would have to be monitored regularly and supernatants harvested when their
antibody activity is still high.
Acknowledgment
We thank C. J. Chesterton (King’s College, London) for sharing with us his enthusiasm
for, and experience of, hybridoma technology in 1981.