Protein Protocols Protein Protocols

An Efficient Method for MAb Production 1135
day. Immunohistochemistry on cultured cells grown in microwells is also feasible,
though we have not used it ourselves. A Transtar 96 apparatus is a rapid and sterile way
to remove 10–50 µL of culture supernatant from all 96 wells of a microwell plate. The
culture plates have to be open inside the hood for only a few seconds. They should
always be wiped with paper tissue soaked in 70% ethanol before opening. Screening
the whole plate, including wells without cell growth, by ELISA avoids possible errors
when trying to keep track of individual wells. If ELISA-positive wells are to be screened
further (e.g., by Western blotting using a multichannel miniblotter), this first 50 µL of
supernatant may be diluted into 100 µL or more of PBS; multiple sampling of fusion
wells should be minimized to avoid contamination. We usually screen by ELISA on d 10,
carry out further screening of ELISA-positive wells on d 11 and 12, and clone about 30
of the best wells on d 12, 13, and 14.
3.6. Cloning by Limiting Dilution
1. Using a plugged Pasteur pipet, transfer the positive clone to a 25-well plate containing
0.5 mL of cloning medium (see Note 11).
2. Take an aliquot of these cells, dilute with an equal volume of 0.1% trypan blue in PBS,
and count cells on at least two chambers of a hemocytometer.
3. Prepare 6 mL of 160 cells/mL and perform serial dilutions to 40 cells/mL and 10 cells/mL.
Plate four rows at 16 cells in 0.1 mL/well, four rows at four cells in 0.1 mL/well, and four
rows at one cell in 0.1 mL/well. Three drops from a plugged Pasteur pipet are about 0.1 mL.
4. Eight to ten days after plating, clones at 1 cell/well are visible, and screening can start
immediately. It is best to screen at least 16–24 wells from each cloning plate. As soon as
positive clones have been identified, they should be cloned a second time, but at 4, 1, and
0.5 cells/well instead of 16, 4, and 1.
5. When these plates are screened again after another 8–10 d, at least one dilution should
have