Collecting and Storing Hybridoma Tissue Culture Supernatants

For most immunochemical methods, tissue culture supernatants will be the most useful source of monoclonal antibodies. The supernatants are not contaminated with high levels of other antibodies, and the concentration is high enough for most assays if used undiluted. This protocol describes the procedure of collecting tissue culture supernatants. When collecting supernatants for antibodies, allow the individual cultures to grow until the hybridomas die. This will allow collection of higher-titer supernatants. In general, antibodies are resistant to the proteases that are released from dying cells, so allowing the cells to die should not affect the quality of the antibodies. If extraneous IgG molecules will alter any of the assays for which the supernatants are being prepared, use medium with fetal bovine serum or use serum-free medium. The yield of this method is ∼20–50 µg of antibody/mL of supernatant. The most common problem encountered in storage of tissue culture supernatants after collection is contamination with bacteria or fungi. This can be prevented by the addition of sodium azide as described.