Hybridoma Screening

Screening

Following fusion and HAT selection, the antibody-producing hybridomas of interest need to be identified. The appropriate screening strategy is one of the most important steps of the process and should be developed prior to the fusion.

11-14 days after fusion the supernatants of the 96-well plates are screened by ELISA for the presence of IgG antibodies directed against the antigen. We process plates by a liquid handling robotic workstation, which allows for highly controlled washing and performance. We may assist in other screening assays (i.e. FACS, capture ELISA/IP) depending on the specific requirements of the screening strategy.

Hybridoma candidates scoring positive in the ELISA screen are expanded in 24-well plates. We cryostore one vial of cells from each positive well to allow sufficient time to further validate the primary supernatant. Usually, more than one clone is growing in a single well. Thus, these primary supernatants originate from oligoclonal hybridomas rather than from a single monoclonal hybridoma.

We collect approx. 4ml of each primary supernatant, confirm the specificity by ELISA, and determine the IgG subclass and titer. Only antibody supernatants with a good titer are considered for further testing. These primary supernatants are delivered to you for further validation in the desired applications (WB, IP, IF, FACS, IHC, etc.). As the primary supernatants are not from single clones, we provide anti-IgG subclass-specific secondary antibodies (HRP-/ biotin- or uncoupled). It is important to use only subclass-specific antibodies for detection as most supernatants contain also IgM antibodie in addition to the specific IgG antibodies. Most commercial secondary antibodies recognize IgG and IgM antibodies and can give rise to false-positive results or high background.

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