Fusion and Screening
The fusion process involves fusing spleen or lymph node cells with a myeloma fusion partner cell line. The Antibody Hybridoma Core utilizes the fusion partner cell line FOX-NY, a nonimmunoglobulin-secreting myeloma cell line that is hypoxanthine-aminopterin-thymidine (HAT) sensitive.
If the mice are immunized by the core, no further action is needed by the investigator prior to fusion. However, mice that are not immunized by the core are required to be certified as "mouse virus-free" prior to fusion. For more information on obtaining this certification, contact the core.
Investigators who need rat or rabbit fusions performed must provide spleens or lymph nodes to the core on the day of the fusion, as the core only immunizes and houses mice.
During the fusion process, 15 96-well microtiter plates — 1,440 wells total — are seeded with the fusion hybrid mixture. Each plate is fed HAT-IMDM selection media and maintained in a carbon dioxide incubator at 37 degrees Celsius. Nine to 14 days after the fusion, hybrid supernatants are tested for the presence of the specific antibody of interest.
Evaluation of hybrids is a critical step. The Antibody Hybridoma Core offers hybridoma, clone and subclone supernate screening by enzyme-linked immunosorbent assay (ELISA), Western blot assay and fluorescence-activated cell sorting (FACS). To reduce costs, investigators may choose to perform all screening of supernates in their own lab.
Hybridoma supernates to be tested may range from 500 to 1,440 samples and will be ready to test by days nine to 14. They could arise over a three- to five-day period or may all be ready on the same day, so it's important for the client lab to be prepared weeks in advance with working specific secondary screening assays of choice. The core provides investigators with 200 microliters of supernate per sample, as well as negative control culture medium.
Enzyme-linked immunosorbent assay (ELISA). ELISA is a solid-phase screening method. As they can be performed in one day, ELISAs provide quick results. The core uses secondary antibody conjugates specific for gamma chain (IgG) subclass isotypes, which are more desirable to obtain. Secondary antibody conjugates specific for mu chain (IgM) subclass isotypes can also be performed upon request.
However, screening by ELISA may not detect antibodies that recognize conformational epitopes of interest. For this reason, the core encourages investigators to perform a secondary screening technique, such as Western blot, dot blot, histology, immunoprecipitation or radioimmunoassay, on candidate ELISA-positive supernates. Investigators must use their own laboratory assay system, which should be perfected a minimum of two weeks prior to fusion, specifically designed for their antigen and experimental application.
Western blot. The Western blot is a technique used to identify candidate hybrids, clones, subclones and monoclonal antibodies of interest that recognize and bind to one or more specific molecular weight proteins of interest. Proteins are run in an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus, with proteins migrating according to molecular weight.
The protein is transferred to a nitrocellulose or polyvinylidene difluoride absorbing membrane sheet. The protein-bound membrane is then incubated with a known or test antibody reagent, followed by a secondary antibody conjugated with a color indicator enzyme or substrate (or radiolabeled isotope). Sources of protein include cell lysates or tissue extracts; a recombinant protein synthesized from in vitro methods may also be used.
- FACS. Since it is used for identifying cell-surface receptor molecules, FACS requires whole-cell suspensions and special cell-sorting equipment to process samples. Fluorescein isothiocyanate (FITC) conjugated to protein A or G binds to the fragment crystallizable region of IgG immunoglobulins, especially IgG2a.