SITE SEARCH

The Washington University School of Medicine Hybridoma Center is under the faculty supervision of Dr. Kathleen Sheehan, Ph.D. and is operated by Research Technicians Misha Hart and Jennie Yeargin. Dr. Sheehan may be contacted at (314) 362-8744 or for information regarding immunization procedures, screening strategies, general operation of the Center or special needs. Misha and Jennie should be contacted at (314) 362-7448 or to schedule fusions and communicate screening results.

1. Fusions are scheduled by calling (314) 362-7448. We recommend that fusions be scheduled at the time animals are initially immunized, this will minimize any delays for the investigators. Fusions are performed on Tuesdays and Thursdays. All animals to be fused must demonstrate a positive serum titer (> 1:1000). Titer results are reviewed by the Director prior to fusion.
   
   
2. Animals (mice or rats) which are the source of immune cells to be fused are to be obtained, immunized, boosted and bled by the investigator (the Armeanian Hamster Colony is maintained by the Hybridoma Center). For B cell fusions the murine myeloma P3X63Ag8.653 is used.
   
   
3. On the scheduled day of the fusion, the investigator will bring the immune animal to the Hybridoma Center - West Building, Room 4708. The animals should be brought to the Center by 9:30 a.m. on the scheduled day of the fusion, unless alternate plans have been previously arranged. The animals should be clearly marked (preferrably ear tagged) and transferred to the Hybridoma Center holding cage. The investigator will return the transport cage to the appropriate animal facility. If the investigator is late, the fusion may be cancelled at the discretion of the Hybridoma Center.
   
   
4. After the fusion is performed, the Hybridoma Center will notify the investigator when to expect supernatants for screening approximately two days in advance. This will generally be 10 to 14 days after the fusion. The investigator will receive replicate 96 well plates containing approximately 120 ul per well of culture supernatant. Researchers should be prepared to screen 300-1000 individual supernatants from the fusion of a single mouse spleen.
   
   
5. Investigators will be given 2 days to notify the Center which supernatants are positive (this limitaion is due to the exponential growth of cells in small culture volumes). A second set of supernatants will be provided to verify antigen-reactive wells. Positive cultures will be expanded and 4 to 7 days later another set of supernatants will be given to the investigator. Only cell lines which have tested positive 2 times will be frozen and subcloned. (Note: Hamster hybridomas are notoriously unstable and will be cloned immediately from original culture wells). In a typical fusion, a maximum of 12 lines will be frozen, with the best 6 to be subcloned. Results from all screenings are expected within 2 days. If this poses a problem for the investigator, the Hybridoma Center can try to accomodate researchers if contacted in advance. Additional lines may be subcloned and frozen for an additional fee.
   
   
6. Select positive clones will be subcloned by limiting dilution to insure clonality and/or select for higher antibody production. These cultures will be tested in a manner similar to the original supernatants. Three subclones from each line will be frozen.
   
   
7. All cell lines generated remain the property of the individual investigator. Researcher may choose to store frozen vials at the Hybridoma Center or all vials can be transferred to the original investigator upon completion of the fusion. The Hybridoma Center services are most successful when there is full communication between the Center and the investigator. For our own records, the Hybridoma Center requests that we be informed as to the success of each particular fusion (in the eyes of the researcher), the utility of individual hybridomas and informed or any publications documenting hybridomas produced in the facility.