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Approximately 10 - 14 days following the fusion, at a time when the majority of culture wells are nearly 50% confluent, supernatants are collected from each well for screening. The investigator will be notified one or two days prior to supernatant harvest. The investigator should be prepared to screen 300 -1000 individual wells. About 125 ?l of supernatant is harvested from each well. Screening results are required within 48 hours to insure that positive culture do not overgrow or die.

Prior to supernatant harvest and screening the investigator should fine tune their screening strategy, using control serum and immune serum. This is the time to be sure that all reagents are available and that proper dilutions of secondary reagents are known. The method of screening should be designed to identify hybridomas secreting antibodies that are specific and will function in the manner desired by the researcher. Common screening protocols include: ELISA, Western Blot Analysis, Immunoprecipitation of radiolabeled protein, Ligand Binding Inhibition, FACS or Bioassay. Dr. Sheehan is available to discuss these methods; a few factors to consider are as follows:

• Are suitable quantities of antigen/cells available to test individual wells, or will samples need to be pooled and then retested?
  
  
• Is the screening assay specific? If an animal is immunized with a fusion protein, can the investigator differentiate between reactivity to the antigen vs. carrier? Has the investigator monitored reactivity to both transfected and non-transfected cells?
  
  
• Can the screening method be completed and analyzed within two days?
  
  
• Is a two tiered screening method needed? One to identify antigen specific hybridomas and another to isolate mAb with specific functions or subclasses.
  
  
• Are the appropriate secondary reagents identified and titered? This is particularly important when screening Armenian hamster fusions.
  
  
• Will non-Ig components in the culture supernatant interfere with the read out of the assay? For example, if a proliferation assay is used, the high concentration of thymidine in the selection medium will prevent incorporation of 3H-thymidine in dividing cells. Often the fetal calf serum present in supernatants at 20% (v/v) will pose a problem.