By John J. Langone, Helen Van Vunakis
FROM THE PREFACE: numerous papers during this quantity care for advances in hybridoma know-how and supplement the bankruptcy through Galfri and Milstein that seemed in quantity seventy three of this sequence. the way to improve the potency of manufacturing wanted fusion items, to simplify the elemental strategies concerned, and to evaluate the specificity and different homes of monoclonal antibodies are mentioned. consultant examples illustrate how a battery of monoclonal antibodies with diverse specificities can be utilized to review a variety of immunochemical and biochemical difficulties. different chapters hide extra techniques that may be used to label antigens and antibodies, separate antigen-antibody complexes, and approach information. a few papers labeled below one heading or contained in one other quantity of the sequence comprise tools that may be utilized in different particular parts. for instance, cytotoxicity checks defined in quantity ninety three can be utilized to monitor supernatant fluids in hybridoma experiments if cytotoxic job is a fascinating estate of the monoclonal antibody
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Extra info for Immunochemical Techniques Part E: Monoclonal Antibodies and General Immunoassay Methods
Sample text
An efficient cell line should be used soon after initiation from frozen stock. 2. The plasmacytoma cells are washed twice and resuspended in DMEM-HEPES. The cell number and viability are determined. 3. Spleen cells and myeloma cells are mixed at a 1:1 ratio of viable cells in a 15-ml round-bottom plastic tube. Between 5 and 25 × 10 6 spleen cells may be fused in a single tube. The procedure is done at room temperature. [2] ENHANCED HYBRIDOMA PRODUCTION 23 4. Centrifuge the tube for 8 min at 300 g and then pour off all the medium.
8-azaguanine). The cells should be in the logarithmic growth phase for optimal results. The cells are subcultured at 2 to 4 x 105 cells per milliliter in fresh medium at 4 days, and again 2 days prior to their use for fusion. Sublines from a single plasmacytoma cell line vary in their capacity to form antibody-secreting hybrids. An efficient cell line should be used soon after initiation from frozen stock. 2. The plasmacytoma cells are washed twice and resuspended in DMEM-HEPES. The cell number and viability are determined.
2] ENHANCED HYBRIDOMA PRODUCTION 19 8. After I0 min. the supernatant is transferred to another 50-ml plastic tube,, leaving the large clumps of debris that have settled out. 9. The cells are washed twice with 50 ml of cold DMEM-HEPES by centrifugation at 4 ° for 10 min at 300 g. 10. After the second wash, the cell pellet is resuspended in 2 ml of cold DMEM-HEPES. It is important that the cell pellet be vigorously resuspended, as clumping can occur. In general, -100 x 106 cells are harvested from a single mouse spleen.