Immunochemical Protocols by Margaret M. Manson

By Margaret M. Manson

Highlights fresh advances in immunochemical innovations and their effect on simple study and medical drugs, combining the forces of specialists in quite a lot of subspecialties to hide a wealth of knowledge.

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P has to be grown in spinner culture to fuse well. Seven to ten days before cells are required, about 5 x lo6 cells, stored frozen in liquid nitrogen as 1-mL aliquots in freezer medium, are thawed quickly at 37OC, diluted with 10 mL of DMEM-10% FCS, centrifuged (500g x 2 min), then resuspended in 100 mL of the same medium and placed in a ZOO-mL spinner flask. Stand for 2 d at 37’C to allow cells to attach to the base of the vessel, then place the spinner flask on a Bellco magnetic stirrer running at about 160 rpm.

Dilute 1:lO (PB/lO) as appropriate. 49. 6, f lMNaC1. 50. , rat hybridomas will grow at Z-3% FCS) and tested for maximum level of MAb. Roller cultures can yield milligram quantities (10-20 mg/L) of either rat or mouse antibodies. 51. Ascites from BALB/c mice or athymic rats. Grow up hybridomas in DMEM-10% FCS in roller culture (essential for rat hybridomas) and inject about 3 x lo6 cells/mouse and 10’ cells/rat into the peritoneal cavity. Harvest ascitic fluid 7-10 d later when tumor growth is visible by swelling of the abdomen.

2. 1. Adsorption of Peptide to Microtiter Plates 1. If using free peptide as the antigenic target, dilute it to a final concentration of 50 mmol/mL in adsorption buffer (roughly a dilution of about 1 in 2000 for a l@mer peptide). If using a peptidecarrier conjugate as the target, dilute the conjugate in adsorption buffer to a pt+tide concentration of about 10 mg/mL. The precise amount of peptide conjugate may need eventually to be titrated to give optimal signals. 2. Add 100 pL of diluted peptide solution to each well.

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