G - Physics – 01 – N
Patent
G - Physics
01
N
G01N 35/00 (2006.01) G01N 33/49 (2006.01) G01N 33/569 (2006.01) G01N 33/577 (2006.01) G01N 33/80 (2006.01)
Patent
CA 2070244
ABSTRACT A rapid method for detection of monoclonal antibody labeled cells using forward light scatter/absorption clinical flow cytometers is disclosed. Calf-intestinal alkaline phosphatase is conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. Red cells are pre-conditioned for lysis during the first incubation period with the alkaline phosphatase conjugated monoclonal antibodies by ammonium-sulfate which is followed by an early fixation of white cells in the presence of dextrose. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer, containiny zinc glycinate, at pH 9.5?0.1, is used as a buffer/substrate to yield stable, insolu- ble and intense purplish precipitates on the surface of the cells labeled with alkaline phosphatase conjugated monoclonal antibodies. The separation of granulocytes and lymphocytes is detected by a forward light scatter/absorption optics. Endoge- nous alkaline phosphatase in granulocytes is inhibited with levamisole. Early mild ixation of the white cells permits incubation at 35°C to 40°C which accelerates each step of the reaction without disrupting the cells throughout the procedure, making the procedure adaptable for use on automated cytometric instrumentation. 1515S
Kim Young Ran
Paseltiner Lynn
Yeh Chien-Kuo
Macrae & Co.
Miles Inc.
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