Signal enhancement in fluorescence microscopy and flow cytometry using fluorescent liposome-antibody conjugates as second layer reagents.
We have developed small unilamellar liposomes (SUV) containing carboxyfluorescein for indirect immunophenotyping of cells by fluorescent microscopy and flow cytometry. Covalently coupled to anti-mouse Ig and anti-human Ig with mouse monoclonal antibodies and human typing antisera respectively, these conjugates can increase sensitivity by a factor of 10 compared to fluorescein isothiocyanate (FITC) conjugates. No increase in background fluorescence was observed. The reagents have been used to detect a low density antigen, the interleukin-2 receptor, on resting T-cells and to overcome the quenching effect of haemoglobin in fluorescent phenotyping of red cells. This has allowed to accurately estimate blood grouping in mixed fields and with small numbers of cells. We have determined the blood group of fetal red cells obtained from a chorionic villous sample at 10 weeks gestation and predicted severe hemolytic disease. This was confirmed by fetal sampling at 19 weeks and several intra-uterine transfusions were undertaken before a successful delivery at 36 weeks. Applications of the method in leucocyte phenotyping and transfusion medicine are discussed.
Gray
A
A
Princess Margaret Hospital, Pathology Department, Swindon, UK.
Huchins
E R
ER
Morgan
J
J
Jaswon
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology; D001789:Blood Group Antigens; D005260:Female; D005312:Fetal Blood; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008081:Liposomes / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D011247:Pregnancy
D000911:Antibodies, Monoclonal; D001789:Blood Group Antigens; D007202:Indicators and Reagents; D008081:Liposomes