Identification of Haemoglobin S in red cells and normoblasts, using fluorescent anti-Hb S antibodies.

Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen. Identification of Hb S in single erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjungated anti-Hb S antibodies with the haemoglobin antigen in fixed blood smears. All the red cells from persons with sickle cell trait and sickle cell anaemia are labelled with the anti-Hb S-FITC, while red cells from persons with normal haemoglobin or other mutant haemoglobin genotypes are not. Erythrocytes from newborns with sickle cell trait bind the anti-Hb S antibody, although the intensity of labelling varies from cell to cell. This method of cellular identification of Hb S preserves the morphological characteristics of the reactive cells and permits observations of the pattern of haemoglobin synthesis among cells of various degrees of maturity. Fluorescent identification of Hb S in fixed blood smears may be applicable to prenatal diagnosis of haemglobinopathies.