Premium
Eosin‐5‐maleimide binding to band 3 and Rh‐related proteins forms the basis of a screening test for hereditary spherocytosis
Author(s) -
King MayJean,
Smythe Jonathan S.,
Mushens Rosey
Publication year - 2004
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.2003.04730.x
Subject(s) - band 3 , hereditary spherocytosis , chemistry , maleimide , spherocytosis , red blood cell , eosin , antibody , microbiology and biotechnology , flow cytometry , polyacrylamide gel electrophoresis , glycoprotein , membrane protein , gel electrophoresis , biochemistry , albumin , immunoprecipitation , monoclonal antibody , membrane , staining , biology , enzyme , immunology , splenectomy , spleen , gene , polymer chemistry , genetics
Summary Flow cytometric analysis of eosin‐5‐maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the ɛ ‐NH 2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh‐associated glycoprotein, both in the M r 40–60 kD region, and the Rh blood group proteins in the 30–32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh‐related integral membrane proteins as well as band 3 in HS red cells.