A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx bl ood group antigen, which is deficient in rare patients with McLeod syndrome . Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immun ized with a synthetic peptide predicted to be located on the second externa l loop of Kx produced a monoclonal antibody called 3E12 which does not reco gnize red cells with common Kell phenotype by agglutination and flow cytome try. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing io nic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L(484 )AQELEKE(491) on the Kx protein and spectrin beta-chain, respectively. To q uantify the relative amount of Kx in Empigen BB extracts of red cell membra nes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K-0 individuals (lacking the Kell glycoprotein) th an in membranes of common individuals, and (ii) that all common individuals , typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cel l membranes. When an erythrocyte membrane detergent extract from one K-0 in dividual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, ind icating that at least the K-0 individual under study may still produce some Kell protein.