Large-scale HPA-1 epidemiological study in scotland: an up-date

The human granulocyte antigen HNA-2a is expressed on a subpopulation of granulocytes in 97% of Caucasians. Alloimmunization against this antigen can cause alloimmune neonatal neutropenia, transfusion-related acute lung injury and immune neutropenia after bone marrow transplantation. Immunochemical studies demonstrated that NB1 allogenic determinants reside on a GPI-linked glycoprotein with apparent molecular weight of 56–64 kDa. To elucidate the primary structure of NB1 protein, we isolated NB1 protein using NB1-specific mab 7D8 by affinity chromatography. Identity of the purified glycoprotein was confirmed by immunoblot using a panel of seven human and two monoclonal NB1-specific antibodies. Determination of molecular mass using MALDI yielded a 50·6 kDa glycoprotein. After removal of N-linked carbohydrates, the molecular mass was reduced to 43·1 kDa. N-terminal amino acid sequencing by Edman degradation yielded a 20 aa-peptide. Rapid amplification of cDNA ends derived from an HPA-2a (+) individual using primers based on N-terminal amino acid sequence resulted in a 1614-bp cDNA, consisting of a 27-bp 5' untranslated region, a 1311-bp open reading frame and a 276-bp 3' untranslated region. The 1311 bp open reading frame encoded for 437 amino acids, of which the first 21 were found to form the signalpeptide. The identity of this transkript was confirmed by the expression of full lengh NB1 cDNA (bp 22–1394) in COS-7 cells.