Subtyping of haptoglobin

Abstract

The haptoglobin molecule consists of α- and β-chains linked with disulfide bridges. The isoelectric heterogeneity observed in the β-chain is due to its content of sialic acid. The α-chain shows genetically determined structural polymorphism, and the most common subtypes 1S, 1F, 2FS, 2SS, 2FF and Johnson may be separated by isoelectric focusing (1,2). A subtyping method is developed which is well suited for large scale haptoglobin subtyping, and which requires no purification of the haptoglobin molecule prior to isofocusing (3): Five μl serum is treated with neuraminidase and reduced. Approximately one third of this mixture is subjected to polyacrylamide gel isoelectric focusing, and the remaining sample may be frozen and applied on a new gel if necessary. The neuraminidase treatment is included in order to diminish background staining, and to avoid interference between the isoelectric band patterns of the α- and β-chains. The Hp band pattern is visualized with an immunoblotting procedure using anti human haptoglobin as the first antibody and a peroxidase conjugated second antibody. In figure 1 is presented an immunoblot showing Hp-subtypes in serum samples from routine cases of disputed paternity.