Lectin Immunoassay for Macrophage-activating Factor (Gc-MAF) Produced by Deglycosylation of Gc-Globulin: Evidence for Noninducible Generation of Gc-MAF

The vitamin D-binding protein, also known as group-specific component (Gc) or Gc-globulin, is a 51.2-kDa polymorphic protein of the α2-macroglobulin fraction of human plasma (1). In human population, three common alleles ( Gc1f , Gc1s , and Gc2 ) and >120 rare variant alleles have been classified by isoelectric focusing (2). Gc1f and Gc1s contain sialic acid residues, whereas Gc2 does not (2). Gc-globulin may have an important role in the activation of macrophages and in osteoclast differentiation from monocytes and thus may control bone morphogenesis and remodeling (3)(4)(5)(6)(7). In humans, deglycosylation of Gc-globulin, involving stepwise removal of β-galactose and sialic acid from the trisaccharide, leaving N -acetyl-galactosamine (GalNAc), produces a potent macrophage-activating factor (Gc-MAF) (3). GalNAc is considered to have a crucial role in the macrophage-activating and osteoclast-differentiating functions of Gc-MAF because the removal of this sugar has been shown to be associated with the loss or impaired function of macrophages (8)(9). In osteopetrotic rat and mice models (6)(7) and in a single human study (4), indirect data suggested a defect in lysophospholipid-inducible Gc-MAF, although direct estimation of this factor in healthy and diseased states has not been performed.Gc-MAF estimation has been hampered by the lack of a suitable detection system for determining the sterically exposed GalNAc, which is critical for the activating properties of Gc-MAF. Because no standard preparation of Gc-MAF (as opposed to Gc-globulin) is available, its presence must be inferred from its properties. We here describe a hybrid sandwich lectin-ELISA for the measurement of the sterically exposed GalNAc (10)(11) generated in vitro from Gc-globulin in the plasma of healthy subjects.Blood was collected from 11 healthy subjects by venipuncture into EDTA sample tubes, and the plasma was separated within 5 min …