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 groupspecific component (Gc) or Gc-globulin, is a 51.2-kDa polymorphic protein of the a2-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 (3–7). In humans, deglycosylation of Gc-globulin, involving stepwise removal of b-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 macrophageactivating and osteoclast-differentiating functions of GcMAF 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 by centrifugation at 300g for 5 min at room temperature (25 °C). The buffy coat layer, containing .70% lymphocytes, was carefully removed under sterile conditions with a Pasteur pipette, excluding red cells as far as possible, and layered into a plastic Petri dish (60 3 15 mm). Plasma (5 mL) was transferred to the Petri dish containing the buffy coat cells, and 0.3 mol/L CaCl2 was added to the plasma sample to obtain a final ionized calcium concentration of 1.5 mmol/L. [Ca] was determined by ion-selective electrode. Lymphocyte confluence was confirmed under a phase contrast microscope. The plasma-white cell preparations were then incubated in 5% CO2 at 37 °C; samples (0.4 mL) were removed at 0, 4, 8, and 16 h and centrifuged, and the supernatants were stored at 4 °C for assay. The study was approved by the local Ethics Committee in accordance with the current revision of the Helsinki Declaration, and all subjects gave their written informed consent. Microtiter plates were prepared for assay by coating with 0.1 mL (1:400 dilution of goat anti-human polyclonal Gc-globulin antiserum) containing ;3.5 mg of IgG (Diasorin) in 0.1 mol/L bicarbonate buffer (composed of 4.24 g/L Na2CO3 and 5.04 g/L NaHCO3, pH 9.6) and incubating overnight at 4 °C. The antiserum was then tipped away, and the unoccupied binding sites on the wells were blocked by washing six times with a solution containing 1 mL/L Tween-20, 0.1 mol/L phosphate buffer, 3.32 g/L Na2HPO4, and 0.57 g/L NaH2PO4, pH 7.4. The plate was then slapped dry over absorbent paper and used for the assay. Incubated plasma samples (0.1 mL) were then added to wells and incubated for 4 h at 4 °C. The samples were then tipped away, the wells were washed six times with Tween-20 solution in phosphate buffer, and the plate was slapped dry. We added 0.1 mL of horseradish peroxidase (HRP)-labeled Helix pomatia lectin (25 ng; SigmaAldrich) in 0.1 mol/L Tris-HCl buffer, pH 7.4, containing 1 mmol/L MnCl2 to the wells and incubated the plates for 1 h at room temperature. The solution was then tipped away, and the wells were washed six times with Tween-20 solution as above and slapped dry. 3,39-5,59Tetramethylbenzidine (TMB; 0.1 mL; Bionostics) was added to the wells and incubated at room temperature for 10 min. The absorbance was then measured at 620 nm on a Multiskan MCC 340 plate reader (Labsystems). In the absence of any standard preparation of Gc-MAF, it was not possible to express the absolute concentration of the factor; therefore, the concentration is expressed in terms of absorbance produced by capture of HRP-labeled lectin. H. pomatia lectin, composed of six subunits and with a molecular mass of 79 kDa, possesses particularly high specificity for terminal N-acetyl-a-d-galactosaminyl residues (12 ). The association constant between GalNAc and H. pomatia lectin is of the same order of magnitude as for other carbohydrate-anticarbohydrate systems, including antibodies and other lectins (12 ). Each assay was carried out in triplicate, and appropriate controls (plasma sample without white blood cells and 5 mmol/L GalNAc) were treated in parallel. The intraassay precision of the lectin sandwich assay was determined using 30 replicates of a plasma-white cell preparation incubated for 16 h. In the absence of a standard preparation of Gc-MAF, an indirect estimation of Gc-MAF was carried out. To estimate the activity of HRP-labeled lectin, a defined quantity of the lectin (100 mL of 0.1, 0.5, 1, and 2 mg/L) was added to microtiter plates. The plates were incubated with TMB at room temperature for 10 min, and the absorbance produced by the enzyme reaction was determined. The reaction conditions, such as temperature, pH, and buffer composition, were kept similar throughout the experiments, and the lectin binding to microtiter plate was assumed to be 90% in all experiments (13 ). For the purpose of making an Technical Briefs