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Technical Briefs |
Department of Clinical Biochemistry, The Medical School, Framlington Place, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom
a author for correspondence: fax 44-0191-222-6227, e-mail h.k.datta{at}ncl.ac.uk
The vitamin D-binding protein, also known as group-specific
component (Gc) or Gc-globulin, is a 51.2-kDa polymorphic protein of the
2-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 ß-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 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 x 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. [Ca2+] 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 µg 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;
Sigma-Aldrich) 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. 3,3'-5,5'-Tetramethylbenzidine (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--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 µmol/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 µL of 0.1, 0.5, 1, and 2 µg/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 estimation of the sensitivity of detection of Gc-MAF, the stoichiometry of the antibody:Gc-MAF:lectin interaction was assumed to be 1:1:1.
Gc-globulin in plasma was measured by an established RIA using the monospecific polyclonal antibody (Diasorin) and Gc-globulin (Calbiochem Novabiochem) (10). The intraassay precision of the RIA was tested by measuring 20 replicate samples diluted at a concentration of 600 mg/L, 16 replicates of a sample at 400 mg/L, and 12 replicate samples at 200 mg/L; the CVs were 6.9%, 8.1%, and 8.6%, respectively. The plasma Gc-globulin concentrations (range, 250–470; mean ± SD, 350 ± 53 mg/L) have been reported previously (9) and were within previously described limits (200–500 mg/L) (14)(15).
The Gc-MAF assay was specific because excess GalNAc (5 µmol/L)
totally blocked binding of the lectin (Fig. 1
A). This indicates that there is a negligible amount of
nonspecific binding of lectin to the matrix and that most of it
specifically binds to GalNAc in Gc-MAF captured by the Gc-globulin
antibodies. Because B- and T-cell surface membrane-bound glycosidases
are crucial for the generation of Gc-MAF, in the absence of white blood
cells no generation of Gc-MAF from Gc globulin is expected. Thus, when
plasma was incubated without white blood cells, Gc-MAF was not
generated (Fig. 1B
).
|
The presence of lymphocyte-enriched buffy coat in the plasma-white cell incubations was associated with a linear generation of Gc-MAF in all subjects, and the omission of lymphocytes from the incubations was associated with failure of in vitro Gc-MAF generation. All control subjects showed basal noninduced generation of Gc-MAF when their lymphocytes were incubated with the plasma Gc-globulin. The rates of Gc-MAF generation varied among 11 male controls; the CV for the increase in absorbance after 16 h was 42%. The generation of activating factor was constant over the first 8 h, with a slight decline thereafter.
The detection limit of the method was assessed approximately from the change in absorbance for various concentrations of lectin-enzyme complex under routine conditions with the assumptions stated above and was compared with the change in absorbance detected during assays. From these experiments, the detection limit of the assay was estimated (mean zero absorbance + 2.5 SD) to be <2 pmol/L. The generation of Gc-MAF from Gc-globulin is achieved by the action of the B- and T-lymphocyte membrane-bound ß-galactosidase and neuraminidase, respectively (4). Because the circulating plasma concentrations of Gc-globulin are known to be relatively high, the activities of the glycosidases are likely to determine the rate of Gc-MAF generation.
It has been reported that Gc-MAF generation was detected indirectly only after induction by lysophosphatidylcholine, a mediator of inflammation (5)(6); however, we observed Gc-MAF generation even in the absence of a mediator of inflammation. Indeed, our method has shown continuous Gc-MAF generation in plasma from healthy adults and the absence of production in a patient with recessive osteopetrosis, but with production demonstrated posttransplantation, coincident with clinical and molecular genetic evidence of engraftment and bone remodeling (9).
The rate of increase in Gc-MAF in the in vitro incubations was estimated to be ~30 fmol · L-1 · min-1 (calculation is based on the fact that 0.1 pmol of HRP-labeled lectin per microtiter well incubated for 1 min produced an absorbance of 0.35; in Gc-MAF assays, 16 h incubation of 0.1 mL of plasma from a healthy adult produced an absorbance of 0.096). Because the lymphocyte-enriched white blood cell preparations used in these experiments were at 10-fold higher concentrations than in circulating blood, we estimate that the rate of generation in vivo is likely to be ~3 fmol · L-1 · min-1. The blood concentrations of Gc-MAF thus are likely to be quite low; therefore; it is unlikely to act as an endocrine factor. In view of its expected increased generation at sites of inflammation, because of the presence of a large number of T- and B-lymphocytes, it is much more likely to act similarly to a paracrine or autocrine agent.
No in vivo or in vitro assays for Gc-MAF have been reported. The current approach in which Gc-MAF generated by lymphocyte enrichment is then demonstrated by lectin-ELISA may enable investigation into the physiology and pathology of this important compound and its effects on bone remodeling.
References
-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1997;82:290-298.
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The following articles in journals at HighWire Press have cited this article:
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  S. Ferrero, D. J. Gillott, P. Anserini, V. Remorgida, K. M. Price, N. Ragni, and J. G. Grudzinskas Vitamin D Binding Protein in Endometriosis Reproductive Sciences, May 1, 2005; 12(4): 272 - 277. [Abstract] [PDF]
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Absorbance 620 nm) corresponds to the change in the
concentration of Gc-MAF.