1 Department of Rheumatology, University Medical Centre and 2 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
Correspondence to: B. Boi
, Department of Rheumatology, University Medical Centre, Vodnikova 62, 1000 Ljubljana, Slovenia. E-mail: borut.bozic{at}guest.arnes.si
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Abstract |
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Methods. Sera from 30 patients with antiphospholipid syndrome and/or systemic lupus erythematosus were selected on the basis of anti-ß2-GPI positivity. Avidity of IgG anti-ß2-GPI was determined by chaotropic ELISA, using increased NaCl concentration during the antibody binding. Immunodetection on nitrocellulose membrane followed reducing or non-reducing sodium dodecyl sulphatepolyacrylamide gel electrophoresis (PAGE) of ß2-GPI. In converted, non-reducing PAGE, the preparation with high-affinity Fab fragments (obtained by papain digestion) was subjected to electrophoresis, while purified ß2-GPI was used as the sample in immunodetection.
Results. Anti-ß2-GPI antibodies of high, low or heterogeneous (low and high) avidity were found in 5/30, 9/30 and 16/30 sera, respectively. The density of ß2-GPI, which was 2030 times higher on the nitrocellulose membrane than on the surface of ELISA plates, was not sufficient for the recognition of the antigen by anti-ß2-GPI: 2/5 high-avidity samples reacted only with non-reduced ß2-GPI, 3/9 low-avidity samples recognized only denatured and reduced ß2-GPI, and 1/16 samples with heterogeneous-avidity antibodies reacted with reduced and non-reduced ß2-GPI.
Conclusions. Our results suggest that neither high density of the antigen nor high avidity of the antibodies (or Fab fragments) alone was sufficient for the binding of anti-ß2-GPI to ß2-GPI. Some conformational modifications and, consequently, exposed neo-epitopes are required for the recognition of ß2-GPI by polyclonal anti-ß2-GPI antibodies.
KEY WORDS: Anti-ß2-glycoprotein I antibodies, Fab fragments, Avidity, Affinity, Antiphospholipid syndrome, Systemic lupus erythematosus
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Introduction |
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Patients and methods |
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Detection of anti-ß2-GPI
Anti-ß2-GPI antibodies were measured by our in-house enzyme-linked immunosorbent assay (ELISA) [11], evaluated through the European Forum on Antiphospholipid Antibodies [12]. Briefly, polystyrene microtitre plates (High Binding; Costar, Cambridge, MA) were coated with ß2-GPI in phosphate-buffered saline (PBS) and incubated with sera diluted 1:100 in PBS containing 0.05% Tween (PBS-Tween). After four washes, 50 µl/well of alkaline phosphatase-conjugated goat anti-human IgG second antibody in PBS-Tween was added. After 30 min of incubation at room temperature and four washes, 100 µl/well of disodium p-nitrophenyl phosphate dissolved at 1 g/l in 1 M diethanolamine buffer (pH 9.8) was added. Following colour development, optical density vs a reagent blank was measured at 405 nm with a Rainbow microtitre plate reader (Tecan, Gröding/Salzburg, Austria). The level of anti-ß2-GPI was derived from the standard curve according to the defined dilutions of monoclonal antibodies [13]. Absorbance values corresponding to the absorbance yielded by monoclonal antibodies at the concentration of 6 µg/l were considered positive.
Chaotropic ELISA assay
Avidity of IgG anti-ß2-GPI was determined by chaotropic ELISA as described recently [11]. The procedure was in principle the same as in the anti-ß2-GPI ELISA, using chaotropic conditions during antibody binding. Samples were diluted in PBSTween containing increasing concentrations of NaCl: 0.15, 0.25, 0.5, 1, 2, 4 and 6 M. The discrimination between anti-ß2GPI with high or low avidity was made arbitrarily, comparing the initial binding at 0.15 M NaCl with binding at 0.5 M NaCl. When the binding at 0.5 M NaCl remained higher than 70% of the initial binding, the presence of high-avidity anti-ß2GPI was declared. When the binding at 0.5 M NaCl decreased to or below 25% of the initial binding, low-avidity anti-ß2-GPI was established. Samples which did not fulfil either of these criteria were considered to be of heterogeneous avidity [11].
Fab fragment preparation
High-avidity anti-ß2-GPI derived from an APS patient by using an affinity G column, as we described recently [11], was the source of Fab fragments. IgG anti-ß2-GPI was digested for 2529 h at 37°C with papain (10 µg of papain for each mg of antibody). The reaction was stopped by iodoacetamide at the final concentration of 75 mM. The efficiency of the digestion was confirmed by 10% non-reducing sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE).
PAGE and immunoblotting
In reducing SDSPAGE, ß2-GPI was diluted in SDS sample buffer, boiled for 5 min at 98°C and applied to vertical polyacrylamide gels (Mini-Protean or Protean II; BioRad, Hercules, CA), using 3% stacking and 10% resolving gels. In non-reducing PAGE, ß2-GPI was diluted in SDS sample buffer without mercaptoethanol and applied to gels without prior boiling.
The transfer of proteins from gels to nitrocellulose membrane and immunodetection were performed according to the standard procedure using goat anti-human IgG alkaline phosphatase (Sigma, St Louis, MO) and bromochloroindolyl phosphate as the detection system [14].
In converted, non-reducing PAGE, the preparation with Fab fragments was subjected to electrophoresis, while ß2-GPI was used as the sample (antigen) in immunodetection. Goat anti-human ß2-GPI IgG (ACSC, Westbury, NY) was used as the bridge antibody to rabbit anti-goat IgG alkaline phosphatase (Sigma) in immunodetection.
Amount of the absorbed antigen
The area concentration on the nitrocellulose membrane was calculated from (i) the amount of ß2-GPI applied to the gel and (ii) the coloured area after the reaction with antibodies and alkaline phosphatase. The area concentration on the microtitre plates was estimated according to (i) the amount of ß2-GPI applied to a single well, (ii) the well surface in contact with ß2-GPI solution, and (iii) the 80% binding efficiency as approximated preliminarily.
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Results |
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Six sera recognized ß2-GPI electrophoretically transferred to nitrocellulose membrane (Fig. 1). The amount of ß2-GPI on the nitrocellulose membrane corresponded to the area concentration of 0.219 µg/mm2, while the amount of the antigen used in the ELISA corresponded to the area concentration of 0.007 µg/mm2.
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High-affinity Fab fragments bound to nitrocellulose membrane (area concentration 1 ng/mm2) could not capture any detectable amount of ß2-GPI free in the solution (at concentrations 28 and 130 mg/l).
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Discussion |
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Anti-ß2-GPI antibodies are generally believed to be of low avidity. The present study was not focused on clinical correlations. However, as we reported recently, thrombosis is the main clinical feature associated with high-avidity anti-ß2-GPI, which was predominantly due to venous thrombosis. Kidney disease, according to the criteria for SLE, was statistically more common in the combined high- plus heterogeneous-avidity anti-ß2-GPI group compared with the low-avidity group [11]. Our results suggested that high-avidity anti-ß2-GPI, detected by ELISA, was clinically more relevant than low-avidity anti-ß2-GPI. Therefore, differences in the binding of high- and low-avidity polyclonal anti-ß2-GPI to ß2-GPI under various conditions were investigated.
The binding of anti-ß2-GPI antibodies to physiological concentrations of ß2-GPI [18] in the fluid phase [5] was weak due to their low avidity. It has been proposed that high density of immobilized antigen or its clustering is required for bivalent or multivalent antibody binding [6]. The density of ß2-GPI is important for low-affinity antibodies, the avidity of which is increased by bivalent binding in vivo and in vitro [5]. It is unlikely that the density of the antigen could have an important influence on the binding of high-avidity antibodies or high-affinity Fab fragments and their subsequent detection in ELISA or immunoblotting.
We demonstrated that, from 30 sera that recognized ß2-GPI bound to oxygenated polystyrene plates, only a limited number recognized ß2-GPI, electrophoretically transferred to a nitrocellulose membrane. Among them, the sera which recognized transferred ß2-GPI in non-reduced form contained high-avidity antibodies, while the sera recognizing the denatured protein contained low-avidity antibodies. It has been hypothesized that anti-ß2-GPI might recognize conformational epitopes on ß2-GPI exposed when ß2-GPI binds to anionic phospholipids or high-binding plates [7, 19, 20]. Therefore, it is possible that conformational changes played an important role in our experiments. The density of ß2-GPI, which was 2030 times higher on the nitrocellulose membrane than on the surface of ELISA plates, was not sufficient for the recognition of the antigen by anti-ß2-GPI. Polyclonal anti-ß2-GPI in sera of APS patients may be directed against a number of different epitopes. Since all our samples contained polyclonal antibodies, it was very unlikely that they would have been oriented only towards one site of the antigen molecule, which could have been hindered due to the protein binding to the nitrocellulose membrane. The importance of ß2-GPI conformation was additionally confirmed by the unsuccessful binding of the physiological concentration of soluble ß2-GPI to high-affinity Fab fragments on the nitrocellulose membrane. The detection by immunoblotting on the nitrocellulose membrane did not seem to add any improvement concerning clinical relevance of anti-ß2-GPI compared with ELISA.
It has also been suggested that both antigen density and conformational changes could explain the disparate behaviour of monoclonal anti-ß2-GPI on different microtitre plates [8]. Our results indicate that neither high density of the antigen nor high avidity of the antibodies (or high affinity of Fab fragments) alone was sufficient for the binding of ß2-GPI and polyclonal anti-ß2-GPI. High density of ß2-GPI seemed to be crucial for the binding of low-avidity anti-ß2-GPI. We believe that some conformational modifications and consequently exposed neo-epitopes are required for the recognition of ß2-GPI by polyclonal anti-ß2-GPI.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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