Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

Patient selection

Thirteen patients were selected from the University College London Hospital (UCLH) and University of Istanbul cohorts of patients with APS, with or without systemic lupus erythematosus (SLE). Patient sera were tested previously for IgG, IgM and IgA aβ2GPI and Anti-Domain I positivity. ² Data on positivity for aCL and lupus anticoagulant (LA) were taken from clinic records. Patients with known IgA positivity were selected for this study.

Preparation of DI and PEG-DI

DI was prepared as previously and PEGylated as described. ¹³ Briefly: protein was produced bacterially in Escherichia Coli and folded manually through dilution. Purification was by Immobilised Metal Affinity Chromatography (IMAC), Cation Exchange Chromatography (CEX) and gel filtration. PEGylation was carried out using TheraPEG^R targeting disulphide bonds. Two conjugates were produced using 20 kDa and 40 kDa poly(ethylene glycol) (PEG). Expression tag was removed by FXa cleavage and protein was dialysed against phosphate-buffered saline before quantification and storage. Concentrations of PEGylated conjugates were expressed in terms of mass of DI.

Serum IgG depletion

Serum samples of IgG/IgA aβ2GPI positive patients were depleted using protein G beads. Serum samples were diluted 1:1 with binding buffer (10 mM NaPO4, 100 mM NaCl, pH 7.4) and applied to 100 µl of protein G beads (Sigma) for 1 h rotating at room temperature. Samples were centrifuged (13,000 g, 10 min, 4℃) and supernatant stored for analysis. Samples were washed with 500 µl of binding buffer, centrifuged and bound IgG was eluted with glycine (0.1 M, pH 2.3) rotating for 1 h before centrifugation and neutralization with 10 µl of Tris (1 M, pH 9.0) per 100 µl of glycine.

Serum IgA purification

IgA antibodies were purified using a peptide M column (Sigma). Purification was as for serum IgG depletion with the following exception: serum was diluted 2:1 with 10 mM sodium phosphate pH 7.2 and loaded onto a 1 ml column.

Direct IgA aβ2GPI ELISA and inhibition ELISA

Direct ELISAs for IgG and IgA aβ2GPI were as previously described. ² The IgA aβ2GPI inhibition ELISA was carried out as in McDonnell et al. ¹³ with the following alterations: secondary antibody was an anti-human IgA (Abcam) at a dilution of 1:10,000 and substrate was applied for 20 min before being stopped. DI was used at concentrations ranging from 25 µg/ml to 100 µg/ml, 20 kDa and 40 kDa PEGylated proteins were used at 50 µg/ml. Purified IgA was used between 12.5 µg/ml and 25 µg/ml of IgA, depleted serum was used at dilutions between 1:12.5 and 1:25 to achieve an optical density (OD) of between 0.4 and 0.8.

Domain I inhibits patient IgA binding to β2GPI in serum in a dose-dependent manner

Initially serum samples from patients (N = 13) were screened for inhibition with increasing doses of DI (0–100 µg/ml). As shown in Figure 1(a) patients clustered into three groups: no or little inhibition (cluster 1, n = 4), low inhibition (<40% inhibition at 100 µg/ml, cluster 2, n = 5) and high inhibition (≥40%inhibition at 100 µg/ml, cluster 3, n = 4). Dose-dependent inhibition for clusters 2 and 3 can be seen in Figure 1(a). Significant differences were seen between clusters 1 and 2 and clusters 1 and 3 (Figure 1(a)). OPEN IN VIEWER

Inhibition with 20 kDa PEG-DI and 40 kDa PEG-DI was carried out at 50 µg/ml. Inhibition can be seen in Figure 1(b), which includes 10 patients from all three clusters. There was significant (p < 0.05) enhancement of inhibition for both PEGylated forms in comparison with non-PEGylated DI.

Another interesting result was the difference in inhibition for patients with high and low IgG and IgA. Patients in cluster 1 with low inhibition also had the lowest levels of both IgG and IgA aβ2GPI (5.08 GBIU and 44.48 ABIU) and aDI (6.02 GDU, 19.92 aDU). Those patients with moderate inhibition in cluster 2 had high IgA (77.08 ABIU, 45.21 ADU) but lower IgG (18.8 GBIU, 9.82 GDU) whereas those in cluster 3 with the highest inhibition had both high IgG and IgA aβ2GPI (64.28 ABIU, 56.63 GBIU) and aDI (53.65 ADU and 52.08 GDU).

To analyse further the high-level inhibition of IgA aβ2GPI binding in serum from patients in cluster 3 we carried out further tests on serum depleted of IgG and on purified IgA samples.

IgG depleted serum retains its IgA aβ2GPI activity, which is inhibited by non-PEG-DI

Samples from three of the four cluster 3 patients (patients 1, 2 and 4 in Figure 1(c)) showed significant IgG aβ2GPI depletion demonstrated by loss of >80% binding in an IgG aβ2GPI ELISA (Supplementary Material Figure 1 online). IgG-depleted samples retained their IgA aβ2GPI activity sufficiently to carry out further inhibition experiments using a dilution that gave an OD between 0.4 and 0.6 (12.5–25 µg/ml) in the absence of inhibitor. Doses of DI ranging between 25 µg/ml and 100 µg/ml showed dose-dependent inhibition of IgA aβ2GPI activity (Figure 1(c)) for patients 1, 2 and 4.

Inhibition of purified IgA aβ2GPI binding

Purified IgA was derived from serum of three patients (1, 3 and 4). Two of these (1 and 4) showed dose-dependent inhibition between 25 and 100 µg/ml of DI. This can be seen in Figure 1(d). These results mirrored the effects of non-PEGylated DI in IgG depleted serum.