Thu0040 using a Novel Bead-Based Immunoassay for Simultaneous Detection of Autoantibodies Against Serum Amyloid A1 and Alpha1 Acid Glycoprotein

Abstract

BackgroundNaturally occurring autoantibodies (Abs) against acute phase proteins (APPs), such as anti-serum amyloid A1 (SAA1) Abs have already been identified in sera of healthy individuals, as well as in patients with autoimmune diseases (1). Currently however, no method exists for simultaneous detection of multiple Abs against APPs.ObjectivesTo develop a bead-based duplex immunoassay for simultaneous detection of IgG anti-SAA1 and anti-alpha 1 acid glycoprotein (AGP) Abs and quantify their levels in sera of healthy blood donors (HBD), and patients with systemic autoimmune diseases, as well as in intravenous immunoglobulin preparation (IVIg).MethodsFluorescently labeled MagPlex microspheres (Luminex Corp) were used to covalently bind SAA1 and AGP. Sera samples (diluted 1:20) were added to a 96-well microtiter plate and incubated with SAA1- and AGP-coupled microspheres for 2h. Subsequently, PE-conjugated anti-human IgG Abs were added to each well, incubated for 30 min, resuspended in PBS-1%BSA and analysed using the MagPix system (Biomedica, GmbH).Abstract THU0040 – Figure 1 Levels of antibodies against SAA1 (A) and AGP (B) in HBD as compared to patients with systemic autoimmune diseases. Shown is median and IQR. 5th and 95th percentile of MFI values in HBD are indicated with dotted lines; *p<0.05; **p<0.01. ?SAA1, serum amyloid A1; AGP, alpha 1 acid glycoprotein; MFI, median fluorescence intensity; HBD, healthy blood donors; GCA, patients with giant cell arteritis; IgAV, immunoglobulin A vasculitis; SLE, systemic lupus erythematosus; SSc, systemic sclerosis; APS, antiphospholipid syndrome; ERA, early rheumatoid arthritis.Abstract THU0040 – Figure 2 Presence of antibodies against SAA1 and AGP in IVIg. ?IVIg, intravenous immunoglobulin G; SAA1, serum amyloid A1; AGP, alpha 1 acid gycoprotein; MFI, median fluorescence intensity.Sera samples were collected from HBD (n=55), patients with giant cell arteritis (GCA; n=30), immunoglobulin A vasculitis (IgAV; n=30), systemic lupus erythematosus (SLE; n=20), systemic sclerosis (SSc; n=27), antiphospholipid syndrome (APS; n=19) and early rheumatoid arthritis (ERA; n=20). Ab levels were determined also in IVIg (Octapharma AG).ResultsWe observed the presence of both, anti-SAA1, and anti-AGP Abs in sera of HBD, as well as in patients with GCA, IgAV, SLE, SSc, APS and ERA.GCA patients had significantly higher anti-SAA1 levels (median (IQR) MFI was 2163 (1113- 3048)) as compared to SSc (816 (480-1462); p<0.01) and ERA (886 (722-1596); p<0.05) patients. Levels of anti-AGP Abs were also significantly higher in sera of GCA patients (495 (298-872)) as compared to ERA (213 (130-338); p<0.01) and APS (264 (154-399); p<0.05) patients. No difference in anti-SAA1 or anti-AGP levels was observed between HBD and the tested groups of patients (Figure 1).Substantial amounts of anti-SAA1 Abs were observed in serially diluted IVIg, while there were ∼4-fold less anti-AGP Abs detected (Figure 2), which corresponds also to the ratio found in HBD (Figure 1).ConclusionSerum IgG Abs against SAA1 and ...