THU0040 USING A NOVEL BEAD-BASED IMMUNOASSAY FOR SIMULTANEOUS DETECTION OF AUTOANTIBODIES AGAINST SERUM AMYLOID A1 AND ALPHA1 ACID GLYCOPROTEIN

Abstract

Background Naturally 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. Objectives To 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). Methods Fluorescently 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). 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). Results We 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 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). Conclusion Serum IgG Abs against SAA1 and AGP present in HBD, patients with systemic autoimmune diseases and in IVIg can be simultaneously quantified using a customized bead-based multiplex assay. These natural autoAbs found in IVIg could be endogenous immune-regulators of the acute phase response. Acknowledgement The authors would like to acknowledge funding from the Slovenian Research Agency (ARRS) for the National Research Program P3-0314. Reference [1] Lakota K, et al. Could antibodies against serum amyloid A function as physiological regulators in humans? Autoimmunity 2011;44: 149-58. Disclosure of Interests None declared