Specific antibodies to soluble alpha-synuclein conformations in intravenous immunoglobulin preparations

Abstract

Alpha-synuclein is the major protein in Lewy bodies, the hallmark pathological finding in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although normally intracellular, it also can be secreted, so extracellular alpha-synuclein may contribute to neuronal injury. Serum antibodies to alpha-synuclein could exert protective effects by increasing alpha-synuclein's movement out of the brain and, if they cross the blood-brain barrier, by inhibiting its neurotoxic effects. The objective of this study was to measure antibody concentrations to alpha-synuclein monomer and soluble oligomers in three intravenous immunoglobulin (IVIG) preparations, Gamunex (Talecris Biotherapeutics), Gammagard (Baxter Healthcare) and Flebogamma (Grifols Biologicals). Antibodies were measured in native IVIG preparations and after antibody-antigen complex dissociation. IVIG's non-specific binding was subtracted from its total binding to alpha-synuclein to calculate specific anti-alpha-synuclein antibody concentrations. Specific antibodies to alpha-synuclein monomer and/or soluble oligomers were detected in all IVIG products. In native IVIG preparations, the highest anti-monomer concentrations were in Gammagard and the highest anti-oligomer concentrations were in Gamunex; the extent to which lot-to-lot variation may have contributed to these differences was not determined. Antibody-antigen complex dissociation had variable effects on these antibody levels. The IVIG preparations did not inhibit alpha-synuclein oligomer formation, although they changed the distribution and intensity of some oligomer bands on Western blots. The presence of antibodies to soluble alpha-synuclein conformations in IVIG preparations suggests that their effects should be studied in animal models of synucleinopathies, as a first step to determine their feasibility as a possible treatment for PD and other synucleinopathies.

Fig. 3

Specific anti-α-synuclein monomer concentrations (means ± standard error of the mean), n = 6 wells/condition; µg of specific antibody/g total protein) in native and antibody–antigen complex-dissociated intravenous immunoglobulin (IVIG) preparations. ^aP < 0·01 versus dissociated Gamunex; ^bP < 0·01 versus dissociated Gammagard; ^cP < 0·01 versus dissociated Flebogamma; ^dP < 0·01 versus native Gamunex; ^eP < 0·01 versus native Gammagard; ^fP < 0·01 versus native Flebogamma (no specific antibodies to α-synuclein monomer were detected in native Gamunex and dissociated Flebogamma).

Fig. 1

Western blot: α-synuclein monomer (lane A; 0·02 µg) and oligomer (lane B; 1·03 µg) preparations. The oligomer preparation was not pure; densitometric analysis indicated that approximately 50% of the total band density in this preparation was due to α-synuclein monomer.

Fig. 2

Assessment of specific versus non-specific immunoreactivity of intravenous immunoglobulin (IVIG) preparations in enzyme-linked immunosorbent assay (ELISA) measuring antibodies to α-synuclein monomer. Data shown are optical density values (means ± standard error of the mean) for binding of native and antibody-antigen-dissociated IVIG preparations to wells coated with α-synuclein monomer or bovine serum albumin (BSA). ^aP < 0·001 versus BSA; ^bP < 0·01 versus BSA; ^cP < 0·05 versus BSA (GX = Gamunex, 1:100 dilution; GG = Gammagard, 1:100 dilution; FG = Flebogamma, 1:50 dilution; n = 2 for binding of dissociated Gammagard to BSA, n = 3 for other conditions).

Fig. 4

Specific anti-α-synuclein oligomer concentrations (means ± standard error of the mean), n = 6 wells/condition; µg of specific antibody/g total protein) in native and antibody–antigen complex-dissociated intravenous immunoglobulin (IVIG) preparations. ^aP < 0·01 versus dissociated Gamunex; ^bP < 0·01 versus dissociated Gammagard; ^cP < 0·05 versus dissociated Flebogamma; ^dP < 0·01 versus native Gamunex; ^eP < 0·01 versus native Gammagard; ^fP < 0·01 versus native Flebogamma; ^gP < 0·05 versus dissociated Gamunex; ^hP < 0·05 versus native Flebogamma.

Fig. 5

Binding curves for wells coated with intravenous immunoglobulin (IVIG) (Gammagard, 1 mg/ml), mouse monoclonal anti-α-synuclein clone syn 211 (1·25 µg/ml), or bovine serum albumin (BSA) (1 mg/ml), followed by increasing concentrations of α-synuclein monomer. Levelling-off of the curve with increasing α-synuclein concentrations was observed with all three conditions.

Fig. 6

Effect of incubation intravenous immunoglobulin (IVIG) preparations on α-synuclein oligomer formation. Western blot shows products resulting from incubation of disaggregated α-synuclein with IVIG products, commercial anti-α-synuclein antibodies, normal mouse IgG or buffer. Lane A: α-synuclein monomer (0·02 µg), prepared as described in text; lanes B–I, α-synuclein oligomer preparations (total α-synuclein loaded in each lane: 1·6 µg) after incubation with the following (duration of incubation: lane B, 1 day; other lanes, 4 days): lanes B and C, phosphate-buffered saline (PBS); lane D, Gammagard (1:50 dilution); lane E, Gamunex (1:50); lane F, Flebogamma (1:25); lane G, rabbit-anti-α-synuclein (1:250); lane H, mouse monoclonal anti-α-synuclein 5C2 (1:50), lane I, MOPC-21 [normal mouse IgG; 1:720 (1·25 µg/ml)].