ELISA method to detect α-synuclein oligomers in cell and animal models

Abstract

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson's disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers.

Fig 1. Specificity and affinity of the MJF14-6-4-2 antibody.

A. Monomers and oligomers were isolated by gel filtration and eluted in two distinct fractions with peak values corresponding to 60 kDa and 800 kDa, respectively (grey broken line). The absorbance profile at 214 nm is indicated at the right ordinate axis. To validate the specificity of the oligomer ELISA we incubated antibody coated wells with the same concentration of monomer and oligomer (9 ng/mL) and used a monoclonal anti-a-syn antibody as second antibody. Bound antibody was detected by anti-mouse HRP and the TMB color change was measured as absorbance at 450 nm. The ELISA gives a strong signal for oligomers whereas there is only a negligible signal from the monomer sample (black bars). Background absorbance was subtracted. The ELISA signal (absorbance at 450 nm) is presented at left ordinate axis. B. We compared the aggregate specificity of the MJF14-6-4-2 antibody with the well characterized polyclonal antibody FILA-1 using dot blotting analysis. Dilutions of monomers (M) and oligomers (O) were immobilized and tested for their antibody binding. The non-conformation dependent monoclonal a-syn antibody (BD) (0.5 μg/ml) demonstrated equal loading of monomer and oligomer. MJF14-6-4-2 (Abcam) (2.2 ng/ml) and FILA-1 (3.5 μg/ml) antibodies display the same extend of selectivity for the aggregated a-syn. C. The affinity of MJF14-6-4-2 antibody was assessed by a direct ELISA on oligomer coated wells. The MJF14-6-4-2 (15.6 ng/ml) antibody was pre-incubated overnight with increasing concentrations of monomer or oligomer before being applied to the oligomer containing wells. The concentration of oligomer that gave 50% inhibition was measured to be 42 ng/ml. The K_D-value of MJF14-6-4-2 was calculated to be 2.9 · 10⁻¹⁰ M based on the assumption that 10 a-syn monomers are required to generate a folding specific MJF14-6-4-2 epitope. Both A and C show one representative experiment of 3. Standard deviations represent three technical replicates.

Fig 2. A-syn oligomer ELISA sensitivity and specificity.

A. Test of three different concentrations (0.125, 0.0625, and 0.03125 μg/ml) of capture antibody MJF14-6-4-2 (MJF) for monomeric and oligomeric a-syn for the oligomer ELISA. 0.0625 μg/ml is chosen as the optimal concentration for MJF14-6-4-2 based on sensitivity and specificity from monomeric a-syn. B. The oligomer ELISA detects oligomeric a-syn in a linear range from 0.25–20 ng/ml oligomer a-syn with the use of MJF14-6-4-2 as capture antibody and the anti-a-syn (BD) as detection antibody (R² = 0.99). Monomeric a-syn is not detected by the oligomer ELISA in this range. Negative control (PBS) shown in light grey. C. The graph from C. zoomed in on the lower part of the X-axis. The oligomer ELISA detects down to 0.25 ng/ml oligomeric a-syn (P = 0.001). D. Determination of inter-assay coefficient of variation (CV). Comparison of results from four different runs is used to determine CV for the oligomer ELISA. CV is calculated for 6 different concentrations of oligomers (1.25 ng/ml– 40 ng/ml) resulting in 6 different CVs values all below 6%—in average 4,5%. E. Recovery by recombinant a-syn oligomers spiked into WT mouse brain synaptosomes. Mouse brain synaptosomes are spiked with different amounts of oligomers (1, 2, 3, and 5 ng/ml). Expected increase in absorbance is calculated from an internal series of controls, and the recovery rate is calculated for each concentration of spiked amount of oligomer. All recovery rates are above 95%—in average 98%. Data are an average of 4 different experiments run in triplicates. Data are compared to unspiked for significance. F. Validation that a-syn oligomer signal is caused by conformational epitopes. Treatment with 8M urea inhibits the a-syn oligomer signal on the oligomer ELISA. Higher concentrated oligomers are treated for 3 hours with 8M urea, and then diluted to 3mM urea before addition to the ELISA. G. To validate that the urea treatment does not affect the binding capability of the ELISA or/and the antibodies used, the experiment was run with longer incubation time, an oligomer concentration of 2.5 ng/ml, and low amounts of urea equivalent to the amount of UREA present in the urea treated oligomers after dilution (urea concentration during running conditions = 3 mM). 8M urea treatment reduces the oligomer signal, whereas 3mM urea treatment does not remove the oligomer signal. “None” represents non-treated a-syn oligomers, whereas background signal from PBS without a-syn is shown by the dotted grey line. Both G. and H. show one representative experiment of 3. Standard deviations represent three technical replicates. Significance for all parts of figure is marked with an asterisk: *p<0.05; **p<0.005. Results were compared using one-way ANOVA followed by Turkey’s post hoc test for multiple comparison.

Fig 3. Validation of total a-syn level by a total a-syn ELISA.

A. Standard a-syn ELISA is used as control with our in-house ASY-1 antibody as capture antibody. The ASY-1 antibody detects monomeric a-syn in linear range up to 2.5 ng/ml (R² = 0.99). The detection is saturated at 10 ng/ml. B. The graph from E. zoomed in on the lower part of the X-axis. The ASY-1 ELISA detects down to 0.16 ng/ml of monomeric a-syn (P = 0.02). Negative control (PBS) shown in light grey.

Fig 4. Detection of oligomeric a-syn in synaptosomes isolated from whole brains of mThy-h-a-syn transgenic mice (ASO) and verification of folding dependent epitopes.

Oligomer a-syn (A.) and total a-syn (B.) levels are measured in whole brain synaptosomes from adult ASO (n = 3) and WT (n = 3) mice that were extracted in detergent containing RIPA buffer and analyzed by ELISA for their a-syn content. A. The oligomer a-syn ELISA reveals increased oligomer levels in the ASO mice compared to the WT mice. The oligomer signal is aggregate specific because no increase is obtained by spiking the sample with increasing amounts of recombinant monomer standards B. The total level of a-syn is increased in ASO mice compared to WT and spiking the sample with the same recombinant monomeric a-syn standard as used in panel A increases the level of total a-syn when more than 5 ng/ml monomer is added. B. Bars in A and B represents standard deviation of three biological replicates (ASO and WT). Significance is marked with an asterisk: *p<0.05; **p<0.005 and indicate in (A.) and (B.) the given sample related to wt. Results were compared using one-way ANOVA followed by Turkey’s post hoc test for multiple comparison. C. Folding specificity of the MJF14 binding to a-syn species in synaptosomes was analyzed by dot blotting in the absence and presence of denaturing 50% formic acid (FA). Dilution of RIPA extracts of ASO mouse brain synaptosomes was applied to the dot blots in the absence and presence of treatment with denaturing 50% formic acid (FA). The filters were tested for binding of MJF14-6-4-2 to detect the aggregate dependent epitope (left panel-oligomer) and ASY-1 to detect total a-syn (right panel-total a-syn). Right dot blot shows the total level of a-syn, which is equal in the two samples treated or untreated with FA. FA treatment removes the epitope for the MJF14-6-4-2 antibody but not the ASY-1 epitopes confirming the folding specificity of the MJF14-6-4-2 epitope.

Fig 5. Time dependent development of a-syn oligomers in a non-mitotic a-syn transgenic cell model.

A-syn SH-SY5Y Tet-off cells were differentiated with retinoic acid to induce a non-mitotic state where after a-syn expression was induced by removal of doxycycline. PBS background is shown by the dotted grey line. A. Measurement of a-syn by the total a-syn ELISA in RIPA extracts demonstrated a linear increase in cellular a-syn that demonstrates approximately a doubling of a-syn from day 5 to day 10. B. Analysis of the RIPA extracts by the oligomer ELISA demonstrates no detectable oligomers after 5 days but a significant increase after 10 days. Bars represents standard deviation of 3 biological experiments (all run in three technical replicates). The Student’s unpaired t-test was used to compare 5 and 10 days. Significance is marked with an asterisk: *p<0.05; **p<0.005. For A., 5 days is also significantly different from PBS background, whereas 5 days in B. cannot be distinguished by PBS.