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Fig. 5 | Molecular Neurodegeneration

Fig. 5

From: Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis

Fig. 5

Anti-SOD1int antibody exclusively recognizes soluble disulfide-crosslinked SOD1 oligomers in vitro. a The antibodies were tested for their specific reactivities to soluble disulfide-crosslinked oligomers (black filled bars) over Cu,Zn-SOD1(WT)S-S (open bars) and E,E-SOD1(A4V)S-S (gray filled bars) by indirect ELISA. Antisera were either affinity-purified with the corresponding peptides (w/o absorption) or first absorbed with SOD1(WT)S-S and then affinity-purified with the peptides (w/ absorption). Anti-SOD148–53 antibody obtained after the absorption exclusively reacted with soluble disulfide-crosslinked oligomers and called anti-SOD1int antibody. b-d The reactivities of b anti-SOD1int, c USOD-like, and d SEDI-like antibody were examined with indirect ELISA. Several forms of SOD1 (WT, A4V, G37R, G85R) with a distinct metallation/disulfide status, soluble disulfide-crosslinked oligomers and insoluble amyloid-like aggregates were prepared and fixed on an ELISA plate. The ELISA signal was represented as a ratio against that obtained using BSA. Three independent experiments were performed to estimate error bars (standard deviation). Fixation of equal amounts of SOD1 proteins on each well of an ELISA plate was confirmed by ELISA using polyclonal anti-SOD1 antibody (FL-154, Santa Cruz Biotechnology), which is shown in Additional file 6: Figure S5

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