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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Aggregate-selective antibody attenuates seeded aggregation but not spontaneously evolving disease in SOD1 ALS model mice

Fig. 1

Mouse monoclonal hSOD1 antibodies specifically target disease associated hSOD1 species. a Graph represents binary epitope mapping patterns of strain A and strain B hSOD1 aggregates formed in vivo (modified from Bergh et al. [6]). Each bar represents the relative immunoreactivity of distinct epitopes of the hSOD1 protein sequence in dot blot assays of filter trapped hSOD1 aggregates from spinal cord homogenates. The aa 57–72 (green) and 131–153 (red) epitopes are available for binding in both A-, and B-aggregate strains, and were selected for the generation of aggregate reactive mouse monoclonal antibodies (mAbs). b Schematic representation of the hSOD1 protein sequence highlighting the epitopes used for immunisation. In the native protein, these epitopes are likely to be engaged in a rigidly folded structure and unable to adapt for antibody binding. In contrast, these epitopes are free and accessible for binding in disease-associated, unfolded/disordered, and aggregated hSOD1 species. c Immunoblots of immunocaptured native and denatured hSOD1 using mAbs immobilized on Dynabeads. To remove any traces of disordered hSOD1 in the native samples we performed two sequential incubations (1′ and 2′) of each sample. Input (1/200th of total input), non-bound (n.b., 1/200th of sample) and immunocaptured (i.c., 1/200th of sample) fractions were immunoblotted using rabbit antibody targeting aa 24–39 of hSOD1 (rbAb αSOD124–39). d Graph showing relative reactivity of αSOD1 mAbs to full-length denatured hSOD1, or short peptides within the aa 57–72 (green bars) or aa 131–153 sequence (red bars) by ELISA. The target sequence of mAb clones 5.7 and 134.2 is within aa 65–72 and both mAb clones 85.11 and 545.2 target an epitope within the aa 143–153 sequence. e Western blots show mAb binding to murine and/or the Tg human SOD1 protein in spinal cord homogenates from C57BL/6J non-Tg control (Wt) and hSOD1G85R Tg mice. SOD1 knockout (KO) spinal cord homogenate was included as negative control. See Additional file 1: Figure S1a for a full view of the filter. f The human SOD1 (hSOD1) peptide sequences used for immunization aligned to the murine SOD1 (mSOD1) epitope sequences. The specific mAb target sequences determined by the short peptide ELISAs are indicated in green and red. Non-conserved amino acids that distinguish murine, from the human sequence are highlighted in black. g SPR sensorgrams showing the binding of three antibodies towards different concentrations of denatured hSOD1. (h) Table summarizing the association rate (Ka), dissociation rates (Kd) and equilibrium dissociation constants (KD) of each mAb. Ka and Kd were calculated with the BIAevaluation software (Biacore AB) selecting Fit kinetics simultaneous Ka/Kd (Global fitting) and 1:1 (Langmuir) interaction model. There was no data obtained from mAb 545.2, possibly due to low affinity. ms−1—millisecond, M—molar, s−1—second

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