HuMabs recognize different regions of the hSOD1 protein.
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(A) Full-length hSOD1 (amino acids 1–153) and various portions of the protein were expressed in bacteria fused to the carboxy-terminus (C-terminus) of thioredoxin (Trx) and containing a C-terminal 6-histidine tag used for purification (Trx-hSOD1-WT-his). Each protein is represented in the figure as a hashed line with the beginning and ending amino acid number listed below the line. The proteins were coated on ELISA plates and binding of HuMabs (listed at the top right) detected with goat-anti-human antibody conjugated to alkaline phosphatase followed by PNPP substrate addition. ELISA results are listed to the right of the schematic; positive recognition is indicated by a plus sign while signals equivalent to background are indicated by a minus sign. HuMabs recognizing only full-length hSOD1 were designated conformation dependent and are noted below with a C. HuMabs with an epitope that mapped to a linear sequence of amino acids are noted below with an L. (B) Minimal linear epitopes were determined with amino-terminal biotin-labeled overlapping peptides coated on streptavidin ELISA plates. Binding of HuMabs was assessed as described in A. The epitopes are noted as a grey box for each linear-epitope HuMab with the amino acids (aa) bound indicated. To distinguish these epitopes throughout the rest of the manuscript each linear-epitope HuMab has a subscripted L for linear followed by the initial amino acid of the epitope.