Fig 1.
Fractionation of polyclonal antibodies.
(A) Illustration depicting the scheme for decomposition of polyclonal antibodies into fractions targeting conformational and linear epitopes. First columns containing the antigen’s protein tag, peptides corresponding to previously mapped linear epitopes, a mix of overlapping peptides covering the antigen sequence and the antigen used for immunization are serially connected. Polyclonal serum is run through the columns where anti-tag antibodies are depleted by the first column, the peptide columns capture antibodies targeting the different linear epitopes and the antigen column binds the remaining antibodies that are targeting conformational epitopes. The columns are then separated from each other and the different antibody fractions are eluted in parallel. (B) An example of results from validation of a polyclonal antibody and antibody fractions towards the target protein tryptophanyl-tRNA synthetase. The left column shows epitope mapping confirming the peptide specificity of the different fractions. The middle column shows the relative antibody amount in each fraction. The right column shows the ability of each antibody fraction to detect a band of expected molecular weight, indicated by an arrow, in Western blots assays with RT-4 and U-251 MG lysates.
Fig 2.
Proportion of antibodies towards linear and conformational epitopes in eight polyclonal sera.
Each protein target is represented in grey with the amino acid length indicated under the bar. Antigens used for immunization are shown above in green with the amino acid lengths shown above. Pie charts to the right of each protein show the relative amount of purified antibodies toward conformational (light blue) and linear (dark blue) epitopes.
Fig 3.
Western blot performance of antibody fractions.
(A) The ability of the different antibody fractions to detect a protein of the expected molecular weight in Western blot is shown in green (positive) or red (negative). The number indicates the relative amount in percent of each antibody fraction and a total of antibody fractions towards linear epitopes that are able to bind the target protein in Western blot. (B) Western blots analysis of eight different protein targets. Detection of protein targets using polyclonal antibody, antibody fraction towards conformational epitopes and one representative antibody fraction towards a linear epitope. Expected molecular weights are indicated by arrows on the right of each set of Western blots. Lysate panels for the target proteins: SYNJ2BP (Marker, RT-4), OTC (Marker, liver), TYMP (Marker, liver), CRABP2 (Marker, LY419677), WARS (Marker, RT-4, U-251 MG), PDXP (Marker, RT-4, liver), CD4 (Marker, U251-MG, plasma, tonsil), EGFR (Marker, A431).
Fig 4.
Structure analysis of epitopes.
3D structures of protein targets with highlighted linear epitopes in green where the corresponding antibody fraction showed bands of correct molecular weight in Western blot analysis and in red where the antibody fraction did not bind the target protein. SYNJ2BP (2eno.pdb), TYMP (2jof.pdb), WARS (1r6t.pdb), CD4 (1wio.pdb), OTC (1oth.pdb), CRABP2 (2g7b.pdb), PDXP (2cft.pdb) and EGFR (3njp.pdb).