Figure 1.
Figure 1. Anti-RARα antibody production in APL mice and patients. (A) Comparative anti-RARα ELISA dose-response curves with a positive mouse serum and an anti-RARα monoclonal antibody control. SA measured by ELISA of a serum from an ATRA-treated mouse serially diluted from 1:50 to 1:10 000 (▵) and from the anti-RARα monoclonal antibody 9αF diluted from 1:10 000 to 1:200 000 (reference curve, □). (B) Time-dependent presence of anti-RARα antibodies in mice with APL. Anti-RARα antibodies were measured by ELISA in 48 mice with APL. On days 15 to 18, all mice were alive, and sera were obtained from only 46 mice, including 23 mice treated by ATRA and 23 treated by placebo. On days 28 to 38, 30 mice were alive, including 24 mice treated by ATRA and 6 treated by placebo. On days 48 to 58, only 13 mice, treated by ATRA, were alive. The box plot in combination with dot plot displays a statistical summary of the data (quartiles and median). Data are expressed as arbitrary units. (C) Time course of the IgG and IgM SA in the anti-RARα ELISA of 1 ATRA-treated APL mouse. For IgG and IgM detection, secondary antibodies specific for mouse IgG and IgM were used in ELISA tests. In 1 ATRA-treated mouse the anti-RARα ELISA was performed in serum collected on days 18, 28, and 38 using specific secondary antibodies for mouse IgG (•) and mouse IgM (▪). Results are expressed as SA. (D) Time-dependent presence of anti-RARα antibodies in APL patient sera. Anti-RARα antibodies (expressed as SA) were measured by ELISA in the serum of 9 patients with APL at diagnosis and after maintenance therapy. ELISA tests were performed in 96-well plates (Nunc; Merck Eurolab, VWR, Fontenay-Sous-Bois, France) coated with either GST or GST-RARα (obtained by fusion of a GST tag to the full-length RARα). After overnight saturation with PBS1X-BSA5%, sera (diluted 1:200 in PBS1X-BSA5%) were incubated 1 hour at room temperature. Peroxidase-conjugated goat anti–human IgG antibody (Sigma-Aldrich, Lyon, France) was added and incubated 1 hour at room temperature, followed by TMB substrate revelation (BD Pharmingen, San Diego, CA). Absorbance was measured at 450 nm (reference filter at 630 nm) using an optical densitometer (Dynatech MR5000; Labsystems, Cergy, France). For each serum, SA was calculated as the difference between duplicates of mean absorbance between GST-RARα and GST. Anti-RARα antibodies were also measured in sera from 18 healthy persons (controls). The box plot in combination with dot plot displays a statistical summary of the data (quartiles and median).

Anti-RARα antibody production in APL mice and patients. (A) Comparative anti-RARα ELISA dose-response curves with a positive mouse serum and an anti-RARα monoclonal antibody control. SA measured by ELISA of a serum from an ATRA-treated mouse serially diluted from 1:50 to 1:10 000 (▵) and from the anti-RARα monoclonal antibody 9αF diluted from 1:10 000 to 1:200 000 (reference curve, □). (B) Time-dependent presence of anti-RARα antibodies in mice with APL. Anti-RARα antibodies were measured by ELISA in 48 mice with APL. On days 15 to 18, all mice were alive, and sera were obtained from only 46 mice, including 23 mice treated by ATRA and 23 treated by placebo. On days 28 to 38, 30 mice were alive, including 24 mice treated by ATRA and 6 treated by placebo. On days 48 to 58, only 13 mice, treated by ATRA, were alive. The box plot in combination with dot plot displays a statistical summary of the data (quartiles and median). Data are expressed as arbitrary units. (C) Time course of the IgG and IgM SA in the anti-RARα ELISA of 1 ATRA-treated APL mouse. For IgG and IgM detection, secondary antibodies specific for mouse IgG and IgM were used in ELISA tests. In 1 ATRA-treated mouse the anti-RARα ELISA was performed in serum collected on days 18, 28, and 38 using specific secondary antibodies for mouse IgG (•) and mouse IgM (▪). Results are expressed as SA. (D) Time-dependent presence of anti-RARα antibodies in APL patient sera. Anti-RARα antibodies (expressed as SA) were measured by ELISA in the serum of 9 patients with APL at diagnosis and after maintenance therapy. ELISA tests were performed in 96-well plates (Nunc; Merck Eurolab, VWR, Fontenay-Sous-Bois, France) coated with either GST or GST-RARα (obtained by fusion of a GST tag to the full-length RARα). After overnight saturation with PBS1X-BSA5%, sera (diluted 1:200 in PBS1X-BSA5%) were incubated 1 hour at room temperature. Peroxidase-conjugated goat anti–human IgG antibody (Sigma-Aldrich, Lyon, France) was added and incubated 1 hour at room temperature, followed by TMB substrate revelation (BD Pharmingen, San Diego, CA). Absorbance was measured at 450 nm (reference filter at 630 nm) using an optical densitometer (Dynatech MR5000; Labsystems, Cergy, France). For each serum, SA was calculated as the difference between duplicates of mean absorbance between GST-RARα and GST. Anti-RARα antibodies were also measured in sera from 18 healthy persons (controls). The box plot in combination with dot plot displays a statistical summary of the data (quartiles and median).

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