A80229ap

Ninety-six-well ELISA plates (Costar) were coated with 8 μg/ml of hIgG1-YTE/KF diluted in PBS, and incubated ON at 4 °C. Plates were blocked with PBS/S for 1 h at RT, and then washed four times with PBS/T. His-tagged hFcRn (10 μg/ml) in pH 5.5 or pH 7.4 PBS/S/T was added and incubated for 1 h at RT. The plates were washed four times with either pH 5.5 or pH 7.4 PBS/T, before serial dilutions (15,000–7.3 ng/ml) of WT HSA or HSA-EQTMVAKP in pH 5.5 or pH 7.4 PBS/S/T were added, and incubated for 1 h at RT. The plates were washed four times as above, before alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat (#A80229AP; Bethyl Laboratories, Inc., 1:5000), diluted (1:3000) in pH 5.5 or pH 7.4 PBS/S/T was added for 1 h at RT. The plates were washed four times and developed as above.

Ninety-six-well ELISA plates (Costar) were coated with 1.0 µg/ml of polyclonal goat anti-HSA (#A1151, Sigma-Aldrich) or polyclonal anti-MSA (#ab19194, Abcam) antibodies, 1.0 µg/ml diluted in PBS, and incubated ON at 4 °C. Plates were blocked with PBS/S ON at 4 °C and washed four times with PBS/T before serial dilutions of MSA or HSA (250.0–0.1 ng/ml) in PBS/S/T were applied in parallel with samples from HERA followed by 2 h incubation at RT. Bound MSA and HSA were detected using horseradish peroxidase-conjugated polyclonal anti-MSA antibody from goat (#ab19195; Abcam, 1:4000) or alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat (#A80229AP; Bethyl Laboratories, Inc., 1:5000). ELISAs were developed by adding 100 µl of 3,3´,5,5´-tetramethylbenzinide solution (Calbiochem) and the reaction was stopped by adding 100 µl of 1 M HCl for MSA, while for HSA 100 µl of the p-nitropenylphospate substrate (Sigma-Aldrich) diluted to 10 µg/ml in diethanolamine buffer was added. The absorbance was measured at 450 or 405 nm using a Sunrise spectrophotometer (TECAN).