LD50 on Rabbit RBC cell line

Activity

Median Lethal Dose (LD50)

ng/mL

Spectrophotometric/Colorimetric Assay

None

None

None

10 μg/mL

450 nm

None

The rabbit red blood cell (RBC) hemolytic assay was performed as described previously (DOI: 10.1128/CVI.05589-11). Briefly, wild-type AT (WT-AT) or AT mutants were serially diluted 2-fold in 50 μL starting at 10 μg/mL and incubated with 50 μL of washed RBC (Pel-Freez) for 1 h at 37°C. Plates were then centrifuged at 1200 rpm for 3 min, and 50 μL of supernatant was transferred to new plates. Nonspecific human IgG1 R347 was used as a negative control (c-IgG). The optical density at 450 nm (OD450) was measured with a spectrophotometer (Molecular Devices). Hemolytic activity of each AT mutant was calculated in hemolytic units per milliliter (HU/mL) as the inverse dilution corresponding to 50% hemolysis, with 10 μg/mL corresponding to 1 HU/mL.