AVS Bio - Antibody Based Assay for PAD activity (ABAP)

The Antibody Based Assay for PAD activity (ABAP) is a solid Enzyme-linked immunosorbent assay (ELISA) for the determination of PAD enzyme activity in cell and tissue lysates. Furthermore it can be used in combination with recombinant PAD enzymes in PAD inhibition assays. Arginine containing peptides have been coated onto strips of a 96-well plate. On each strip, well H is coated with a deiminated arginine-containing peptide, serving as a positive control. During the first incubation with recombinant PAD enzymes or cell/tissue lysates containing PAD enzymes, arginines are being deiminated. After incubation with PAD containing solutions, the excess enzyme is washed away and a monoclonal detection antibody specific for deiminated arginine is added to the wells. After incubation wells are washed and incubated with a HRP-labelled polyclonal anti-mouse immunoglobulin antibody. Development of the plate using HRP substrate results in a staining reaction that is directly proportional to the amount of arginines that have been deiminated. The optical density measured in a plate reader can be directly correlated to the enzyme activity of the control enzyme present in the test, resulting in a quantitation of the PAD enzyme activity. The ABAP was validated using recombinant human PAD4 (Figure 1) and mouse tissue lysates of a variety of PAD expressing tissues (Figure 2).