Anti rabbit alkaline phosphatase conjugated secondary antibody

Purified human Cp (Vital Products) was coated on Nunc MaxiSorp® flat-bottom 96 well plates (Nalgene). 100 nM solution of purified human MPO (Lee Biosolutions), either alone or preincubated with excess amount of free Cp or ascorbate oxidase (Sigma), was added into wells and incubated for 1 hour at room temperature. Bound MPO was detected by incubating plate with rabbit anti-human MPO antibody (DAKO), anti-rabbit alkaline phosphatase-conjugated secondary antibody (Abcam), and alkaline phosphatase substrate solution (Sigma), respectively. The absorbance was read on microplate reader at 405 nm.

Each of the 87 peptides spanning the entire amino acid sequence of human Cp was designed at 24 residues long and to overlap by 12 residues with preceding and 12 residues with following peptide. The peptides were synthesized (Mimotopes) and coated on 96 well plates (Nalgene). A total of 87 plates were designed and used for each individual peptide by coating one half of the 96-well plate with peptide and the other half with Cp as a control. MPO, either alone or pre-incubated Cp or AO, was added to the wells. Bound MPO was detected by incubating the plates with rabbit anti-human MPO antibody (DAKO), anti-rabbit alkaline phosphatase-conjugated secondary antibody (Abcam), and alkaline phosphatase substrate solution (Sigma), respectively. The absorbance was read by microplate reader at 405 nm.