Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library

• We performed biopanning of phage displayed-peptide library to identify peptides that selectively bind to myoglobin and successfully identified myoglobin-binding peptides.
• Cross binding activity of the myoglobin-binding peptides to other cardiac biomarkers, such as troponin I, was minimal.
• We set up a sandwich assays for detection of myoglobin using clinical serum, where the selected peptides effectively captured and detected myoglobin.
• Both peptide–peptide and antibody based sandwich assays were similar in terms of sensitivity and specificity.
• Such biomarker-binding peptides with a high affinity would be a cost effective alternative to antibodies as a diagnostics.

Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd = 57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd = 125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide–peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.