Identification of LPS Peptide Mimics using Phage Display

Identification of the LPS peptide mimics was performed by using a 7-mer Phage display peptide library referred to as Ph.D.-7 (New England BioLabs, Ipswich, MA). Panning was performed according to manufactures specifications using a LPS specific antibody as the target. The LPS antibody (Abcam, Cambridge, MA, CAT# ab35654) used for the panning was a monoclonal antibody, which was produced by vaccinating mice with whole Escherichia coli O111B4J5 cells. Although it is not known by the manufacturer as to which structural component of LPS is recognized by the antibody, it is specific to LPS and has been used in various publications [31] (link), [32] (link). Briefly, 1 µg of the LPS antibody was absorbed to one well of a 96 well plate in a total volume of 100 µl of PBS overnight at 4°C. Phages (2×10¹¹) were added to the well and allowed to adhere at room temperature after which non-binding phages were washed away and bound phages eluted. Eluted phages were subsequently amplified and used for further panning so as to enrich for LPS antibody specific phages. The panning procedure described was repeated three times. Lastly, twelve random phage clones were selected from three rounds of panning and specificity to LPS antibody (black bars) was confirmed by ELISA using HSP70 antibody (ENZO life sciences, Farmingdale, NY) (white bars) as a negative control. The species for both the anti-LPS and anti-HSP70 antibodies used were mouse. Bound phages were detected by using a HRP labeled anti-M13 phage antibody (Abcam) followed by adding the HRP substrate SIGMAFAST™ OPD (Sigma, St. Louis, MO) and absorbance measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA). The panning, titer determination, and ELISA experiments were performed according to the manufacture's protocol. Once their reactivity was determined, the DNA encoding the peptide sequence from each positively reacting phage clone was purified using QIAprep Spin M13 Kit (Qiagen, Valencia, CA) and were sequenced by Genewiz (Genewiz, South Plainfield, NJ) using the primers supplied in the kit (New England BioLabs). The LPS peptide mimics sequenced were synthesized by Genemed Synthesis and their purity was greater than 95% (Genemed Synthesis, San Antonio, TX).