BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3272

Peptides:

Sequences

Affinity Values

LMSTCTWLDECFRPQ(6)
PTPPCHRGDECQPLW(5)
SRPHCYPMDDCHPLW(4)
WSGMCESQWCKDFA(4)
TPHTCKLLDECVPLW(3)
TPTVCTWLDECPPWS(1)
HTPWCSIADPCLWEL(1)
YHEPCLWATACPTTP(1)
MQDMCNDDSCPLWS (1)
CTHPCEPPPLWPIAP(1)
PGPQCPYPAELWCTQ(1)

[1.025]
[0.703]
[1.137]
[1.039]
[2.925]
[1.052]
[1.597]
[0.464]
[0.481]
[2.082]
[0.336]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (competitive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 24356816

Target Name:

Anti-CD43 monoclonal antibody UN1

Template Name:

Leukosialin

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

f88-Cys5 phage display library (X4CX5CX4)

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

Optical density (OD) at 405 nm (OD405) and 620 nm (OD620) were measured by an ELISA reader (Tecan), and values were expressed and shown as difference between OD405 and OD620.

Brief Description:

The UN1 mAb (10 μg) was linked to streptavidin-conjugated magnetic beads (Promega), which had been coated with 10 μg of goat anti-mouse IgG (Fc-speciﬁc) biotin-conjugated Ab (Sigma-Aldrich) in 200 μL of beads suspension. A total of 3.0e10 transducing units of phages from the library were added and incubated for 16 hours at 4 °C. After extensive washing, bound phages were eluted with 0.1 mol/L HCl/glycine buffer, pH 2.2, 1 mg/mL bovine serum albumin (BSA), and neutralized with 1 mol/L Tris, pH9.1. Eluted phages were ampliﬁed by infection of K91BK bacteria, and puriﬁed from plaques for a second round of afﬁnity selection. Phage colonies were transferred according to an ordered grid on a lawn of K91BK cells on Luria-Bertani (LB) agar plates supplemented with 1 mmol/L isopropyl b-D-1-thiogalactopyranoside. Nitrocellulose ﬁlters were layered onto these plates and incubated overnight at 37 °C. Filters were blocked for 2 hours with blocking buffer(1 × PBS, 5% non-fat dry milk, 0.1% NP40, 0.01% NaN3) at room temperature and incubated O/N at 4 °C with the UN1 mAb (1 μg/mL) in blocking buffer. Then, filters were washed with washing buffer (1 × PBS, 0.1% NP40) and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Fc speciﬁc) secondary antibody (SigmaAldrich) at the dilution of 1:5000 for 4 hours at 4 °C. After extensive washing, filters were incubated in developing solution (1-Step NBT/BCIP, Pierce, Thermo Scientiﬁc). Collected phage supernatants from positive clones were further analyzed by ELISA.

BiopanningDataSet Comments:

By screening a phage-displayed random peptide library, we identified the phagotope 2/165 with the sequence of TPHTCKLLDECVPLW as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy.

Curator:

Bifang He

Curation Date:

2018-01-19

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