BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3549

Peptides:

Sequences	Affinity Values
SQSGPTIFYNLA(13) AESRRPFFEPFM(2) FNPSFAKMGNSL(1) SALTSAVSGSAA(1)	[2.203160±0.009388] [2.833720±0.009388]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 32808238

Target Name:

Anti-epstein-barr virus (EBV) multiple sclerosis(MS) brain IgG antibodies (MS 95–2)

Template Name:

Epstein-barr virus (EBV)

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-12 phage display library (X12)

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

Purified MS brain 95–2 IgG in 0.1 M carbonate buffer (50 μl, 200 μg/ml) was coated onto wells of ELISA plates overnight at 4 °C. The wells were then blocked with 3% BSA for 2 h followed by incubation with purified phage 3–2 and 3–3 (5 × 109/well) for 1 h. After washing with 0.05% Tween-20/Tris buffered saline (TBST), the wells were incubated with a 1:500 dilution of mouse anti-M13 IgG-HRP (New England BioLabs) antibody for 1 h, followed by incubation with peroxidase substrate ABTS (Vector Laboratories, Burlingame, CA) for 20 min. The optical absorbance was measured at 415 nm with a Microplate Manager (BioRad, Hercules, CA). All samples were tested in duplicate and the ELISA was repeated at least one additional time. OD415 were reproduced from Figure 3A and shown.

Brief Description:

Wells were blocked with 3% BSA in Tris-buffed saline (TBS) for 2 h, washed 6 times with 0.1% Tween20–TBS (TBST), and the phage peptide library (1.5e11 phage in 100 μl of TBST) was added to the wells and incubated for 2 h at room temperature. Wells were washed 20–30 times with TBST, and bound phage were eluted with 100 μl of 0.2 M glycine, pH 2.2/0.1% BSA for 10 min. The phage-containing solution was immediately neutralized with 15 μl of 1 M Tris, pH 9.5, and eluted phage were titered and reamplified. Amplified phage (e11 pfu) from the first round of panning was applied to additional rounds of panning using the same conditions as in the first pan, except that the concentration of Tween20 in the washing buffer was increased to 0.5%. After the third round of panning, affinity-selected phage were titered. Single plaques were amplified and the peptides were sequenced.

BiopanningDataSet Comments:

We screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays.

Curator:

Chunchao Pu

Curation Date:

2020-12-29

First |

Prev |

3549

| Next

| Last