Array-in-well binding assay for multiparameter screening of phage displayed antibodies

Susan Pérez-Gamarra, Liisa Hattara, Gaurav Batra, Petri Saviranta, Urpo Lamminmäki (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

4 Citations (Scopus)

Abstract

Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10 × 10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.
Original languageEnglish
Pages (from-to)43-50
Number of pages8
JournalMethods: A Companion to Methods in Enzymology
Volume116
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Fingerprint

Bacteriophages
Assays
Screening
Antibodies
Epitopes
Epitope Mapping
Immunoglobulin Fragments
Clone Cells
Display devices
Antigens
Single-Chain Antibodies
Streptavidin
Horseradish Peroxidase
Immunoassay
Immobilization
HIV-1
Anti-Idiotypic Antibodies
Binders
Amplification
Enzyme-Linked Immunosorbent Assay

Keywords

  • antibody discovery
  • antibody phage library
  • epitope mapping
  • overlapping peptide libraries
  • protein array

Cite this

Pérez-Gamarra, Susan ; Hattara, Liisa ; Batra, Gaurav ; Saviranta, Petri ; Lamminmäki, Urpo. / Array-in-well binding assay for multiparameter screening of phage displayed antibodies. In: Methods: A Companion to Methods in Enzymology. 2017 ; Vol. 116. pp. 43-50.
@article{a6c9b1e04276453f873c380ef2eda795,
title = "Array-in-well binding assay for multiparameter screening of phage displayed antibodies",
abstract = "Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10 × 10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.",
keywords = "antibody discovery, antibody phage library, epitope mapping, overlapping peptide libraries, protein array",
author = "Susan P{\'e}rez-Gamarra and Liisa Hattara and Gaurav Batra and Petri Saviranta and Urpo Lamminm{\"a}ki",
year = "2017",
doi = "10.1016/j.ymeth.2016.12.004",
language = "English",
volume = "116",
pages = "43--50",
journal = "Methods: A Companion to Methods in Enzymology",
issn = "1046-2023",
publisher = "Academic Press",

}

Array-in-well binding assay for multiparameter screening of phage displayed antibodies. / Pérez-Gamarra, Susan; Hattara, Liisa; Batra, Gaurav; Saviranta, Petri; Lamminmäki, Urpo (Corresponding Author).

In: Methods: A Companion to Methods in Enzymology, Vol. 116, 2017, p. 43-50.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Array-in-well binding assay for multiparameter screening of phage displayed antibodies

AU - Pérez-Gamarra, Susan

AU - Hattara, Liisa

AU - Batra, Gaurav

AU - Saviranta, Petri

AU - Lamminmäki, Urpo

PY - 2017

Y1 - 2017

N2 - Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10 × 10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.

AB - Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10 × 10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.

KW - antibody discovery

KW - antibody phage library

KW - epitope mapping

KW - overlapping peptide libraries

KW - protein array

UR - http://www.scopus.com/inward/record.url?scp=85008214363&partnerID=8YFLogxK

U2 - 10.1016/j.ymeth.2016.12.004

DO - 10.1016/j.ymeth.2016.12.004

M3 - Article

VL - 116

SP - 43

EP - 50

JO - Methods: A Companion to Methods in Enzymology

JF - Methods: A Companion to Methods in Enzymology

SN - 1046-2023

ER -