Antibody arrays for high-throughput screening of antibody–antigen interactions

Abstract

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.

Protein Expression Profiling of Breast Cancer Cells by Dissociable Antibody Microarray (DAMA) Staining

“…As the protein expression profiles for each cell line were determined at least twice, there are two independent DAMA staining images for each spot: two for the sample cell line (S1 and S2) and two for the reference cell line (R1 and R2). Therefore, four different sets of MRAT values, MRAT (1,1) , MRAT (1,2) , MRAT (2,1) and MRAT (2,2) , corresponding to the intensity ratios of S1 to R1, S1 to R2, S2 to R1, and S2 to R2, respectively, were obtained for every spot(i, j) (Fig. 2a).…”

“…DISCUSSION DAMA staining is a novel platform for protein microarray analysis that can determine both expression and subcellular localization profiles of hundreds of proteins in targeted cell and tissue samples. 2 We developed this unique technology for protein expression profiling and developed a program, DAMAPEP, for intensity integration and data analysis. We demonstrated the application of this technology for potential biomarker identification by comparing the expression profiles of 312 proteins among 10 different breast cell lines.…”

“…Protein microarrays have recently been attracting great attention for their potential use in high throughput studies of protein function (1)(2)(3)(4)(5)(6). The ultimate goal of developing this technology is to construct ordered arrays of individual proteins for biochemical study at the molecular level.…”

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer

“…Bait molecules are printed onto a variety of substrate surfaces, including membranes [26], modified glass surfaces [25], and hydrogels [27]. Proteins and antibodies are generally immobilized via non-covalent adsorption or covalent coupling to amine-reactive surfaces.…”

Perspectives for systematic in vitro antibody generation

“…Current large-scale strategies for the screening of enriched phage display libraries are based on the concept of the arrayed library (Lennon and Lehrach, 1991). Using picking and spotting technology (Eickhoff et al, 2002), thousands of antibody-producing clones can be picked and gridded on membranes as bacterial clone arrays, and screened for specific binding with labelled selection targets (De Wildt et al, 2000). Most commonly, however, single colonies are picked into 96-or 384-well microtitre plates to generate antibody phage producing monoclonal cultures, and subsequent test of antibody specificity is done by enzyme linked immunosorbant assay (ELISA) on solid phase immobilised selection targets.…”