Seeing Better Through a MIST: Evaluation of Monoclonal Recombinant Antibody Fragments on Microarrays

Anal Chem. 2004 May 15;76(10):2916-21. doi: 10.1021/ac035357a.

Abstract

Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / metabolism
  • Automation
  • Calmodulin / genetics
  • Combinatorial Chemistry Techniques / methods*
  • Cyclophilins / genetics
  • Enzyme-Linked Immunosorbent Assay / instrumentation
  • Enzyme-Linked Immunosorbent Assay / methods
  • Homeodomain Proteins / genetics
  • Humans
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / metabolism
  • Polyubiquitin / genetics
  • Protein Array Analysis / methods*
  • Proteomics / methods

Substances

  • Antibodies, Monoclonal
  • Calmodulin
  • Homeodomain Proteins
  • Immunoglobulin Fragments
  • Polyubiquitin
  • Cyclophilins