Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays.
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.
Angenendt
Philipp
P
Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. angenend@ molgen.mpg.de
Wilde
Jeannine
J
Kijanka
Gregor
G
Baars
Sabine
S
Cahill
Dolores J
DJ
Kreutzberger
Jürgen
J
Lehrach
Hans
H
Konthur
Zoltán
Z
Glökler
Jörn
J
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001331:Automation; D002147:Calmodulin / Q000235:genetics; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D021983:Cyclophilins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025821:Polyubiquitin / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D018398:Homeodomain Proteins; D007128:Immunoglobulin Fragments; D025821:Polyubiquitin; D021983:Cyclophilins