title: Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen.
abstract: BACKGROUND
  Antibodies constitute a powerful tool to study protein function, protein localization and protein-protein interactions, as well as for diagnostic and therapeutic purposes. High-throughput antibody development requires faster methodologies with lower antigen consumption.
  
  
  RESULTS
  Here, we describe a novel methodology to select human monoclonal recombinant antibodies by combining in vitro protein expression, phage display antibody libraries and antibody microarrays. The application of this combination of methodologies permitted us to generate human single-chain variable fragments (scFvs) against two proteins: green fluorescent protein (GFP) and thioredoxin (Trx) in a short time, using as low as 5 μg of purified protein. These scFvs showed specific reactivity against their respective targets and worked well by ELISA and western blot. The scFvs were able to recognise as low as 31 ng of protein of their respective targets by western blot.
  
  
  CONCLUSIONS
  This work describes a novel and miniaturized methodology to obtain human monoclonal recombinant antibodies against any target in a shorter time than other methodologies using only 5 μg of protein. The protocol could be easily adapted to a high-throughput procedure for antibody production.
authors.affiliation: Functional Proteomics Laboratory, Centro de Investigaciones Biológicas (CIB-CSIC), Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Babel
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Barderas
authors.forename: Alberto
authors.initials: A
authors.lastname: Peláez-García
authors.forename: J Ignacio
authors.initials: JI
authors.lastname: Casal
mesh_terms: D000941:Antigens / Q000276:immunology*; D049452:Green Fluorescent Proteins / Q000276:immunology; D006801:Humans; D019151:Peptide Library; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013879:Thioredoxins / Q000276:immunology
chemical_list: D000941:Antigens; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins; D013879:Thioredoxins