title: HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species.
abstract: BACKGROUND
  The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories.
  
  
  RESULTS
  HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets.
  
  
  CONCLUSIONS
  HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.
authors.affiliation: Icosagen Cell Factory OÜ, Eerika tee 1, Õssu village, Ülenurme parish, Tartumaa, 61713, Estonia. gaily.kivi@icosagen.ee.|University of Tartu, Institute of Technology, Nooruse1, Tartu, 50411, Estonia. gaily.kivi@icosagen.ee.
authors.forename: Gaily
authors.initials: G
authors.lastname: Kivi
authors.affiliation: Icosagen Cell Factory OÜ, Eerika tee 1, Õssu village, Ülenurme parish, Tartumaa, 61713, Estonia. kaupo.teesalu@icosagen.ee.
authors.forename: Kaupo
authors.initials: K
authors.lastname: Teesalu
authors.affiliation: Estonian Biocentre, Evolutionary Biology Group, Tartu, 51010, Estonia. jyri.parik@gmail.com.
authors.forename: Jüri
authors.initials: J
authors.lastname: Parik
authors.affiliation: Icosagen Cell Factory OÜ, Eerika tee 1, Õssu village, Ülenurme parish, Tartumaa, 61713, Estonia. elen.kontkar@gmail.com.
authors.forename: Elen
authors.initials: E
authors.lastname: Kontkar
authors.affiliation: University of Tartu, Institute of Technology, Nooruse1, Tartu, 50411, Estonia. mart.ustav.jr@ut.ee.
authors.forename: Mart
authors.initials: M
authors.lastname: Ustav
authors.affiliation: University of Tartu, Institute of Technology, Nooruse1, Tartu, 50411, Estonia. Liis.Noodla@entsik.ee.
authors.forename: Liis
authors.initials: L
authors.lastname: Noodla
authors.affiliation: Icosagen Cell Factory OÜ, Eerika tee 1, Õssu village, Ülenurme parish, Tartumaa, 61713, Estonia. mart.ustav@icosagen.ee.|University of Tartu, Institute of Technology, Nooruse1, Tartu, 50411, Estonia. mart.ustav@icosagen.ee.|Estonian Academy of Sciences, Kohtu 6, Tallinn, 10130, Estonia. mart.ustav@icosagen.ee.
authors.forename: Mart
authors.initials: M
authors.lastname: Ustav
authors.affiliation: Icosagen Cell Factory OÜ, Eerika tee 1, Õssu village, Ülenurme parish, Tartumaa, 61713, Estonia. andres.mannik@icosagen.ee.
authors.forename: Andres
authors.initials: A
authors.lastname: Männik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D002645:Chickens; D003584:Cytological Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D019151:Peptide Library; D011994:Recombinant Proteins