title: Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae.
abstract: Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.
authors.affiliation: Unilever Research Vlaardingen, The Netherlands. leon.frenken@unilever.com
authors.forename: L G
authors.initials: LG
authors.lastname: Frenken
authors.forename: R H
authors.initials: RH
authors.lastname: van der Linden
authors.forename: P W
authors.initials: PW
authors.lastname: Hermans
authors.forename: J W
authors.initials: JW
authors.lastname: Bos
authors.forename: R C
authors.initials: RC
authors.lastname: Ruuls
authors.forename: B
authors.initials: B
authors.lastname: de Geus
authors.forename: C T
authors.initials: CT
authors.lastname: Verrips
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D000946:Antigens, Fungal / Q000276:immunology; D001709:Biotechnology / Q000379:methods; D002161:Camelids, New World / Q000276:immunology*; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000276:immunology; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014227:Triazines / Q000737:chemistry / Q000276:immunology
chemical_list: D000946:Antigens, Fungal; D006063:Chorionic Gonadotropin; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D012333:RNA, Messenger; D014227:Triazines; C003213:Procion Brilliant Red M-2BS; C042516:reactive red 120 dye