title: Generation of llama single-domain antibodies against methotrexate, a prototypical hapten.
abstract: Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.
authors.affiliation: Inserm, Université de Nantes, Nantes Atlantique Universités, U601, Département de Recherche en Cancérologie, 9 quai Moncousu, F-44000 Nantes, France.
authors.forename: Nidia
authors.initials: N
authors.lastname: Alvarez-Rueda
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Behar
authors.forename: Virginie
authors.initials: V
authors.lastname: Ferré
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.forename: Françoise
authors.initials: F
authors.lastname: Roquet
authors.forename: Louis
authors.initials: L
authors.lastname: Gastinel
authors.forename: Catherine
authors.initials: C
authors.lastname: Jacquot
authors.forename: Jacques
authors.initials: J
authors.lastname: Aubry
authors.forename: Daniel
authors.initials: D
authors.lastname: Baty
authors.forename: Jacques
authors.initials: J
authors.lastname: Barbet
authors.forename: Stéphane
authors.initials: S
authors.lastname: Birklé
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002161:Camelids, New World / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006241:Haptens / Q000276:immunology; D008727:Methotrexate / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D006241:Haptens; D019151:Peptide Library; D011994:Recombinant Proteins; D008727:Methotrexate