"Cleavable" hapten-biotin conjugates: preparation and use for the generation of anti-steroid single-domain antibody fragments.
Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17beta [E(2)], cortisol, and 17alpha-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E(2)-biotin conjugate was used to generate anti-E(2) single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V(H) domain of a mouse anti-E(2) antibody, and these products were expressed as phagemid particles that were reacted with the E(2)-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.
Kobayashi
Norihiro
N
Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan. no-kobay@kobepharma-u.ac.jp
Oyama
Hiroyuki
H
Nakano
Masanori
M
Kanda
Tatsuaki
T
Banzono
Erika
E
Kato
Yoshinori
Y
Karibe
Tsuyoshi
T
Nishio
Tadashi
T
Goto
Junichi
J
D019326:17-alpha-Hydroxyprogesterone / Q000276:immunology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin; D004958:Estradiol / Q000276:immunology*; D006241:Haptens / Q000276:immunology*; D006854:Hydrocortisone / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D015202:Protein Engineering
D006241:Haptens; D007128:Immunoglobulin Fragments; D004958:Estradiol; D019326:17-alpha-Hydroxyprogesterone; D001710:Biotin; D006854:Hydrocortisone