“Cleavable” hapten–biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

• Published in: Analytical biochemistry Vol. 387; no. 2; pp. 257 - 266
• Main Authors: Kobayashi, Norihiro, Oyama, Hiroyuki, Nakano, Masanori, Kanda, Tatsuaki, Banzono, Erika, Kato, Yoshinori, Karibe, Tsuyoshi, Nishio, Tadashi, Goto, Junichi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2009
• Subjects: 17-alpha-Hydroxyprogesterone - immunology; Amino Acid Sequence; Antibody engineering; Base Sequence; Biotin; Estradiol - immunology; Estradiol-17β; Hapten; Haptens - immunology; Hydrocortisone - immunology; Immunoglobulin Fragments - biosynthesis; Molecular Sequence Data; Phage display; Protein Engineering; Single-domain antibody fragment; Steroid–biotin conjugate; Estradiol-17β; Steroid–biotin conjugate; Phage display; Antibody engineering; Single-domain antibody fragment; Hapten
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.01.004

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-17β [E 2], cortisol, and 17α-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.