“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments |
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Authors: | Norihiro Kobayashi Hiroyuki Oyama Tatsuaki Kanda Yoshinori Kato Tadashi Nishio Junichi Goto |
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Affiliation: | a Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan b Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan c Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo 105-8512, Japan d Department of Pharmaceutical Sciences, Tohoku University Hospital, Aoba-ku, Sendai 980-8574, Japan |
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Abstract: | 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 [E2], 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 E2-biotin conjugate was used to generate anti-E2 single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the VH domain of a mouse anti-E2 antibody, and these products were expressed as phagemid particles that were reacted with the E2-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. |
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Keywords: | Antibody engineering Phage display Hapten Estradiol-17β Steroid-biotin conjugate Single-domain antibody fragment |
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