“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-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.