No abstract

Cortisol levels in bodily fluids represent a useful index for pituitary adrenal function, and thus practical anti-cortisol antibodies are required. We have studied "antibody-breeding" approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A "wild-type" single-chain Fv fragment (wt-scFv; K a , 3.4 10 8 M 1 ) was prepared by bacterial expression of a fusion gene combining the V H and V L genes for this antibody. Then, random point mutations were generated separately in V H or V L by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity (K a 1.2 10 10 M 1 ). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its V L sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated doseresponse curves with measuring range ca. 0.03-0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.Key words antibody; single-chain Fv fragment; cortisol; in vitro evolution; affinity maturation; enzymelinked immunosorbent assay Immunoassays are essential tools for monitoring various biomarkers in bodily fluids because of the excellent specificity conferred by antigen−antibody reactions. 1) Currently, most diagnostic antibodies are produced by B-cell hybridoma technology, 2,3) which generates "native" antibodies (in vivo antibodies) induced in animals by immunization as cloned products ensuring constant binding abilities. However, the limited B-cell clone repertoire in mammals often prevents the generation of antibodies with practical performance. In particular, the equilibrium affinity constant (K a ) of antibodies against small biomarkers (i.e., haptenic compounds) rarely exceeds the 10 10 M −1 range. 4,5)We have employed an "antibody-breeding" approach to overcome this limitation inherent to native antibodies and to enable subfemtomole detection of small molecules. 5) Using this approach, the antigen-binding affinity can be enhanced by in vitro mutagenesis and subsequent selection of improved species (i.e., in vitro affinity maturation).3,5-9) Typically, the antibody of interest is converted to the corresponding singlechain Fv fragment (scFv) [10][11][12] or Fab fragment (Fab) by ...

In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings.

Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ 9 -tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with K a 6.2 10 7 M 1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with K a , 1.1 10 7 M 1 was prepared by bacterial expression of a fusion gene combining the V H and V L genes for Ab-THC#33. Subsequently, random point mutations in V H and V L were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (K a 1.1 10 8 M 1 ) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the V H -complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers. A "wild-type" scFv (scFv-wt) with a K value of 2.7 × 10 M (at 4 °C) was prepared by linking the V and V domains in a mouse anti-cotinine antibody. "One-shot" random mutagenesis on the scFv-wt gene by error-prone PCR generated mutant scFv genes, which were expressed on phage particles. Repeated panning directed toward mutants with slower off-rates selected scFv clones that showed improved sensitivity in an ELISA system. One of these mutants (scFv#m1-54) with five amino acid substitutions showed more than a 40-fold enhanced K (1.2 × 10 M at 4 °C) and, thus, was used to monitor human urinary cotinine. A limited amount of soluble scFv was reacted with urine specimens (or cotinine standards) at 4 °C for 120 min in microwells on which cotinine residues had been immobilized. The midpoint of the dose-response curves under optimized conditions (0.27 ng/assay) was more than 100-fold lower than the ELISA results obtained using scFv-wt. The limit of detection (8.4 pg/assay) corresponded to 0.17 ng/mL urinary cotinine, which was satisfactorily low for testing the threshold levels for passive smoke exposure. The assay values for volunteers correlated with the values determined using a commercial assay kit. This study evidently showed the potential of a molecular breeding approach, in which simple in vitro evolution might generate superior antibody reagents as cloned proteins, overcoming the limited molecular diversity inherent to conventional immunization-based antibodies.