“Breeding” Diagnostic Antibodies for Higher Assay Performance: A 250-Fold Affinity-Matured Antibody Mutant Targeting a Small Biomarker

Conventional immunization-based antibodies are not always applicable to monitor trace amounts of clinical biomarkers due to insufficient antigen-binding affinity. The development of in vitro affinity maturation for native antibodies has paved the way to solve this problem, but there has been minimal success in obtaining improved antibody mutants for small molecule biomarkers (haptens). We here describe a “molecular breeding” approach, in which a practical anti-hapten antibody mutant has successfully been generated from a prototype antibody with poor binding properties. Random point mutations were introduced by error-prone polymerase chain reaction in the <i>V</i>_<i>H</i> and <i>V</i>_<i>L</i> genes of a second-generation mutant of a single-chain Fv fragment (scFv) that binds estradiol-17β (E₂), whose parent mouse Fab fragment showed poor affinity for E₂ (<i>K</i>_a = 5.2 × 10⁷ M^–1). The resulting <i>scFv</i> gene library was expressed on filamentous phage particles. Phage clones with strong E₂ binding were isolated with dissociation-independent methods using newly developed reagents. The binding characteristics and clinical applicability of the soluble scFvs prepared from the selected clones were examined in a competitive enzyme-linked immunosorbent assay (ELISA). We finally obtained a third-generation scFv mutant, scFv#m3-<i>a</i>18 (11 amino acid substitutions), which showed ∼250-fold greater <i>K</i>_a (1.3 × 10¹⁰ M^–1) than the parent Fab, and yielded sensitive ELISA dose–response curves for E₂ (limit of detection < 0.5 pg/assay) with practical specificity. The average human serum E₂ levels, determined after acetonitrile extraction, were compatible with reported reference ranges. The present results will prompt a new era for preparing diagnostic reagents

Anti-Idiotype scFv–Enzyme Fusion Proteins: A Clonable Analyte-Mimicking Probe for Standardized Immunoassays Targeting Small Biomarkers

Most immunoassays use probes that convert concentrations of analytes into signal intensity. To prepare the probes, analytes are usually linked to a reporter protein (e.g., enzymes) with the aid of chemical reagents. However, these conventional methods yield a mixture of heterogeneous products and consequently reduce assay performance. “Clonable” homogeneous probes, i.e., recombinant molecules in which a target protein is genetically fused to a reporter with a defined coupling ratio, are now available for analyzing protein biomarker concentrations. Here, we have expanded this strategy to measure small biomarkers (haptens) by genetically fusing proteinaceous molecules that mimic target haptens with enzymes. 11-Deoxycortisol (11-DC) was chosen as the model hapten, and the β-type anti-idiotype antibodies (βId-Abs) that recognize the paratope of anti-hapten antibodies were used as the target hapten mimic. The <i>V_H</i> and <i>V_L</i> genes of a βId-Ab, targeting a mouse anti-11-DC antibody (CET-M8), were assembled to encode a single-chain Fv fragment (βId-scFv), which was then fused with a gene encoding a variant of alkaline phosphatase. The product, βId-scFv-ALP′ protein, had satisfactory enzyme activity and bound to CET-M8 in a competitive manner with 11-DC. A colorimetric enzyme-linked immunosorbent assay (ELISA) for 11-DC, based on the competitive reaction between the analyte and βId-scFv-ALP′ against immobilized CET-M8, was found to be sensitive (limit of detection = 22 pg/assay) and specific (cross-reactivity with cortisol, 0.24%) for clinical use and could be used to determine serum 11-DC levels after a simple solvent extraction. The anti-idiotype scFv–enzyme fusion proteins proposed here can be prepared reproducibly as homogeneous products with a 1:1 coupling ratio and would facilitate standardization of immunoassays for small biomarkers

<i>Gaussia</i> Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers

In this study, we show the utility of <i>Gaussia</i> 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 (V_H and V_L) were cloned and combined to encode a single-chain Fv fragment (scFv). <i>scFv</i> was then linked to the wild-type <i>GLuc</i> gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several <i>Escherichia coli</i> 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

Characteristics of Natural Gas Hydrates Occurring in Pore-Spaces of Marine Sediments Collected from the Eastern Nankai Trough, off Japan

Pore-space gas hydrates sampled from the eastern Nankai Trough area off of Japan were minutely characterized using several instrumental techniques. Gas chromatographic results indicated that the natural gas in the sediment samples studied comprises mainly CH₄. The concentrations of minor components varied according to depth. The powder X-ray diffraction patterns showed that the pore-space hydrates were of structure I (sI); the lattice constants were 1.183−1.207 nm. Both ¹³C NMR and Raman spectra confirmed that CH₄ molecules were encaged in sI hydrate lattice. The average cage occupancies were calculated, respectively, from the Raman data as 0.83 for small cages and 0.97 for large cages. The hydration numbers were determined as 6.1−6.2

One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity

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 <i>K</i>_a value of 2.7 × 10⁷ M^–1 (at 4 °C) was prepared by linking the V_H and V_L domains in a mouse anti-cotinine antibody. “One-shot” random mutagenesis on the <i>scFv-wt</i> gene by error-prone PCR generated mutant <i>scFv</i> 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 <i>K</i>_a (1.2 × 10⁹ M^–1 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