Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

[A Novel System for Discovering High-affinity Antibody Mutants That Enables Immunoassays with Higher Sensitivities -Development and Application of Clonal Array Profiling (CAP)].

Antibody engineering is a powerful method used to generate high-affinity antibodies that enables sensitive immunoassays. It is commonly performed with the following steps: First, antibody fragments (e.g., single-chain Fv fragments; scFvs) with various mutations are displayed on the surface of filamentous bacteriophages to generate a diverse scFv-phages library. Then, rare clones with improved affinities are selected from the library via "panning" against target antigens immobilized on solid phases. However, this process often fails because of biased proliferation of scFv-phage clones and competition with large excesses of clones with weaker affinities. To overcome these limitations, we developed a clonal array profiling (CAP) method in which scFv-phage members in a library are individually examined for their antigen-binding ability. The advantage of CAP over conventional panning was evident in a comparative study that explored a library of anti-cortisol scFvs. CAP isolated five scFv mutants with >30-fold enhanced affinity (Ka) compared with wild-type scFv, enabling >11-fold more sensitive immunoassays. In contrast, no clones showing >5-fold higher Ka were found via panning. Considering the unique features of the primary structures of the improved scFvs found via CAP, we constructed new anti-cortisol scFv libraries where amino acid substitutions or insertions were introduced into the heavy-chain framework region 1 (VH-FR1). As expected, we obtained 21 mutants with >15-fold enhanced affinities. This VH-FR1-targeting mutagenesis also succeeded in generating affinity-matured scFvs against psilocybin, a hallucinogenic compound found in some mushrooms, which could be applied for developing on-site identification systems for hallucinogenic mushrooms, e.g. as immunochromatography devices.

Retrieving Dissociation-Resistant Antibody Mutants: An Efficient Strategy for Developing Immunoassays with Improved Sensitivities.

Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, "panning" has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17-31-fold improved Ka from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; Ka, 3.6 × 108 M-1), but their Ka did not exceed 1.1 × 1010 M-1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with "ordinary affinities," dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33-56-fold increased Ka (1.2-2.0 × 1010 M-1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18-51-fold higher sensitivity than the assay performed using wt-scFv.

Purification of Emu IgY for Therapeutic and Diagnostic Use Based on Monoclonal Secondary Antibodies Specific to Emu IgY.

The emu is the second largest ratite; thus, their sera and egg yolks, obtained after immunization, could provide therapeutic and diagnostically important immunoglobulins with improved production efficiency. Reliable purification tools are required to establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to the immunoglobulin Y (IgY) class. Therefore, we generated a monoclonal secondary antibody specific to emu IgY. Initially, we immunized an emu with bovine serum albumin multiply haptenized with 2,4-dinitrophenyl (DNP) groups. Polyclonal emu anti-DNP antibodies were partially purified using conventional precipitation method and used as antigen for immunizing a BALB/c mouse. Splenocytes were fused with myeloma cells and a hybridoma clone secreting a desirable secondary antibody (mAb#2-16) was established. The secondary antibody bound specifically to emu-derived IgY, distinguishing IgYs from chicken, duck, ostrich, quail, and turkey, as well as human IgGs. Affinity columns immobilizing the mAb#2-16 antibodies enabled purification of emu IgY fractions from sera and egg yolks via simple protocols, with which we succeeded in producing IgYs specific to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein with a practical binding ability. We expect that the presented purification method, and the secondary antibody produced in this study, will facilitate the utilization of emus as a novel source of therapeutic and diagnostic antibodies.

Derivatization-assisted immunoassays: application for group-specific detection of potent methamphetamine and amphetamine enantiomers.

Reliable and feasible tools for detecting (S)-methamphetamine [(S)-MAP] and (S)-amphetamine [(S)-AP] are required for regulating their illicit circulation. Antibodies that react equally to these stimulants are desirable for this purpose, but have been difficult to generate because of the crucial difference between their characteristic structures: i.e., N-methylamino (MAP) and amino (AP) groups. Furthermore, their small molecular masses (Mr < 150) have hampered the generation of high-affinity antibodies. To overcome these problems, we converted (S)-MAP and -AP into their 2-(trimethylsilyl)ethyl carbamate forms, Teoc-(S)-MAP and -AP, respectively, as surrogate analytes. The Teoc-derivatization not only increases their molecular masses, but also masks their structural differences. We generated a novel monoclonal antibody that showed a satisfactory affinity to Teoc-(S)-MAP residues (Kd = 13 nM as the IgG form) and developed a competitive enzyme-linked immunosorbent assay (ELISA) using microplates containing immobilized Teoc-(S)-MAP residues. Almost overlapping dose-response curves were obtained for Teoc-(S)-MAP and -AP, with the limit of detection of 0.078 and 0.10 ng per assay, respectively. A fixed amount of test powder sample (1 mg) was derivatized with Teoc-O-succinimidyl for 5 min, and subjected to ELISA using Teoc-(S)-MAP as the calibration standard. Under this protocol, (S)-MAP and -AP were converted to their Teoc derivatives with 30% and 34% yield, respectively, determined using ELISA as "Teoc-(S)-MAP equivalent," being distinguished from the derivatization products of (R)-MAP, (R)-AP, ephedrine, (S)-methylenedioxymethamphetamine, tyramine, dopamine, and ß-alanine. This ELISA detected as little as 10 µg of (S)-MAP and -AP, and (S)-MAP in urine obtained from (S)-MAP-administered rats. Immunochromatography devices were also developed using gold nanoparticles coated with the monoclonal antibody, with which 0.10 mg of (S)-MAP and -AP was detected by the naked eye. We conclude that the present derivatization-assisted immunoassays may be useful for the detection of (S)-MAP and/or -AP in early stage screening of suspicious substances.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17ß by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17ß (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Derivatization-assisted enzyme-linked immunosorbent assay for identifying hallucinogenic mushrooms with enhanced sensitivity.

A sensitive immunochemical method for identifying hallucinogenic mushrooms (magic mushrooms) is required for regulating their illicit use. We have previously generated a monoclonal antibody (mAb) that targets psilocin (Psi), the major psychoactive compound in hallucinogenic mushrooms, and developed an enzyme-linked immunosorbent assay (ELISA). However, this ELISA failed to achieve the expected low-picomole-range sensitivity, as a result of insufficient affinity of the mAb to Psi. It is recognized that haptenic antigens with a larger molecular mass tend to induce antibodies with higher affinities. Thus, we herein report a "derivatization-assisted ELISA," in which the "real analyte" Psi was determined as a "surrogate analyte," the tert-butyldimethylsilyl ether analog thereof (TBS/Psi) having a 1.6-fold greater molecular mass (Mr 318.53) than Psi. A novel mAb against TBS/Psi, prepared by immunizing mice with a TBS/Psi-albumin conjugate showed a 69-fold higher affinity to TBS/Psi residues (Ka = 3.6 × 107 M-1 as IgG) than that of our previous mAb against Psi. This mAb consequently enabled a competitive ELISA for measuring TBS/Psi with the desired sensitivity: the dose-response curve midpoint (12.1 pmol per assay) was >100-fold lower than that of the previous ELISA for determining Psi. Extracts of dried mushroom powders were mixed with TBS triflate for 30 min at room temperature, converting Psi into TBS/Psi in approximately 50% yield. The reaction mixture was then subjected to an ELISA using the anti-TBS/Psi mAb to determine TBS/Psi. Psilocybe cubensis, a species of hallucinogenic mushrooms, gave rise to positive signals, indicating the presence of Psi therein in the expected quantity, while no detectable response was observed for four kinds of edible mushrooms available in the markets.

NanoLuc luciferase as a suitable fusion partner of recombinant antibody fragments for developing sensitive luminescent immunoassays.

Enzyme-linked immunosorbent assays (ELISAs) are essential for monitoring various biomarkers. Competitive and noncompetitive (sandwich) assay formats are used to determine hapten and macromolecule levels, respectively. Both formats require more sensitive detection of reporter enzymes for greater assay sensitivities. We previously reported the utility of wild-type Gaussia luciferase (wtGLuc) as a fusion partner with antibody single-chain Fv fragments (scFvs) for developing sensitive luminescent ELISAs. Here, we evaluated utility of NanoLuc luciferase (NLuc), a recently developed luciferase, as fusion partner with scFvs from the view of comparison with wtGLuc and a mutant of alkaline phosphatase (ALP'). Thyroxine (T4) and T4-labeled albumin were chosen as model haptenic and macromolecular antigens, respectively. An in-house-prepared anti-T4 scFv was fused with NLuc, wtGLuc, or ALP'. The scFv-NLuc fusion protein showed 47-fold and 29-fold lower limit of detection [LOD; 59 zmol (per assay)] than the wtGLuc- and ALP'-fusions, respectively. In a competitive T4 ELISA, the NLuc-fusion showed 9.3- and 6.3-fold lower LOD, (0.67 pg) than the wtGLuc- and ALP'-fusions, respectively, with a higher specificity in clinical applications. A typical colorimetric ELISA using a peroxidase-labeled second antibody showed 70-fold higher LOD than NLuc-based ELISA. Another advantage of the NLuc-fusion was shown in the sandwich assays; the LOD of T4-labeled albumin (5.0 fmol) was >6-fold lower than that of the other luminescent ELISAs. In an additional sandwich assay developed to count bacteriophage particles, NLuc enabled more sensitive determination than wtGLuc, whereas ALP' showed nearly equivalent performance. Its slowest alteration rate for light intensity after starting the enzyme reaction should enable robust batch-by-batch assay operations. Thus, we concluded that scFv-NLuc fusions serve as suitable probes in various types of immunoassays and may facilitate higher sensitivities with practical specificities.

The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants.

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two "designed" site-directed mutagenesis (A and B) targeted the N-terminal 1-10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (Ka, 3.6 × 108 M-1). Mutagenesis A substituted the amino acids at the position 1-3, 5-7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1-6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17-31-fold increased affinity with > 109 M-1 Ka values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest Ka value, 1.1 × 1010 M-1.

Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms.

Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20-20 or 0.040-2.0 µg/assay (limit of detection was 0.14 or 0.029 µg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants.

"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32-63-fold improved Ka values (> 1010 M-1), compared with the wild-type scFv (Ka = 3.8 × 108 M-1), none of which could be recovered via conventional panning procedures operated for the entire library.

Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity.

"Antibody-breeding" has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17ß with 11 amino acid substitutions and a >100-fold improved affinity constant (Ka = 1.19 × 1010 M-1) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M-1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a "high rate of return" in terms of selecting mutants with dramatically enhanced affinities. The "high risk" of generating a tremendous excess of "junk mutants" can be overcome with the efficient selection systems that we developed.

Antibodies and Engineered Antibody Fragments against M13 Filamentous Phage to Facilitate Phage-Display-Based Molecular Breeding.

Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 107-1010 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.

A Single-Step "Breeding" Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity.

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; Ka, 3.4×108 M-1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for this antibody. Then, random point mutations were generated separately in VH or VL 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 (Ka 1.2×1010 M-1). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its VL sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response 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.

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 Ka value of 2.7 × 107 M-1 (at 4 °C) was prepared by linking the VH and VL 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 Ka (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.

Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

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.