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.

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.

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.

PM Q-probe: A fluorescent binding protein that converts many antibodies to a fluorescent biosensor.

Quenchbody (Q-body) is a fluorescent biosensor in which a fluorescent dye is tagged near the antigen binding site of an antibody. The fluorescence of the dye is quenched by the tryptophan residues present in the variable region of the antibody, and is recovered when the antigen binds. Q-bodies have been prepared using recombinant DNA technology by introducing one or more tag sequence(s) at either the N-terminal of the Fab or the single chain variable region fragment of the antibody, and labeling the tag with a fluorescent dye. However, preparation of recombinant antibody fragments is time-consuming and the performance of the Q-body is unpredictable. Here we report an antibody-binding quenching probe made from protein M from Mycoplasma genitalium that can transform the IgG antibody into an immunosensor. By using bacterially expressed and purified protein M and labeling the C-terminal cysteine-containing tag, we prepared a TAMRA-labeled PM Q-probe. When the Q-probe was incubated with Fab or IgG recognizing the bone Gla protein, the fluorescence of the probe was quenched and subsequently recovered by the adding of antigens in a dose-dependent manner. We also succeeded in detecting several small biomarkers with nanomolar sensitivity, including thyroxine extracted from human serum. The clone found to be suitable for the detection of cortisol was confirmed to work as a recombinant Q-body as well, which also worked in 50% human serum. The results suggest that the Q-probe can quickly convert an IgG to a biosensor, which will be useful in rapid diagnosis of small biomarkers.

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.

Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use.

Ketamine (KT) is a chiral anesthetic agent, (R)- and (S)-enantiomers of which differ in their pharmacological properties. KT has become one of the most commonly used illicit drugs in the world, thus, rapid and feasible on-site testing is required to crack down on the illicit use. Although immunochemical approach with specific antibodies is promising for this purpose, in practice anti-KT antibodies are difficult to obtain. We here disclose generation of monoclonal antibodies against KT. Mice were immunized with either (a) commercially-available or (b) in-house-prepared KT-albumin conjugates. Splenocytes from these mouse groups (a and b) were separately fused with P3/NS1/1-Ag4-1 myeloma cells. After standard screening and cloning, we established 5 hybridoma clones: 2 were derived from group-a mice [generating Ab-KT(a)#2 and #37] and 3 were from group-b mice [generating Ab-KT(b)#9, #13, and #45]. These antibodies exhibited practical performance in competitive enzyme-linked immunosorbent assay systems. When (±)-KT·hydrochloride (HCl) was used as the competitor, dose-response curves showed midpoint values of 30 and 70 ng/assay (a-series antibodies) and 2.0-3.0 ng/assay (b-series antibodies). Remarkably, the a-series antibodies were specific for (S)-KT·HCl, while the b-series antibodies were specific for (R)-KT·HCl. Ab-KT(a)#2 (Ka, 7.5×107 M-1) and Ab-KT(b)#45 (Ka, 7.7×108 M-1) exhibited the highest enantioselectivity for each group, and cross-reactivity with the (R)- and (S)-antipodes was 1.3 and 1.7%, respectively. The hybridomas established here are also valuable as a source of genetic information for the anti-KT antibodies, which is required for progressing to next-generation technologies using genetically engineered antibodies.

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies.

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1-43 or 44-65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44-65 restored the immunoreactivity of mAbs whose epitope being within residues 44-65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

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.

Simultaneous determination of histamine and prostaglandin D2 using an LC-ESI-MS/MS method with positive/negative ion-switching ionization modes: application to the study of anti-allergic flavonoids on the degranulation of KU812 cells.

A new method for simultaneous determination of histamine and prostaglandin D(2) (PGD(2)) by liquid chromatography-electrospray ionization tandem mass spectrometry operated in positive and negative ionization switching modes was developed and validated without a previous derivatization step. This method was used to measure histamine and PGD(2) release following degranulation of KU812 human basophilic cells, using pyrazol and d(4)-PGD(2) as internal standards. Analyses were performed on a liquid chromatography system employing a Cosmosil 5C(18) PAQ column and an isocratic elution with mixed solution of methanol-water (7:3, v/v) with 0.0015% trifluoroacetic acid at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer operating in selected reaction monitoring mode simultaneously monitored using the following transitions: positive m/z 112/95 for histamine and negative m/z 351/271 for PGD(2). The retention times of histamine and pyrazol were 4.2 and 5.0 min, respectively. PGD(2) and d(4)-PGD(2) had retention times of 8.5 min. The limits of detection were 0.3 and 0.5 ng/mL for histamine and PGD(2), respectively. The relative standard deviations of the retention time and peak area for histamine were between 1.6% and 7.7%, and were 1.2% and 7.8% for PGD(2). This method was used to evaluate the anti-allergic effects of 26 flavonoids and sodium cromoglicate which are first-line anti-allergic drugs. Of these compounds, baicalein and morin were the most potent inhibitors.

Correlation between cytotoxic activities and reduction potentials of heterocyclic quinones.

To search for possible anti-tumor agents or anti-tumor promoters among natural or synthetic products, we used cyclic voltammetry to determine the reduction-oxidation potentials of heterocyclic quinones in phosphate buffer at pH 7.2. We determined the growth inhibitory- and cytotoxic activities of 12 heterocyclic quinone anti-tumor agent candidates against a panel of 39 human cancer cell lines (JFCR39). The average concentrations of the heterocyclic quinones required for 50% growth inhibition (GI(50)) against JFCR39 ranged from 0.045 to 13.2 µM, and the 50% lethal concentration (LC(50)) against JFCR39 ranged from 0.398 to 77.7 µM. The average values of GI(50) or LC(50) of the heterocyclic quinones correlated significantly with their reduction potentials. These results suggested that reduction-oxidation potentials could be a useful method for the discovery of novel antitumor agents.

Correlation between reduction potentials and inhibitions of Epstein-Barr virus activation by anthraquinone derivatives.

As a continuation of studies using natural and synthetic products as cancer chemopreventive agents, we used cyclic voltammetry to examine the reduction-oxidation potentials of methylated emodin derivatives prepared from emodin in phosphate buffer at pH 7.2. A good correlation was found between the inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation and the reduction potential of methylated emodin derivatives. Furthermore, there was significant correlation between EBV-EA activation and the reduction potential of 35 anthraquinone derivatives including methylated emodin derivatives. It was further shown that the correlation could be enhanced by including LUMO energy and the number of hydroxy groups as additional parameters.

Correlation between oxidation potentials and inhibitory effects on Epstein-Barr virus activation of flavonoids.

The oxidation potentials of fifteen flavonoids in phosphate buffer at pH 7.2 were determined by cyclic voltammetry. A good correlation was found between these oxidation potentials and the ability of flavonoids to inhibit Epstein-Barr virus early antigen (EBV-EA) activation. Furthermore, multiple regression analysis revealed that the solvent-accessible surface area (SASA) was a useful parameter for estimating the inhibitory effects of flavonoids on EBV-EA activation.

Correlation between reduction potentials and inhibitory effects on Epstein-Barr virus activation by emodin derivatives.

As a continuation of studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction-oxidation potentials of hydroxylated emodin derivatives prepared from emodin in phosphate buffer at pH 7.2 using cyclic voltammetry. A significant correlation was found between the reduction potentials and number of the hydroxyl groups and the inhibitory effects of the hydroxylated emodin derivatives on Epstein-Barr virus early antigen activation. The electronic properties, i.e. LUMO energy and atomic charges of carbon at the 9-position (C(9)) and oxygen at the 11-position (O(11)), may also be useful for estimating the inhibitory effect on EBV-EA activation.

Correlation between reduction potentials and inhibitory effects on Epstein-Barr virus activation of poly-substituted anthraquinones.

As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction potentials of some poly-substituted anthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the reduction potentials and the inhibitory effects of the poly-substituted anthraquinones on Epstein-Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing log P as an additional parameter.

Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones.

As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some 2-azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the 2-azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has been further shown that the correlation can be enhanced by introducing an electronic properties, i.e. the atomic charges at the C5 and O12 atoms in the quinone skeleton ring and the HOMO energy as additional parameters.

Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of naphthoquinones.

As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some naphthoquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the naphthoquinones on Epstein-Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing an electronic property, i.e. the atomic charges at the C(4) and O(10) atoms in the quinone skeleton ring as additional parameters.

Chemopreventive effects of emodin and cassiamin B in mouse skin carcinogenesis.

In continuation of our works of natural and synthetic products as cancer chemopreventive agents, we have examined emodin and cassiamin B, which were isolated from Cassia siamea. These compounds exhibited the remarkable anti-tumor promoting effect on two-stage carcinogenesis test of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter by both topical application. Furthermore, emodin exhibited potent inhibitory activity on two-stage carcinogenesis test of mouse skin tumors induced by nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide as an initiator and TPA as a promoter.