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

Morita, Izumi; Oyama, Hiroyuki; Kanda, Yui; Yasuo, M; Aya, Ito; Toyota, Masahiro; Hayashi, Yoshinori; Yokoyama, Takeshi; Kobayashi, N.

doi:10.1248/bpb.b17-00762

Cited by 9 publications

(10 citation statements)

References 33 publications

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“…Dissociation rate constants ( k d ) of the soluble scFvs obtained by the ORD selection were determined by the BLI 24 at 25 °C using BLItz (ForteBio), a BLI sensor. Streptavidin-coated biosensor tips, which were saturated with a biotin-labeled CS-BSA, prepared by reaction with EZ-Link NHS-LC-Biotin (Thermo Fisher Scientific) 44 , 45 , were dipped into 4.0-µL scFv solutions in G-PBS (50–1,600 nmol/L). Association of the scFvs with the cortisol residues was monitored for 120 s, and then dissociation was measured for 600 s in G-PBS.…”

Section: Methodsmentioning

confidence: 99%

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

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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"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 (~ 10 5 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 K a values (> 10 10 M −1 ), compared with the wild-type scFv ( K a = 3.8 × 10 8 M −1 ), none of which could be recovered via conventional panning procedures operated for the entire library.

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Section: Methodsmentioning

confidence: 99%

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

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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“…Production and Partial Purification of Emu Polyclonal Immunoglobulins 4-(2,4-Dinitrophenylamino) butyric acid (CAS 10466-75-8) was coupled with bovine serum albumin (BSA) using the N-hydroxysuccinimide ester (NHS) method 11) to prepare an immunogenic conjugate (DNP-BSA; coupling molar ratio 11 : 1) (Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

“…After three days, splenocytes obtained from the mouse were fused with P3/NS1/1-Ag4-1 (NS1) myeloma cells. 11,13) The hybridomas producing antibodies that are estimated to be specific to emu IgYs (eIgYs) were selected, expanded, and then cloned. The established clones were cultured for 7-10 d, and the antibody (mAb#2-16) secreted in the supernatant was precipitated by adding 50%-saturated (NH 4 ) 2 SO 4 and affinity-purified with an in-house-prepared emu-Ig-immobilized column to remove bovine immunoglobulins present in the hybridoma culture media (Fig.…”

Section: Generation Of Monoclonal Secondary Antibody Specific To Emu Igymentioning

confidence: 99%

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

Yamaki

Ohta

Kobayashi

et al. 2022

Biological & Pharmaceutical Bulletin

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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.

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“…S1 †). [13][14][15][16] The serum titer of antibodies against the hapten moieties was examined using ELISA (described in ESI †), and four mice that gave rise to favorable responses received intraperitoneal and intrasplenic injections 17 of the conjugate. Aer 3 days, splenocytes from these mice were fused with P3/NS1/1-Ag4-1 (NS1) myeloma cells 18 (Health Science Research Resources Bank; Sennan, Osaka, Japan) as described previously.…”

Section: Generation Of Mabs Via Cell Fusionmentioning

confidence: 99%

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

et al. 2022

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

Abstract: Ketamine (KT) is a chiral anesthetic agent, (R)-and (S)-

Cited by 9 publications

References 33 publications

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

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

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

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

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