Anti-estradiol-17β single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display

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

“…After removing the solvent, the residue was chromatographed on a silica gel (CHCl 3 /MeOH=15 : 1) to isolate ester (1) as a colorless oil (40.3 mg 17) Crude ester 3 (6.6 mg, 16 µmol) was reacted with BSA (Sigma-Aldrich) (15 mg, 0.23 µmol) in a mixture of 1,4-dioxane (1.5 mL), PB (1.5 mL), and pyridine (1.0 mL) with stirring at room temperature for 3 h, then overnight at 4°C. The reaction mixture was treated as described previously (protein precipitation with acetone and subsequent dialysis), 18) and the desired conjugate KT-BSA(b) was obtained as a 1.0-mg/mL solution in saline. The KT:albumin-coupling molar ratio was determined to be 28, based on titration of the residual amino groups with sodium 2,4,6-trinitrobenzene sulfonate.…”

“…After 3 d, splenocytes were collected from the mice and cell fusion 22) with P3/NS1/1-Ag4-1 (NS1) myeloma cells 23) was performed according to our standard procedure. 18,24,25) Briefly, splenocytes (1-3×10 8 cells) were fused with 1/5 number of NS1 cells using a 40% polyethylene glycol 4000 solution containing dimethyl sulfoxide and poly-L-arginine. Fused cells were cultured for approximately 2 weeks at 37°C in HAT (hypoxanthine-aminopterin-thymidine) medium supplemented with 10% briclone (DS Pharma Biomedical, Osaka, Japan) under 5% CO 2 /95% air.…”

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

“…Colonies were then scraped into 2×YT medium containing 15% glycerol (1.5 mL) and a small aliquot was used for bacteriophage rescue with the aid of VCSM13 helper phage (Agilent Technologies), as described previously. 13,23,24) The resulting phages were used in the next round of selection.…”

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

“…To date, we have produced affinity-matured scFv mutants binding to small biomarkers [e.g., estradiol-17β (E 2 ) [12][13][14] , cotinine 15 , cortisol 16 , and Δ 9 -tetrahydrocannabinol (THC) 17 ] to establish more sensitive immunochemical assay systems. Actually, these mutants enabled 3-100-fold higher sensitivity in competitive enzyme-linked immunosorbent assays (ELISAs), compared with the corresponding parental scFvs (Fig.…”

“…1). Because these scFvs were generated via mutagenesis based on error-prone polymerase chain reaction (PCR) experiments [12][13][14][15][16][17][18] , the missense mutations (causing amino acid substitutions) were introduced randomly. Consequently, some of them might function as "key mutations" for increasing in the affinity, whereas other mutations might be "junk mutations" that contribute little, nothing, or even decrease the affinity.…”