Two-Step in Vitro Antibody Affinity Maturation Enables Estradiol-17β Assays with More than 10-Fold Higher Sensitivity

Kobayashi, N.; Oyama, Hiroyuki; Kato, Yoshinori; Goto, Junichi; Söderlind, Eskil; Borrebaeck, Carl

doi:10.1021/ac902283n

Cited by 52 publications

(147 citation statements)

References 64 publications

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“…14,16) The primers used were THC#33V H -Rev and THC#33V H -For (for amplifying V H ) and THC#33V L -Rev and THC#33V L -For (for amplifying V L ), respectively ( Table 1). Amplification products were gel-purified and spliced by overlap extension PCR 13,14) to generate a gene encoding "wild-type" scFv (wt-scFv), which was then subcloned into the NcoI-SalI site in pEXmide 5, 25) and propagated in Escherichia coli (E. coli) XL1-Blue cells (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…14,20) The pEXmide 5 containing the wtscFv gene (1 ng) was mixed in a buffer solution (100 µL) with or without 0.10 mM MnCl 2 , THC#33V H -Rev and THC#33V HFor (or THC#33V L -Rev and THC#33V L -For) primers (Table 1) (0.10 nmol each), AmpliTaq DNA polymerase (Applied Biosystems) (5 U), and either of the following unbalanced deoxyribonucleotide triphosphates (dNTPs): 0.10 µmol of each dNTP except deoxyadenosine triphosphate (dATP) (0.020 µmol) or 0.10 µmol of each dNTP except deoxyguanosine triphosphate (dGTP) (0.020 µmol). This mixture was amplified for 35 cycles at 95°C (1 min), 50°C (1 min), and 72°C (3 min), followed by a 10 min extension at 72°C.…”

Section: Methodsmentioning

confidence: 99%

“…The mutated V H and V L genes that were amplified using the same unbalanced dNTPs (the products obtained with 0 and 0.10 mM MnCl 2 were combined) were spliced to produce scFv genes. 13,14) The two gene libraries of the mutated scFv ("dATP-diminished V H /V L " and "dGTPdiminished V H /V L " combinations) were separately ligated into the pEXmide 5, 25) and transformed into XL1-Blue cells. The resulting bacterial libraries were used to rescue phage particles, which were partially purified and examined as follows.…”

Section: Methodsmentioning

confidence: 99%

“…1/6 as molecular mass, but which often retain the antigen-binding capacity of the parent antibodies. [10][11][12][13][14] These scFvs can be expressed from a single transcript, and thus can be fused with a variety of functional proteins, such as enzymes and fluorescent proteins, to generate "clonable labeled antibodies" with distinct properties. [15][16][17] Q-body, a family of innovative antibody reagents particularly suitable for on-site testing was recently developed.…”

Section: 6)mentioning

confidence: 99%

“…Thus we established novel hybridoma clones producing usable antibodies against THC, amplified the genes encoding the variable regions thereof, assembled the scFv, and improved its affinity for THC via in vitro affinity maturation, i.e., "molecular breeding." 14,20) …”

Section: 6)mentioning

confidence: 99%

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Antibody Fragments for On-Site Testing of Cannabinoids Generated <i>via in Vitro</i> Affinity Maturation

Morita

Oyama

Yasuo

et al. 2017

Biological & Pharmaceutical Bulletin

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Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ 9 -tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with K a 6.2 10 7 M 1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with K a , 1.1 10 7 M 1 was prepared by bacterial expression of a fusion gene combining the V H and V L genes for Ab-THC#33. Subsequently, random point mutations in V H and V L were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (K a 1.1 10 8 M 1 ) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the V H -complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

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