Development of an immunoassay for fluvoxamine detection using a recombinant single-chain variable fragment antibody

Sasao, Ako; Takaki, Michiyo; Ohtsu, Yuki; Mishima, Satoko; Yonemitsu, Kosei; Morioka, Hiroshi; Nishitani, Yoko

doi:10.1007/s11419-017-0358-9

Cited by 1 publication

(6 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[5][6][7] In our previous study, we challenged ourselves to develop a drug screening method for psychotropic drugs using a recombinant antibody such as a single chain variable fragment (scFv). 8 Both the parental and scFv versions of the anti-FLV antibody we developed showed strong FLV binding activities and we demonstrated that anti-FLV scFv could be applied as a tool to screen for FLV. Recombinant antibodies have several advantages, such as (a) they can be produced using in vitro expression systems which is cost-effective and (b) mutations using genetic engineering techniques can be introduced for molecular enhancement of antigen-binding performance.…”

Section: Introductionmentioning

confidence: 81%

“…Fluvoxamine maleate and ovalbumin (OVA) were obtained from Sigma‐Aldrich (St Louis, MO, USA). Fluvoxamine conjugated ovalbumin (OVA‐FLV) was prepared and used as immobilized antigen for enzyme‐linked immunosorbent assays (ELISAs) as described previously . ATTO520‐C2‐maleimide (ATTO520) was obtained from ATTO‐TEC (Siegen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…A V H ‐V L type anti‐FLV scFv gene with an internal (G 4 S) 3 linker sequence was amplified from pETCF‐HL using primers 1 and 2 (Table ). The resulting PCR product was inserted into Nde I/ Xho I‐digested FLVscFv plasmid using Ligation high Ver.…”

Section: Methodsmentioning

confidence: 99%

“…14 Reactivity of anti-FLV scFv against FLV was assayed using an ELISA described previously, with slight modifications. 8 In brief, 1.0 μg OVA-FLV was coated in each well of a 96-well microtiter plate. The plate was then blocked with 0.5% gelatin.…”

Section: Preparation Of Scfv Proteinsmentioning

confidence: 99%

“…In our previous study, we challenged ourselves to develop a drug screening method for psychotropic drugs using a recombinant antibody such as a single chain variable fragment (scFv) . Both the parental and scFv versions of the anti‐FLV antibody we developed showed strong FLV binding activities and we demonstrated that anti‐FLV scFv could be applied as a tool to screen for FLV.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Development of a fluvoxamine detection system using a Quenchbody, a novel fluorescent biosensor

Sasao

Takaki

Jeong

et al. 2018

Drug Testing and Analysis

Self Cite

View full text Add to dashboard Cite

The misuse of psychotropic drugs intended for medical treatment represents a recent worldwide public health concern. Quenchbody (Q‐body) is a novel fluoroimmunosensor that can detect an antigen immediately without additional reagents or washing steps. Here, we describe creating Q‐bodies for the detection of the antidepressant fluvoxamine (FLV) and determining optimal conditions to achieve the highest fluorescence intensity (FI). We prepared five Q‐bodies with the fluorophore labeled at either the N‐ or C‐ terminus and with different linker lengths. Fluorescence was measurable within minutes, indicating the interaction of Q‐bodies with FLV. The normalized FI (FI ratio) of the N‐terminus labeled Q‐body increased approximately 1.5‐fold upon FLV addition; Q‐bodies labeled at the C‐terminus did not significantly increase FI. Among the fluorescence dyes used in this study, Rhodamine 6G labeled Q‐body showed the best FI ratio. EC50 values of the N‐terminus labeled Q‐bodies were similar (23.2–224nM) regardless of linker length or labeling dye. We examined whether the Q‐body could be applicable to serum matrix instead of phosphate‐buffered saline. The intact serum interfered strongly with the Q‐body fluorescence. However, the FI ratios of the Q‐body for FLV‐spiked serum filtrate, for which proteins were removed by filtration, showed a dose‐dependency for detecting FLV levels. Deproteinization, which does not interfere with Q‐body fluorescence measurements, is likely necessary to detect serum FLV with high sensitivity. This study demonstrates the potential of Q‐body probes as a tool towards developing creative immunoassay applications.

show abstract