Development of an immunoaffinity chromatographic adsorbent using a moderate affinity, anti-salbutamol chicken scFv for high sensitivity LC-MS/MS detection of salbutamol in porcine urine

• Published in: Microchemical journal 2024-10, Vol.205, p.111246, Article 111246
• Main Authors: Warren, Lee, Ali, Syed Atif, Leow, Chiuan Yee, Tan, Soo Choon, Leow, Chiuan Herng
• Format: Article
• Language: English
• Subjects: Chicken scFv; LC-MS/MS; Moderate affinity antibody; Salbutamol; scFv-immunoaffinity chromatography
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.111246

[Display omitted] •Developed a proof-of-concept scFv-based immunochromatographic (IAC) assay to detect a widely misused substance, salbutamol.•The IAC assay demonstrated the application of moderate-affinity antibodies for high-sensitivity detection platforms. Widespread misuse of salbutamol as an athletic performance enhancer and livestock growth promoter necessitates its detection in biological matrices. Antibodies remain important immunodiagnostic reagents in the detection of salbutamol. Previously, a novel anti-salbutamol chicken scFv (clone OP2B1C4) was generated from a SAL-KLH-immunized chicken antibody phage display library. This scFv (KD = ∼0.592 μM) was used to develop an indirect competitive (ic) ELISA capable of detecting salbutamol at the high ng/mL to low μg/mL levels for human doping urinalysis but was incapable of low ng/mL detection. In this study, the same scFv OP2B1C4 was used to develop a prototype immunoaffinity chromatographic (IAC) adsorbent for higher sensitivity applications. The scFv was immobilized on CNBr-activated Sepharose 4B and the scFv-IAC was profiled for its binding capacity and recovery. From three consecutive runs, estimated binding capacity was > 7 μg with elution recoveries of 59.43 %, 69.34 % and 73.8 %. When used as a clean-up SPE prior to LC-MS/MS analysis, the scFv-IAC could isolate salbutamol at a concentration of 1 ng/mL (∼100-fold lower than ic-ELISA LOD) in salbutamol-spiked porcine urines with relative recoveries ranging from 76.96 % − 99.85 %. The results highlight the potential of often-neglected moderate-affinity antibodies for high-sensitivity applications through extension onto IAC-based immunodiagnostic platforms.