One-Step Detection of Aflatoxin-B1 Using scFv-Alkaline Phosphatase-Fusion Selected from Human Phage Display Antibody Library

Abstract

A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1. Such an antibody has major potential applications in therapy and diagnostics. To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent. The performance of this reagent was further characterized, demonstrating its efficacy. The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form. The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries. Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets. Equally, the approach demonstrated should also be applicable for other haptens and larger antigens.

Generation of recombinant scFv antibody against Ochratoxin A (OTA)

“…Biopanning was performed according to Rangnoi et al (2011) using Yamo I.3 (Pansri et al 2009) and rabbit Bozmix library, which were constructed as described above. One well of Immuno 96 microWellTM plate (Nunc, Denmark) was coated with 25 µg of OTA-BSA conjugate in PBS buffer and incubated at 4°C for overnight.…”

“…The expression of soluble scFv antibody was performed as previously described (Rangnoi et al 2011). Approximately 10-20 µL of phage particles were mixed with 500 µL of log-phase (OD 600 = 0.4) E. coli HB2151 (nonsuppressor host), and incubated at 37°C for 30 min without shaking.…”

“…The scFv-AP was constructed as previously described (Rangnoi et al 2011). Briefly, the scFv gene from the phagemid vector was digested with Nco I and Not I restriction enzymes and sub-cloned into pKP300∆III, an alkaline phosphatase fusion vector (Kay and Hoess 1996).…”

“…The scFv-AP ELISA was performed as previously described (Rangnoi et al 2011). Briefly, triplicate wells of microtiter plate (Immuno 96 Wells TM , Nunc, Denmark) were immobilized with 2 µg of OTA-BSA in 100 µL PBS or 1% (w/v) BSA in PBS (a negative control).…”

“…Analytical methods such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography (GC) (Turner et al 2009) have been used to control OTA that contaminates both food and feed in agricultural products but these techniques are expensive and timeconsuming (Zou et al 2016). Immunological analysis, especially enzyme-linked immunosorbent assay (ELISA), which uses monoclonal or polyclonal antibodies, has several advantages including high sensitivity, high specificity and higher simplicity compared with instrumental methods (Rangnoi et al 2011;Qiu et al 2015). However, the cost for the production of antibodies is still relatively high.…”