A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I + J phagemid libraries

Ricin toxin (RT), extracted from castor bean, is a type II ribosomeinactivating protein (RIP). As a biothreat agent, there is no effective antidote for RT to date, but recent advances in antibody research may have meet this need. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with RT activity for further clinical use. The purified RT component was used in phage biopanning to select ricin-bound HuscFv-displayed phage clones from synthetic (Tomlinson I + J) phage display libraries. The selected HuscFv, named JE11, showed an almost 100% protective effect to HeLa cells by mixing with RT at a 1:1000 ratio in vitro. The antibody showed an 83% protection against injection of 2 × LD 50 RT dose mice within 72 h. This research has therapeutic potential for the diagnosis and treatment of RT poisoning.

Section: Expression and Purification Of The Je11 Scfvmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Neutralization and binding activity of a human single-chain antibody to ricin toxin

Chang

Dong

et al. 2019

“…The pellet was suspended in 100 mL 2×TY (containing 100 μg/mL ampicillin, 50 μg/mL kanamycin and 0.1% glucose) and shaking at 30°C, 250 rpm overnight. The next day, the phage was precipitated according to the protocol of Tomlinson (I + J) library (de Wildt, Mundy, Gorick, & Tomlinson, 2000;Ossysek et al, 2015).…”

Section: Construction Of Chain-shuffled Librariesmentioning

confidence: 99%

Generation of panels of anti-idiotypic single-chain variable fragments mimicking Cry2Aa toxin using the chain shuffling technique

Liu

Lin

et al. 2018

Here, we firstly reported to generate panels of anti-idiotypic singlechain variable fragments (scFvs) of Bacillus thuringiensis Cry2Aa toxin by chain shuffling method. Light-and heavy-chain-shuffled libraries based on two parental clones were constructed. The signal of the light-chain-shuffled library was 4.2-fold higher than the heavy one. Solely after one round panning, eight mutants were rapidly isolated from the chain-shuffled libraries. The apparent affinity of the best clones was 2.65 × 10 6 M −1 , a 2.59-fold improvement over the parental clone. The binding abilities between anti-idiotypic scFvs with brush border membrane vesicle of Plutella xylostella larvae were also determined. This work demonstrated that chain shuffling could rapidly generate panels of antibodies with similar binding abilities but of the distinct idiotypic composition. The serials of mutants can be used to map the binding epitope of the receptors in target insects instead of Cry2Aa toxin.

“…The digested products were purified again and then ligated using T4 DNA ligation enzyme at 16°C for 16 h. The ligation product was introduced into JM109 competent cells and grown at 37°C on selective LB-Agar plates containing 100 μg/mL ampicillin. Positive clones were characterized through BamHI and SalI enzyme digestions and sequence analysis (Ossysek, Uchański, Kulesza, Bzowska, & Klaus, 2015;Zhang, He, Zhao, Wang, & Jin, 2016).…”

Section: Construction Of a Plasmid Encoding The Scfv-ap Fusion Proteinmentioning

confidence: 99%

Production and characterization of a single-chain Fv antibody–alkaline phosphatase fusion protein specific for ampicillin

Wei

Zhang

et al. 2018

Anti-AMP scFv were prepared by cloning variable region genes (VH and VL) from hybridoma cell line which secretes antibodies against AMP, and phoA gene was cloned from Escherichia coli strain K12 chromosomal DNA. The resulting scFv and phoA gene were inserted into the expression vector pBV220 to express the scFv-AP fusion protein in E. coli strain BL21. The purified scFv-AP fusion protein was used to develop an ic-CLEIA method for detection of AMP. The limit of detection (IC 15) was 0.11 ± 0.004 ng/mL and the IC 50 was 0.75 ± 0.04 ng/mL, respectively. The assay was 3 times as sensitive as the corresponding ic-ELISA based on the same fusion protein. IC-CLEIA was used to analyse AMP spiked milk samples, and the validation was confirmed by HPLC. Good correlation between ic-CLEIA and HPLC data was obtained (R 2 = 0.9934). It indicated that the developed method can apply to effectively monitor food safety.