Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

Abstract

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named “Ecobody technology”. The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the VH and VL genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) ‘Zipbody’ as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (K D = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology

“…His-tag purification of the refolded protein yielded about 8.5 mg of purified 22 G Zipbody from 1 L of LB medium (Table 1). The refolded and purified Zipbody had Hc-Lc association and the binding activity was sufficiently high (KD = 469 pM, data not shown) when compared with that of natural rabbit mAbs, which have KD values of a few hundred pM [17]. Interestingly two mutants in which particular Cys residues were removed also showed the ELISA signals after refolding (Figure 4), indicating Lc and Hc might be associated with LZ interaction.…”

“…Reverse transcription from the single cell, 1st PCR, and 2nd PCR were serially carried out as described previously [17]. In the case of human mAbs, variable regions of kappa and lambda types of Lc, and gamma and mu of Hc were separately amplified.…”

“…To produce the Zipbody with an SKIK tag, Hc and Lc were coexpressed in E. coli Shuffle T7 Express (New England Biolabs, Ipswich, MA, USA) as the host, grown in 50 mL LB medium supplemented with 100 µg/mL ampicillin with 1 mM IPTG induction at 16 °C for 24 h, as described in our previous study [17]. The inclusion bodies were refolded by the stepwise dialysis method using guanidinium hydrochloride [20].…”

“…Together with Zipbody and the SKIK peptide tag technologies, for improvement of Fab formation and protein production in CFPS, we have developed an improved SICREX system renewed as ‘Ecobody technology’ [17]. Here, we demonstrate a 2-day protocol to complete screening of antigen-specific mAbs from single B cells of rabbits and Epstein-Barr Virus (EBV) infected human B cells.…”

Monoclonal antibody against human Tim‐3 enhances antiviral immune response

“…Many methods have been developed to obtain antibodies, such as hybridoma technology, which allows for the production of mAbs directed against antigens of interest . Recently, antibody library technology and single B cell PCR antibody technology have also been used. Here, we used hybridoma technology to develop monoclonal antibodies against human Tim‐3.…”