Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Abstract

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the United States, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the national “863” scheme, China, for financial support.

Figures and Tables

Figure 1 (A) DNA and amino acid sequence of the human anti-IgE scFv. IGHV1-2*02 and IGKV1D-12*02 germline sequences are also shown for comparison. Complementarity determining regions (CDRs) are indicated. Residues different to the germline sequences are shown while the identical residues are marked by -. (B) A diagram showing the essential elements of the construct for E. coli expression. T7, T7 promoter; Pel B, the leader sequence; scFv, single-chain antibody fragment. The (His)₆-tag and transcription and translation termination region are also indicated.

Figure 2 scFv expression in E. coli. (A) Coomassie blue stain. Lane 1, un-induced total cell (prior to IPTG induction); lane 2, induced total cell (at time of the harvest); Lane 3, periplasmic extract; Lane 4, flow through mixture from Ni-sepharose column; Lane 5, washes; Lane 6, elution from Ni-sepharose column. (B) Western blotting of (A) using anti-His antibody as the probe. The samples are in the same order as in (A). The scFv is indicated by arrows. M, the standard markers (KDa).

Figure 3 ELISA analysis. (A) Binding of scFv on IgE coated wells. The amount of scFv used in the dilution was shown as uM. (B) Competitive inhibition assay.

Figure 4 Biacore analysis. The scFv was injected at various concentrations as indicated by different lines.