One-step immunoassay for Interleukin-4 (IL4) Detection Using IL4-Receptor Mimotopes Screened from Fv-Antibody Library

One-step immunoassay for IL4 was developed using a hypervariable part of immunoglobulin G (Fv-antibody) and switching-peptides. Conventional immunoassays require multistep procedures, whereas one-step immunoassays do not require additional steps. Fv-antibodies comprise three complementarity-determining regions (CDRs) and three frame regions (FRs). An Fv-antibody library was constructed using site-directed mutagenesis of the CDR3 region (with 11 amino acid residues) and expressed on the outer membrane of Escherichia coliusing autodisplay technology. Two IL4 receptor (IL4r) mimotopes, designated Fv-1 and Fv-2, were screened from the Fv-antibody library using the anti-IL4r antibody. These mimotopes were expressed as soluble proteins with three CDRs and four FRs. Using the surface plasmon resonance (SPR) biosensor, the binding affinities of the mimotopes Fv-1 and Fv-2 to the anti-IL4r antibody were 39.5 and 48.8 nM, respectively. Using HEK-Blue IL4 cells, the expressed IL4r mimotopes were analyzed to determine whether they compete with IL4r for the binding of IL4. The inhibition constants of mimotopes were 2.3 and 1.9 μM for Fv-1 and Fv-2, respectively. The interaction between mimotopes and IL4r (binding affinity and amino acids) was analyzed and compared with that of IL4 using a docking simulation. The interaction between mimotopes and IL4 occurred at binding sites on IL4r. A complex of expressed mimotopes and switching-peptides (fluorescence-labeled) was used for detecting IL4. The binding of IL4 to the complex resulted in the quantitative release of switching-peptides. IL4 detection was within the clinically required IL4 concentration range.