| Products/Services Used | Details | Operation |
|---|---|---|
| Codon Optimization | Renilla luciferease fusion protein constructs were synthesized for the Fel d 1 component of cat allergen by GenScript Biotech (Piscataway, NJ) using the amino acid sequence previously published 10. Codons were optimized for expression in mammalian cells. Constructs were transfected into FreeStyle™ 293-F cells (ThermoFisher, Waltham, MA). Fusion proteins were isolated after 48-72 hours of cell culture by cell lysis in lysis buffer composed of 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 50% glycerol and protease inhibitors. Anti-IgE beads were created for use in the LIPS assay by coupling goat anti-human IgE (SeraCare KPL, Gaithersburg, MD) to Ultralink beads (ThermoFisher, Waltham, MA). | Get A Quote |
Current assays to detect allergen-specific IgE have constraints related to obtaining pure, conformationally active allergen, variability in allergen extracts, sample volume required, and turnaround time. The luciferase immunoprecipitation systems (LIPS) immunoassay is a fast, sensitive assay created using recombinant antigens that requires a low specimen volume. These assays can also be easily modified to detect multiple antigens and antibody isotypes. Here, we demonstrate the use of LIPS assays as an innovative method to quantitatively measure allergen component-specific IgE in small sample volumes. Sera from healthy volunteers, helminth-infected adults, and peanut-allergic children were screened for IgE to ca... More
