Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

BACKGROUND: German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. OBJECTIVE: Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. METHODS: Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. RESULTS: Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. CONCLUSIONS: An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.