Presented at:
2006 Annual Meeting of the American Academy of Allergy, Asthma and Immunology
Miami, FL, March 3-7, 2006
Validation of a Functional Assay of IgE-Facilitated CD23-Dependent Allergen Binding to B Cells to Monitor Clinical Efficacy of Immunotherapy
Shamji MH, Wilcock LK, Larche M, Francis JN, Durham SR
Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, UK
Rationale: ‘Blocking’ IgG antibodies induced by allergen immunotherapy can inhibit IgE-facilitated allergen binding to B-cells in a simple flow-cytometric assay. (Wachholz,P. et al., JACI 2003,12:915-922). We aimed to validate this assay according to the guidelines from the International Conference on Harmonisation (ICH) to determine its value in monitoring successful immunotherapy.
Methods: Atopic serum (IgE >100KU/ml) was pre-incubated with allergen (Phleum pratense). The allergen-IgE complexes were then incubated with EBV-transformed B cells at 4°C and surface binding was detected by flow cytometry. IgE, allergen and CD23 dependency was assessed; some experiments were performed in multiple replicates to determine within and between assay variability. In a separate experiment serum from 33 post-immunotherapy patients were included in the pre-incubation step, the results were analysed following ICH guidelines.
Results: Inactivation of IgE prevented IgE/allergen complexes from binding to EBV-transformed B cells, binding of complexes also was inhibited by blocking antibodies to CD23 and was allergen-concentration dependent. The intra-assay imprecision was 2% and inter-assay imprecision was 10%. Post-immunotherapy serum inhibited binding of allergen/IgE complexes, we determined the cut-off for this assay as 36.6% inhibition for which the clinical sensitivity was 95% and specificity was 100%.
Conclusion: We have confirmed that this assay is IgE, allergen and CD23-dependent. The assay is reproducible and provides potential for monitoring the clinical efficacy of grass pollen-specific immunotherapy. Whether the assay is either surrogate or predictive of efficacy remains to be determined.