The aim of this study was to characterise and validate an assay that could be utilised as a relevant test of inhibitory activity from sera obtained from patients with severe allergic rhinitis treated with allergen-specific immunotherapy. The IgE-Facilitated Allergen Binding (IgE-FAB) assay is a simple flow cytometric method that detects allergen-IgE complexes bound to low affinity FcεRII (CD23) on the surface of EBV-transformed B cells (Wachholz et al., 2003
We have confirmed that inhibition of allergen-IgE binding is optimal at low allergen concentrations (van der Heijden et al., 1993
; Wachholz et al., 2003
). Formation of large allergen-IgE complexes, in this context, is dependent on the ratio of specific-IgE antibody to Phl p (allergen) in the system. In the presence of excess allergen, formation of large IgE-allergen complexes is inhibited because of a limited number of binding sites on IgE antibody (allergen excess). However, at low concentrations of allergen, multiple IgE antibodies can be cross-linked by binding to allergen to form large IgE-allergen complexes (Wachholz et al., 2004
We have also shown that binding of allergen-IgE complexes to B cells is IgE and CD23 dependent. By heat-denaturing IgE (Demeulemester et al
., 1996) within indicator sera we no longer observed allergen-IgE binding. There was a significant correlation between IgE-facilitated allergen binding and allergen-specific IgE levels as measured by RAST (van der Heijden et al., 1995
). Pre-treating the EBV-transformed B cells with monoclonal anti-CD23 antibody resulted in complete inhibition of allergen-IgE complexes binding to EBV-transformed B cells. Wachholz and colleagues showed only a partial inhibition of IgE-FAB using an anti-CD23 antibody (Wachholz et al., 2003
). This may have been attributable to the use of different clones of anti-CD23 antibody between the two studies.
The IgE-FAB assay was highly reproducible. The assay was also robust and unaffected by repeat freeze-thawing of serum samples or the age of cells in culture. Using the ROC curve analysis and a cut off 68.1% relative binding, we were able to define the clinical sensitivity and specificity, positive predictive value and negative predictive value (NPV) of this assay. The results show that the assay reliably detects inhibitory activity of blocking antibodies in the actively treated group. Importantly it does not give false positive results in patients who have not received treatment.
Although the read-out of the IgE-FAB assay does not measure the antigen presenting capacity of B cells to T cells, this assay has previously been shown to be representative of this process (Wachholz et al., 2003
). Therefore IgE-FAB assay can provide an alternative model of facilitated allergen presentation without using T cell culture techniques that are difficult to quality control. In terms of quality control, the acceptance criteria of the IgE-FAB assay were centred on the use of assay controls for IgE-FAB (atopic serum of known allergen binding) and inhibition of IgE-FAB (post immunotherapy serum of known inhibitory quality). These controls were run in every experiment and were used to monitor assay performance (bias and imprecision). Quality Assurance protocol was adhered to by the use of Standard Operating Procedures (SOPs) to prevent operator-related variability and Good Laboratory Practice (GLP) was followed at all times.
Patients receiving allergen-specific immunotherapy have been shown to have increased concentrations of allergen-specific IgG4 antibodies in their serum (Gehlhar et al., 1999
; Jutel et al., 2003
; Nouri-Aria et al., 2004
; Djurup et al., 1987
). However assays that detect only immunoreactive IgG4 are unlikely to predict clinical efficacy (Djurup et al., 1987
; Muller et al., 1989
; Ewan et al., 1993
; Bodtger et al. 2005
). Therefore, measuring IgG4 concentration is not suitable for monitoring clinical efficacy of immunotherapy. Despite this lack of correlation, previous reports have demonstrated that fractionated IgG4 antibodies in serum from patients who received grass pollen immunotherapy were responsible for the inhibition of IgE-FAB to B cells (Nouri-Aria et al., 2004
; Wachholz et al., 2003
). This suggests a functional role of IgG4 in inhibition of IgE-FAB, which may be due to changes in their specificity and/or affinity. Another role of allergen-specific IgG4 antibodies induced by immunotherapy is their ability to block allergen-induced IgE-dependent histamine release by basophils confirming the functional blocking activity of these antibodies (Ball et al., 1999
; Lambin et al., 1993
These data have characterised the assay using grass pollen as an allergen. This assay could also be easily adapted for use with other common allergens, in conjunction with a suitable indicator serum containing high levels of allergen-specific IgE, to test for specific inhibitory antibodies induced by therapeutic protocols. In summary, the FAP assay is reproducible, robust, sensitive and specific and has potential to be used as a tool for monitoring inhibitory antibody responses induced by allergen-specific immunotherapy. It requires straightforward methodology and it can easily be introduced into specialist immunology laboratories investigating inhibitory antibody responses induced during allergen immunotherapy.