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Allergen specific antibody binding characteristics and longitudinal serological changes to purified peanut allergens
Project Code: T07018
Regional Immunology Department, St Mary's Hospital
It has been reported that compared to other food allergies clinical sensitivity to peanut frequently continues into adulthood. At present, the reasons for peanut allergy persisting in the majority of individuals while resolving in a minority have not been identified. This study was undertaken to investigate if one characteristic of antibodies “affinity” is different in those individuals with declining specific antibody responses to purified peanut allergens. The work was divided into four phases.
In the first phase a cohort of seventy patients was identified from the FSA funded database with clinical sensitivity to peanut and a minimum of three samples per patient collected over 2-5 years. Results of routine total and specific IgE to peanut determined at the time blood was taken were available from the database.
In the second phase, all sera were retrospectively investigated for any changes in specific antibody concentration to purified peanut allergens by chemiluminescent immunoassay.
In the third phase apparent antibody affinity for purified peanut Ara h2 was determined for the final sample from each patient using ammonium thiocyanate titration. Additional affinity work investigated the binding of radio-labelled Ara h2
In the fourth and final part patients sera were analysed for differences in their dissociation rate constants against three purified peanut allergens using Biacore® optical methodology.
For the cohort of seventy patients a frequent rise in peanut specific antibody levels occurred in children under 12 years at the time of the final sample. This contrasted with a gradual decline in older individuals. When assessed by Pharmacia Immunocap® the overall results show a downward trend in peanut specific IgE in half the individuals tested.
Retrospectively analysis of the samples, to detect changes in specific IgE against purified peanut Ara h2, differed from the Immunocap® findings with more stability, including a lower percentage with declining antibody levels. Corresponding results for IgG anti-Ara are characterized by both greater variation between occasions and far higher levels (fifty-fold) than specific IgE.
To determine if patients with declining specific antibody levels have a different affinity, the patient cohort was divided into three groups with rising, falling or fluctuating antibody levels to Ara h2. No difference in median affinity was observed for individuals with either fluctuating or falling antibody levels. In contrast a significant increase in affinity was detected for a small number of young children with rising anti-Ara h2 levels.
When the IgE affinity index for Ara h2 was compared at the start and end of the study a highly significant correlation was obtained. This indicates that for the majority of individuals tested antibody affinity for a major peanut allergen is stable over the period studied.
For the cohort studied median affinity index of IgG and IgE anti-Ara h2 are comparable. This correspondence between IgE and IgG results did not hold for individual patients and prevented IgE affinity being extrapolated from IgG affinity measurements.
The affinity values of IgE anti-Ara h2 obtained by direct allergen binding studies show the antibodies to be predominantly high affinity. This corresponds to a median association constant (Ka) of 1.14x109 (Kd = 8.84x10-10) for the thirty-five samples suitable for investigation.
Using Biacore® the results show the IgG median dissociation rates for two peanut allergens Ara h1 (kd = 0.7x10-4) and Ara h2 (1.0x10-4) are slow whereas antibodies to PNA detached twice as rapidly (kd = 2.0 x10-4) indicating the latter antibodies are low affinity. Biacore® methodology was not sufficiently sensitive to determine IgE dissociation rates for the same samples.
Additional ammonium thiocyanate studies for both IgE and IgG, show that the median affinity for PNA is lower than for either major peanut allergen. This finding was further investigated by immunoblotting. Preliminary results appear to confirm antibodies to minor allergenic bands bind with a lower apparent affinity than antibodies to the major peanut determinants.
Finally, the possibility antibody affinity is one factor that determines the strength of the in vivo SPT response was assessed. No simple association was observed between the standard peanut SPT response index and antibody affinity to purified Ara h2.
The study demonstrates apparent affinity of IgE antibodies is characteristically high for both major peanut allergens investigated, Ara h1 and Ara h2, and shows little tendency to vary over the period studied. These high affinity IgE antibodies appear to be retained regardless of any detectable fall in specific IgE and affinity is not directly related to antibody concentration. At the end of the study the highest affinity for Ara h2 occurred in young children with rising anti-Ara h2 levels. For the individuals studied no simple correlation was observed between antibody affinity for Ara h2 and the peanut SPT reaction index, although specific SPT data to purified Ara h2 was unavailable.
IgG antibodies to purified peanut do not correlate with IgE antibody levels, and were found to fluctuate widely between occasions. The results emphasize a significant association between high levels of IgG to Ara h1 and Ara h2 and high affinity IgG antibodies against these major peanut allergens. The presence of high affinity IgG specific antibodies suggests that continued exposure to trace levels of peanut allergens may occur even in a group actively avoiding peanut. While the extent to which IgG may influence the in vivo IgE response was not specifically investigated, it was observed that a high ratio of IgE anti-Ara h2 / IgG anti-Ara h2 is correlated with a strong SPT response to peanut.
The lower affinity antibodies directed against minor peanut allergens (PNA), will be less well detected by immunoassay as IgE antibody levels fall. This factor may account for the decline in IgE antibody levels to peanut ImmunoCAP® with a mixture of allergens, compared to the more stable levels observed against a single allergen Ara h2. For this reason monitoring of IgE antibodies levels over time to a whole peanut extract is likely to differ from the response to purified Ara h1 and Ara h2. The length of this study is at present insufficient to determine if the patients antibody profiles will change in accordance with this predicted pattern.
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