• Jon Lalko

    Student thesis: Phd


    A common characteristic of all chemical allergens, both respiratory and skin allergens, is the ability to form stable associations with proteins; the resulting hapten-protein complex being sufficient to provoke an immune response. There is evidence to suggest that selective binding of chemicals with proteins or peptides may impact on the quality of immune response that will develop. In the investigations described here, we have taken a reductionist approach to protein reactivity and evaluated the binding characteristics of 20 of the most commonly reported chemical respiratory allergens towards defined peptides with a single reactive amino acid of interest. The hypothesis is that it is possible to identify and characterize different forms of chemical allergens as a function of preferential peptide binding.Utilizing the standardized reaction conditions of a direct peptide reactivity assay (DPRA), the reactivity of respiratory allergens for cysteine and lysine peptides was evaluated. Activity in the DPRA is reported as the percent depletion of peptide following 24 h incubation. An important and intriguing observation was that, when compared with skin sensitizers, chemical respiratory allergens exhibited a preferential reactivity for lysine. This preference was characterized quantitatively as a ratio of the mean depletion of lysine compared with cysteine (Lys:Cys ratio). The Lys:Cys ratio was observed to be robust and reproducible over time.A limitation of many in chemico methods for hazard identification is the lack of a metabolic component that allows for the identification of pro-haptens. In order to address this limitation, reported here is the use of the peroxidase peptide reactivity assay (PPRA), which utilizes a horseradish peroxidase/hydrogen peroxide (HRP/P) enzymatic system as a proxy for oxidative metabolism. Additionally, reactivity in the PPRA is characterized after a 24 h reaction time utilizing a concentration-response model (thus, permitting consideration of dose-response relationships defined as an EC15 value). Unexpectedly, the preferences for lysine observed with chemical respiratory allergens in the DPRA were lost or blunted in PPRA. The EC15 values demonstrated that relative reactivity between chemical respiratory allergens varied by up to 4 orders of magnitude. The identification of quantitative differences in reactivity could prove useful as a guide to evaluate potency in the future, should reliable metrics become available.To characterize the selectivity of binding by chemical respiratory allergens, the DPRA was modified to allow for the evaluation of reactivity to histidine, tyrosine and arginine. Confirming our previous observations, each of the respiratory sensitizers was observed to react to both lysine and cysteine, with in most instances, a preference for the former. Reactive promiscuity was a function of the other peptides with histidine being the most reactive followed by arginine and tyrosine. To model more complex reactive conditions, a novel modification was made to the DPRA to allow competition for lysine and cysteine to be assessed in a single reaction mixture. The results of these competitive reactivity experiments identified a range of binding patterns to lysine and cysteine that in some cases resulted in different binding being expressed.At present, there are no methods available to reliably identify potential chemical respiratory allergens. The work presented here has demonstrated that respiratory allergens can be identified as potential sensitizers based on their ability to react with lysine and cysteine. More importantly, the balance of reactivity to these two peptides can provide a means of discriminating between respiratory and skin sensitizers.
    Date of Award3 Jan 2013
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorIan Kimber (Supervisor) & Rebecca Dearman (Supervisor)


    • Skin sensitizers
    • Respiratory allergens
    • Peptide reactivity
    • Hazard identification

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