Direct peptide reactivity assay

A prevalidation study carried out by Cosmetics Europe on this optimized protocol showed that the SkinEthic HCE method consistently discriminated UN GHS classified from non-classified test substances within and between laboratories [47]. A combination of the two exposure times in a testing strategy that allocates test substances to the short or long exposure time depending on their intrinsic chemical reactivity increased the overall accuracy under GHS. The chemical reactivity, i.e., electrophilic potential to react with cysteine- or lysine-containing peptides, was measured by the direct peptide reactivity assay (DPRA) [48]. Reactive substances (peptide depletion is >5.95 % relative to the control) were allocated to the short exposure time, whereas non-reactive substances (depletion <5.95 %) are allocated to the long exposure time. 

Advances in the understanding of the immunobiology of skin sensitization have led to the establishment of predictive in vivo tests which not only identify sensitizing hazards but also characterize their potency. Recently, appreciation of the underlying biology has also resulted in the development of mechanistically based in vitro alternatives which offer the prospect of the replacement of current in vivo methods. Assays under active validation include the Direct Peptide Reactivity Assay (DPRA), the human Cell Line Activation Test (h-CLAT), and KeratinoSens. None of the methods have a sufficient level of accuracy or freedom from applicability domain limitations to allow them to act as a standalone replacement. Consequently, it will be necessary to consider how to deploy these assays, perhaps in combination and/or in a structured assessment of skin sensitization hazard, to ensure at least the same level of predictive accuracy as the in vivo methods. However, a challenge remains the capacity of these methods to provide potency information on skin-sensitizing chemicals has yet to be assessed. This is an essential requirement for future risk assessment without use of animal models if we are to retain the same level of human health protection that is currently delivered. 

Key words Skin sensitization, Contact allergy, Allergic contact dermatitis, Local lymph node assay, In vitro alternatives, Direct peptide reactivity assay, KeratinoSens, Human cell line activation test... 

Extensive reviews concerning the opportunities for the development of in vitro sensitization methods already exist [41-44], These reviews show that essentially all of the methods address one or other of the key mechanistic steps in the induction of skin sensitization—and these are nicely represented in the OECD adverse outcome pathway description [45], From this large body of work, three methods have emerged whose initial promise has been substantiated by demonstration not only of their predictive merits but also by verification of their robustness in terms of inter-laboratory transferability and within and between laboratory reproducibility [46]. The three methods are the direct peptide reactivity assay (DPRA) [47, 48], KeratinoSens [49, 50], and the human Cell Line Activation Test (h-CLAT) [51-53]. The first of these, the DPRA, addresses the question of chemical reactivity, the second investigates an aspect of keratinocyte activation... 

ECVAM (2015b) https //eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations/eurl-ecvam-recommendation-on-the-direct-peptide-reactivity-assay-dpra (accessed on October 14, 2015). 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Antisera raised against insulin show some cross-reactivity with proinsuUn but not with C-peptide. Specificity is not a problem in healthy individuals because the low proinsulin concentrations do not appreciably affect the absolute values of insulin. In certain situations (e.g., islet cell tmnors and diabetic individuals), proinsulin is present at higher concentrations and direct assay of plasma may falsely overestimate the true insulin concentration. Because proinsulin has very low activity, incorrect conclusions regarding the availability of biologically active insulin may be reached in patients with diabetes. The magnitude of the error depends on the concentration of proinsulin and the extent of cross-reactivity of the... 

Beta-endorphin is a frequently measured opioid peptide. Immunoassay is the method of choice for the analysis of plasma (3-endorphin. Both RIAs and direct IRMAs have been developed for this purpose. Commercial reagent kits are widely available, and many commercial reference laboratories offer P-endorphin assays. The concentrations of P-endorphin are usually very low to undetectable in normal subjects, and it may be necessary to use extraction procedures to detect meaningful concentrations in plasma. The specificity of commercial antibodies for p-endorphin relative to p-LPH varies widely in some immunoassays, 50% cross-reactivity is seen with p-LPH. With polyclonal antibodies, results may be spuriously high owing to crossreactivity with serum immunoglobulin G (e.g., in patients with immunoglobulin G myeloma). 

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