A proposed screen for evaluating low-molecular-weight chemicals as potential respiratory allergens

Allergic asthma can result when reactive low-molecular-weight chemicals (LMWCs) haptenate carrier proteins to form immunogenic conjugates, which then induce specific allergic antibodies. As part of an overall assessment process for evaluating the allergenic potential of LMWCs, an in vitro test for detecting the covalent derivatization of proteins by LMWCs was developed. In the assay, globulin-free serum albumins were incubated with increasing concentrations of a given LMWC and the mixtures separated via reversed-phase high-performance liquid chromatography (HPLC). Derivatization was monitored by shifts in the retention time of native versus modified protein. Retention time shifts were seen for most haptens when incubated with human serum albumin at a 50:1 (hapten:protein) starting molar ratio. Some haptens changed the retention time of the protein at a 5:1 initial ratio. Almost all chemicals that non-covalently bind to proteins did not change the protein retention time, even when incubated at 1500:1 molar ratios. The screen correctly identified 12/14 known human allergenic haptens and 23/24 non-allergenic LMWCs. It cannot detect sensitizers which must be metabolized into reactive haptens. This screen can be incorporated into an overall risk assessment approach for evaluating chemicals as respiratory allergens.