Two
proteases,
Esperase and
Alcalase, derived from Bacillus licheniformis and B. subtilis, respectively, are used in laundry products. In testing for the prevalence of
IgE antibodies to these
enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these
antigens. The first problem was the propensity of the
allergen,
Esperase, to undergo
autolysis, suggesting that solid-phase
Esperase might also lose reactivity through degradation. Treatment of
Esperase with
phenylmethylsulfonyl fluoride stabilized the
enzyme and permitted the synthesis of a stable solid-phase
antigen. The second problem was the finding that sera reactive with
Esperase in the RAST were also reactive with
Savinase, an
enzyme from B. licheniformis to which the workers were not exposed. Immunochemical analyses of the three
enzymes with specific rabbit
antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent. RAST inhibition demonstrated that solid-phase
Esperase possessed unique allergenic determinants in that the reactivity of
IgE antibodies was inhibited by low concentrations of
Esperase and only by very high concentrations of
Alcalase and
Savinase. In contrast, the reactivity of solid-phase
Alcalase was occasionally inhibited equally well by
Esperase and
Alcalase. Most strikingly, the reaction of
IgE antibodies with solid-phase
Savinase was always inhibited by comparable quantities of
Esperase,
Alcalase, and
Savinase. Thus, the establishment of the RAST for these
proteases appears to require the use of
phenylmethylsulfonyl fluoride to retard
autolysis, and the results must be interpreted with caution because
IgE antibodies in certain sera demonstrate cross-reactivity with
Alcalase and
Savinase.