Monoclonal antibodies against Dermatophagoides group I allergens as pseudo-cystatins blocking the catalytic site of cysteine proteinases.

The enzyme allergens Der p I and Der f I produced by the house dust mites Dermatophagoides pteronyssinus and D. farinae display partial sequence homology with other members of the cysteine proteinase superfamily. We report that certain widely used mouse mAbs against these Group I allergens indeed crossreact with the plant enzymes papain, bromelain and ficin. The recognition sites of these anti Group I mAbs comprise conformational and thermolabile epitopes involved in molding the catalytic center of the proteinases. Thus, the mAbs inhibit the enzymatic hydrolysis of specific chromogenic substrates by the Group I allergens, while specific cysteine proteinase inhibitors abolish the recognition of the enzymes by the mAbs. Similarly, activation of the thiol-proteases with L-cysteine abrogates their binding in the two-site mAb system, indicating that the mAbs recognize a proenzyme conformational peptide epitope. It follows that mAb-based assays for mite Group I components can neither detect the allergens after inactivation, nor in their fully activated forms.

Evaluation of the potency of allergenic extracts by inhibition of IgG-antibody binding.

A method is described for the in vitro potency evaluation of allergenic extracts by using their capacity of binding to specific human IgG antibodies. The results obtained with this inhibition-type enzyme immunoassay are compared with the analyses by the customary method of IgE antibody binding. A large series of allergenic protein fractions from the pollen of the Gramineae, Olea europea, Parietaria judaica, and from the dust mite Dermatophagoides pteronyssinus as well as from cat dander and peanuts were examined for inhibition of specific antibodies of both IgG and IgE isotypes. Potency evaluation by inhibition assays for IgE- and IgG-binding showed a significant correlation for the points of 50% inhibition (r = 0.92, p = 0.0001), but not for the slopes of the inhibition curves, i.e. the respective antibody avidities. Evidence is provided that the strict relationship between IgE- and IgG-inhibition by allergens could not be explained by possible cross-contaminations of the anti IgE- or anti IgG-reagents employed in the immunoassays. It is concluded that the inhibition of IgG-antibody binding presents a fast, reliable and low-cost alternative for the potency control of allergens used in clinical practice.