The use of phage-peptide libraries to define the epitope specificity of a mouse monoclonal anti-Der p 1 antibody representative of a major component of the human immunoglobulin E anti-Der p 1 response (1)
More than 80% of individuals who are sensitive to the dust mite Dermatophagoides pteronyssinus produce immunoglo- bulin (Ig) E antibodies to Der p 1, the most significant domestic allergen. The model shows that Der p 1 consists of two domains. One domain consists of mainly a- helices formed by residues Thr21-Tyr116 and the crossing over of the C-terminal portion Val219-Leu222. The N-terminal portion Thr1-Arg20 crosses over into the other domain, which consists mainly of beta-pleated sheets formed by residues Cys117-Tyr218.
The vast majority of clones showed binding to mAb 2C7, with 0.820 being the maximum OD405 value obtained.
Eighteen clones, giving OD405 values of 0.750 or above, were DNA sequenced. Examination of these sequences revealed five motifs. Examination of these sequences revealed five motifs. Collectively, sequence comparison shows that the identified motif corresponds with Leu147-Gln160 in the native Der p 1 sequence.
The specificity of clones representative of these sequences was ascertained by titration of the PEG precipitate against mAb 2C
The biological relevance of clones 3 and 27, which are potentially constrained, was ascertained by ELISA inhibition against native Der p 1; when tested at the same titre non-constrained clones showed no inhibition.
Alpha-helices and beta-sheets comprise the core of the Der p 1 molecule, and that the outer surface comprises mainly the interconnecting loops, of which Ser92-Ser114 and Val140-Tyr169 are the longest. The Der p 1 epitope, Leu147-Gln160, identified by mAb 2C7 is therefore part of one of these loops (Val140-Tyr169) which is located on the surface of the domain comprising mainly beta-sheets. Obtained using a concentration of Der p 1 giving 50% inhibition (0.6 mg/mL). To achieve this degree of inhibition, 100-fold molar excess of chymopapain and 183-fold molar excess of papain were needed. On the other hand, actinidin showed no inhibition even at 600-fold molar excess.
The scaffold of the epitope recognized by mAb 2C7 is formed by nonacces- sible, non-polar aromatic (Phe150) and aliphatic (Leu147, Ile158 and Ile159) residues, while the remaining amino acids form the exposed surface of the epitope, with Arg151 and Arg156 being the most accessible amino acids. The charge distribution of the identified epitope is such that large accessible residues, namely Arg151, His152 and Arg156, form the central part of the epitope.
The epitope also contains two small negatively charged residues, namely Asp148 and Asp154.