Enhanced Diagnostic Potential of Glutamine Glutamic Acid Substituted Gliadin Analogous Peptides

Screening of phage-displayed peptides revealed PEQ as important motif recognized by gliadin antibodies of CD patients [157]. Therefore, we investigated all possible nonapeptides bearing a central PQQ from representative sequences of a-, 7-, and o-type gliadins (for sequences see [169-171], respectively) and their glutamine-glutamic acid substituted peptide counterparts. Altogether we studied 51 different PQQ-containing sequences in a luminescence assay [127]. Binding of IgA from sera of patients with CD was increased when PQQ was substituted by PEQ in nearly all peptides (Fig. 2).

Peptide No. 10 20 30 40 50

Fig. 2. Binding of IgA from sera of CD patients (positive for endomysium antibodies) to native and glutamine-glutamic acid substituted gliadin peptides. All 51 nonapeptides bearing a central PQQ motif from a-, 7-, and o-type gliadin and the corresponding 51 substituted peptides with central PEQ were synthesized onto cellulose membranes. Peptides numbered consecutively from N- to C-terminus first of a-, then of 7-, and finally of o-type gliadin. Binding of IgA detected by means of a luminescence assay applying antihuman IgA conjugated with peroxidase and luminol as substrate [127]. Top: recognition of the peptides by 41 human sera (means). Black columns: peptides with central PQQ. Gray columns: peptides with central PEQ. Bottom: reactivity of a representative serum (upper line peptides with central PQQ, lower line corresponding peptides with central PEQ).

Peptide No. 10 20 30 40 50

Fig. 2. Binding of IgA from sera of CD patients (positive for endomysium antibodies) to native and glutamine-glutamic acid substituted gliadin peptides. All 51 nonapeptides bearing a central PQQ motif from a-, 7-, and o-type gliadin and the corresponding 51 substituted peptides with central PEQ were synthesized onto cellulose membranes. Peptides numbered consecutively from N- to C-terminus first of a-, then of 7-, and finally of o-type gliadin. Binding of IgA detected by means of a luminescence assay applying antihuman IgA conjugated with peroxidase and luminol as substrate [127]. Top: recognition of the peptides by 41 human sera (means). Black columns: peptides with central PQQ. Gray columns: peptides with central PEQ. Bottom: reactivity of a representative serum (upper line peptides with central PQQ, lower line corresponding peptides with central PEQ).

A common feature of the PEQ-containing peptides, which are strongly bound by CD patients' antibodies, was a C-terminal PFP. Of the 51 glutamine-glutamic acid substituted nonapeptides studied, the sequences FSQPEQPFP, SQQPEQPFP, PQQPEQPFP, PLQPEQPFP, PIQPEQPFP, and TQQPEQPFP were recognized by the highest number of CD sera. Interestingly, each contained the common heptapeptide QPEQPFP. Because 10% CD patients were nonreactive with the above peptides, we investigated other peptides that may be used to complement the assay. Although the pep-tide PEQLPQFEE was recognized only by about one-half of CD patients, it detected several of the patients not detected when the QPEQPFP-containing peptides were used. Thus, for diagnostic purpose, PEQLPQFEE should be used in combination with a QPEQPFP-containing nonapeptide. If PLQPEQPFP and PEQLPQFEE were used within one assay a diagnostic accuracy of 93.8% was obtained. This accuracy was substantially higher than that of gliadin antibodies. It should be stressed that the control group in our study included 14 patients with high gliadin antibody concentration. Only three of these controls were also positive for peptide antibodies, a finding that underscores the specificity of the test [127]. Of course, further variation of the amino acid sequence of peptides could even improve sensitivity and specificity of the peptide antibody test. For routine use, however, the technique has to be simplified and transferred from a luminescent-based assay performed on cellulose membranes to a microtiter plate-based format.

Later, a commercial assay was developed using deamidated gliadin peptides for antibody detection in CD patients (INOVA Diagnostics, San Diego, CA) [172]. Although peptide length was reported to be less than 30 amino acids, the exact amino acid sequence was not disclosed. The authors concluded that the new ELISA was superior in sensitivity and specificity to the standard gliadin antibody format and agreed much better with assays that determined antibodies against tTG and the endomysium [173]. Due to lack of clinical information, the new assay was only correlated to autoanti-body assay data. It could be shown that the results of the peptide test showed 97% concordance with results for anti-endomysium and anti-tTG antibody assays. More importantly, of the 56 sera negative for autoantibodies but positive for conventional gliadin antibodies, 54 (96%) were also negative for antibodies against deamidated gliadin peptides. Furthermore, the IgG-based version of the assay was found to detect all cases of IgA-deficient CD patients

  • 172]. The diagnostic value of the test was further substantiated by investigating sera of biopsy proven adult CD patients vs sera of non-CD controls
  • 173]. These data indicated that the new deamidated gliadin peptide assay was an effective and reliable tool for the accurate diagnosis CD in clinical practice [174]. In addition, we were able to develop an ELISA version of the test applying our glutamine-glutamic acid substituted peptides described above [127] with high diagnostic accuracy [175].

However, the question remains, why a new ELISA test when the assay for tTG antibodies already approaches a diagnostic accuracy of ~ 100%? It should be stressed that the new peptide ELISA neither is a substitute for already existing autoantibody tests nor is it measuring the same antibody species by another method (as is the case for assays for antibodies against endomysium and tTG). The new peptide ELISA measures a new species of antibodies different from conventional gliadin antibodies and autoantibodies against tTG. Therefore, the new peptide ELISA might provide new and additionally useful information on pathogenesis of CD. However, further investigations will need to determine if the new peptide ELISA is better suited for diagnosis in small children in whom autoantibodies are still absent [148, 149]. In addition, the peptide assay may prove to be more stable and yield better reproducibility vs tests using large (recombinant) proteins, for example tTG, in which a native conformation appears crucial for proper antigenicity [147, 176]. The peptide assay may be additionally beneficial if it avoids nonspecific binding to antibodies against tTG (human recombinant), commonly observed in liver disease [140, 141, 145, 146].

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