Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes.
To enable discovery of serum antibodies indicative of disease and simultaneously develop reagents suitable for diagnosis, in vitro directed evolution was applied to identify consensus peptides recognized by patients' serum antibodies. Bacterial cell-displayed peptide libraries were quantitatively screened for binders to serum antibodies from patients with celiac disease (CD), using cell-sorting instrumentation to identify two distinct consensus epitope families specific to CD patients (PEQ and (E)/DxFV(Y)/FQ). Evolution of the (E)/DxFV(Y)/FQ consensus epitope identified a celiac-specific epitope, distinct from the two CD hallmark antigens tissue transglutaminase-2 and deamidated gliadin, exhibiting 71% sensitivity and 99% specificity (n = 231). Expansion of the first-generation PEQ consensus epitope via in vitro evolution yielded octapeptides QPEQAFPE and PFPEQxFP that identified ω- and γ-gliadins, and their deamidated forms, as immunodominant B-cell epitopes in wheat and related cereal proteins. The evolved octapeptides, but not first-generation peptides, discriminated one-way blinded CD and non-CD sera (n = 78) with exceptional accuracy, yielding 100% sensitivity and 98% specificity. Because this method, termed antibody diagnostics via evolution of peptides, does not require prior knowledge of pathobiology, it may be broadly useful for de novo discovery of antibody biomarkers and reagents for their detection.
Department of Chemical Engineering and Center for Bioengineering, Biomolecular Science and Engineering Program, University of California, Santa Barbara, CA 93106.
John T
JT
Ballew
Joseph A
JA
Murray
Pekka
P
Collin
Markku
M
Mäki
Martin F
MF
Kagnoff
Katri
K
Kaukinen
Patrick S
PS
Daugherty
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000906:Antibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood*; D002446:Celiac Disease / Q000175:diagnosis* / Q000276:immunology; D015331:Cohort Studies; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D000939:Epitopes / Q000235:genetics; D005260:Female; D005387:Finland; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008875:Middle Aged; D008910:Minnesota; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2
D000906:Antibodies; D015415:Biomarkers; D017931:DNA Primers; D000939:Epitopes; D010455:Peptides; C000717608:TGM2 protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2