Autoantibodies and Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 355

cDNA library screening and phage display:

Display protein by phage Biopanning for

informative antigens

Pooled sera
from patients

Probe library display for
recognized antigens

Phage array screening

Cell fractionation:

Print fractions on
multiple slides

200 400 600 80010001200
m/z

Ce l l l y s a t e

Multidimensional
fractionation in
solution or gel

Identify proteins in
informative fractions

Probe with sera

Blot 2D gels

Protein microarray:

Probe with sera Determine sensitivity and

specificity of antigens

Protein arrays (left) or bead arrays
(right) with candidate antigens

Peptide display:

Print peptide arrays

Display peptides by phage Probe with sera Determine sensitivity and

specificity of peptides

FIGURE 1 Methods of antigen identification. In traditional library screening (top row), cDNA libraries from target tissue
are expressed by phage, blotted on a membrane or panned in solution, and probed with patient sera. Con rmation of

sensitivity and speci city requires recombinant expression of protein for ELISA analysis. Cell fractionation (second row)

involved separation of cell lysates by two-dimensional or three-dimensional separations in solution phase or in gel phase,
followed by probing with patient sera. Positive fractions are identi rmed with ELISA.

Protein microarrays (third row) can be printed using recombinant proteins or proteins translated in situ, on slides or on bead
arrays. Because the arrays are addressable, no further antigen identi

ed by mass spectrometry and con

cation is required. Similarly, peptides (bottom) can be
printed on slides or displayed by T7 phage for rapid serum screening. (Modi ed from reference #109, reprinted with permission.)