Table 1 Proteomics technologies for autoantibody profiling: selected published studies

Antigen microarrays

Robotic attachment of antigens in ordered arrays on membranes and derivatized microscope slides

Secondary antibody; chemiluminescence

5000+

Demonstrate sensitive and specific detection of autoantibodies in serum on planar arrays

Protein microarrays

Robotic attachment of antigens in ordered arrays on derivatized microscope slides

Direct labeling of samples with fluorescent markers for comparative analysis

10,000+

Comparative analysis requires fluorescent labeling of individual samples; 50% of antigens detected

Antigen microarrays

Robotic attachment of antigens on derivatized microscope slides

Secondary antibody; fluorescence; comparative analysis with direct fluoresecent labeling of samples

10,000+

Detection of autoantibodies characteristic of eight autoimmune rheumatic diseases, including autoantibodies against proteins, peptides, nucleic acids, and macromolecular complexes

Bead microarrays (LabMAP™; cytometric bead array)

Antigens conjugated to sets of spectrally resolvable fluorescent beads

Fluorescence; analysis of individual beads using a flow cytometer

64 per well; 5000+ per 96-well plate

Fluid-phase; commercial development by Luminex, and Becton–Dickinson

Nanobarcodes™ particle technology

Attachment of antigens to addressable multimetal microrods encoded with submicrometer metal stripes

Light microscopy; fluorescence; mass spectrometry

80,000 using three distinct metals

Fluid-phase; Commercial development by SurroMed

Arrayed proteins from cDNA expression libraries

Expression and purification of polypeptides encoded in a cDNA expression library in microtiter plates, followed by robotic attachment to PVDF filters

Chemiluminescence

10,000+

Performing autoantigen discovery; bacterial expression of autoantigens does not confer post-translational modifications

Protein in situ array

Protein array generated in situ using PCR and a cell-free transcription/ translation expression system

Colorimetric

96 per plate

Probably less robust than other systems

Photolithography-generated peptide arrays

In situ synthesis of peptides by photolithography

Fluorescence

10,000+

Linear peptide epitopes only; not under active development

Microarrays of cells expressing defined cDNAs

Robotic printing of cDNA in expression vectors on slides followed by incubation with adherent mammalian cells

Fluorescence

10,000+

Mammalian expression system confers certain post-translational modifications

Protein arrays of living transformants; modified yeast two-hybrid screen

Robotic delivery of yeast transformants expressing yeast open reading frames fused to an activating domain

Colorimetric

Performed in 384-well microtiter plates

Arrays of yeast expressing fusion proteins

'Line immunoassay'

Electrophoresis of antigens and transfer to nitrocellulose membranes (western blot of purified antigens)

Chemiluminescense

< 50

Not high-throughput; commercial development by Innogenetics

'Universal protein array'

Dot-blots of purified antigens on nitrocellulose membranes

Secondary antibody; radioactivity

< 200

Requires large quantities of purified antigen and serum samples

'Lab-on-a-chip', microfluidics

Microchannels etched in solid supports; electrokinetic, electro-osmotic, electrophoretic, or pressure-driven flow

Fluorescence; UV light absorption

N/A

Fluid-phase assay; low-affinity binding detectable; kinetics can be calculated; commercial development by Caliper, Aclara, and Fluidigm

Peptides on pins (Multipin™)

In situ synthesis of peptides on polyethylene pins

Colorimeteric

96 per plate

Linear epitopes only; strip and re-use peptides on pins for subsequent experiments