Bead arrays enable multiplexed analysis of biomolecular interactions. The LabMAP™ system of Luminex (Austin, Texas, USA) utilizes 64 sets of spectrally resolvable fluorescent beads. Each set can be conjugated to a distinct antigen (or antibody or oligonucleotide). Following incubation with the test sample, analysis is performed using a flow cytometer. Further multiplexing is achieved by analysis of multiple wells in microtiter plates, each with beads conjugated to different sets of antigens.

The eTAG™ assay of Aclara (Mountain View, California, USA) utilizes eTAG™ reporters that are fluorescent labels with unique and well-defined electrophoretic mobilities. Each eTAG™ label is coupled to an antigen (or another biological probe) via cleavable linkages. When an autoantibody binds to an eTAG™ reporter-labeled antigen, the coupling linkage is cleaved and the eTAG™ is released. Mixtures of eTAGs™ are readily separated and analyzed by capillary electrophoresis.

SurroMed (Mountain View, California, USA) is developing a system based on addressable multimetal microrods intrinsically encoded with submicrometer stripes 8, termed Nanobarcodes™ particle technology. Using three different metals, 80,000 distinctive striping patterns are possible 8. This far exceeds the complexity of fluorescence-based bead and tag systems.

Microfluidics utilizes microchannels for analysis of antigen–autoantibody interactions. Small quantities of biomolecules are separately introduced into a network of microchannels and subjected to electrokinetic, electro-osmotic, electrophoretic or pressure-driven flow, mixing and separation. Binding events, reflected by changes in mobility, are measured by UV absorption or fluorescent detection. Real-time millisecond quantitation of binding kinetics and detection of low-affinity interactions are among the important advantages of this system.

Several groups have described arrays of living cells expressing transformed or transfected cDNA 910. Such systems could be easily adapted for autoantibody profiling.

Methods to fabricate arrays on planar surfaces include stamping, ink jetting, capillary spotting, contact printing, and in situ synthesis. Commonly used solid supports include: nitrocellulose, nylon and polyvinylidene difluoride membranes; poly-L-lysine-coated, silane-treated, and other derivatized glass microscope slides; and glass microscope slides coated with gelatin, acrylamide and other coatings.

Membrane-based systems include low-density dot blot arrays on nitrocellulose membranes 11, autoantigens electrophoretically separated prior to transfer to membranes 12, and spotting of cDNA expression-library-produced proteins onto polyvinylidene difluoride filters 1314. The generation of arrays of polypeptides derived from cDNA expression libraries by Büssow and colleagues provides an elegant system for autoantigen discovery 1314. cDNAs are expressed and their protein products purified in vitro, following which purified proteins are robotically arrayed. On identification of autoantibody targets, their corresponding cDNAs are readily sequenced to genetically identify autoantigens. Walter et al. describe use of one such cDNA library, a human fetal brain cDNA expression library, for autoantigen discovery in inflammatory bowel disease 15.

Other workers are developing protein arrays on derivatized microscope slides. Joos et al. have demonstrated sensitive and specific autoantibody detection using microarrays containing serial dilutions of 18 antigens 16. Haab et al. generated protein arrays to characterize 115 purified antigen–antibody pairs, demonstrating that 50% of the arrayed antigens and 20% of the arrayed antibodies where detectable when immobilized 7. Some cognate ligands were detected at concentrations as low as 1 ng/dl 7.

We have modified and refined the experimental protocol introduced by Haab et al. 7 to develop spotted antigen arrays for analysis of autoantibody responses 17. We applied this technology to analyze the autoreactive B-cell response in patients with autoimmune diseases including systemic lupus erythematosus (SLE), scleroderma, and mixed connective tissue disease 17.

Our antigen array technology utilizes a robotic arrayer to attach proteins, protein complexes, peptides, nucleic acids, and other biomolecules in an ordered array on poly-L-lysine-coated microscopic slides (Fig. 1) 17. Approximately 1 nl of solution containing 200 pg antigen is deposited on each array to produce antigen features measuring 100–200μm in diameter. Individual arrays are incubated with serum from patients or controls, followed by fluorescently labeled secondary antibody. We typically use 1:150 dilutions of human or animal serum to probe arrays, requiring 2 μl serum per array under standard protocols and only 0.15 μl serum per array when employing cover slips 17. Other biological fluids such as cerebrospinal fluid, synovial fluid, and tissue eluates may also be used (our unpublished observations).

Arrays are scanned using a fluorescence-based digital scanning device. Algorithms are available for nearest-neighbor (cluster) 18 and statistical analysis 19 of the data. Detailed protocols are presented both in our earlier work 17 and online 20. Information for construction of robotic arrayers is also available 21.

Antigen arrays proved to be fourfold to eightfold more sensitive than conventional ELISA analysis for detection of autoantibodies specific for five recombinant autoantigens 17. Moreover, antigen arrays demonstrated linear detection of antibody concentrations over a 3-log range 17.

Our 'connective tissue disease' arrays contain 200 distinct proteins, peptides, nucleic acids, and protein complexes targeted in a host of autoimmune diseases, including SLE, polymyositis, limited and diffuse scleroderma, primary biliary sclerosis, and Sjögren's disease (Fig. 1) 17. Specific antigens include Ro, La, histone proteins, Jo-1, heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins, Smith ribonucleoproteins (Sm/RNP), topoisomerase I, centromere protein B, thyroglobulin, thyroid peroxidase, RNA polymerase, cardiolipin, pyruvate dehydrogenase, serine–arginine splicing factors, and DNA.

We developed 'synovial proteome' arrays to study autoimmune arthritis involving synovial joints, including rheumatoid arthritis (RA) and its animal models. Our 'synovial proteome' arrays contain 650 candidate RA autoantigens, including deiminated fibrin, citrulline-modified filaggrin and fibrinogen peptides, vimentin, the endoplasmic chaperone BiP, glucose-6-phosphate isomerase, hnRNP A2/B1, collagens and overlapping peptides derived from several of these proteins.

Our 'myelin proteome' arrays contain 500 proteins and peptides derived from the myelin sheath, the target of the autoimmune response in multiple sclerosis and in experimental autoimmune encephalomyelitis (EAE). These myelin antigens include myelin basic protein, proteolipid protein, myelin-associated glycoprotein, myelin oligodendrocytic glycoprotein, golli-myelin basic protein, oligodendrocyte-specific protein, cyclic nucleotide phosphodiesterase and overlapping peptides derived from these proteins. We are utilizing our 'myelin proteome' arrays to characterize the autoantibody response in EAE serum, multiple sclerosis patient serum and cerebral spinal fluid, and to guide selection of antigen-specific therapies in relapsing EAE 22.

We are constructing 'islet cell proteome' arrays containing glutamic acid decarboxylase, IA-2, insulin and additional candidate autoantigens in insulin-dependent diabetes mellitus (IDDM).

Autoantibodies have diagnostic utility for several autoimmune diseases. Such diseases include myasthenia gravis (antiacetylcholine receptor antibody), Grave's disease (antithyroid hormone receptor antibody), and SLE (combination of antinuclear antibodies, plus anti-DNA or anti-Sm antibodies). Furthermore, in T-cell-mediated IDDM, the presence of combinations of autoantibodies against at least two islet antigens, including insulin, glutamic acid decarboxylase, and IA-2, are diagnostic for or predictive of future development of IDDM 23. The presence of autoantibodies against a single islet antigen has minimal clinical value. The clinical utility of autoantibodies in IDDM suggests that autoantibody profiles may have diagnostic utility for other T-cell-mediated diseases, such as RA and multiple sclerosis.

Intermolecular and intramolecular epitope spreading of the autoreactive B-cell response is associated with progression to overt clinical disease in human and murine SLE 2425 and in IDDM 23. Proteomics technologies are ideally suited to monitoring epitope spreading. Epitope spreading of the autoantibody response may represent a common harbinger of more severe and progressive autoimmunity, providing the clinician with valuable prognostic information to guide the use of nonspecific disease-modifying therapies.

Spotted antigen microarrays can identify antigen-specific autoantibody isotypes 17. Th1-type immune responses, associated with production of interferon-γ and interleukin-12, generate antibodies of isotypes capable of fixing complement and causing tissue injury 26. The ability to characterize isotype usage may facilitate the identification of offending autoantigens, based on determination of autoantigens against which autoantibodies of pathogenic isotypes are directed. Moreover, microarray isotype analysis may provide insight into both B-cell and T-cell autoimmunity because not only T cells, but also effector B cells, have been implicated in the reciprocal regulation of polarized Th1 versus Th2 cytokine production 27. Therapeutic deviation of immune responses from Th1 to Th2 cytokine production has been associated with efficacious treatment of Th1-mediated immune disease 2829.

Proteomics technologies can be applied to discover novel autoantigens utilizing cDNA expression libraries 1314, peptide libraries, or arrayed fractions of autoimmune-target tissues. Once candidate autoantigens are identified, proteomics technologies can rigorously characterize the sensitivity and specificity of autoantibodies directed against candidate antigens in cohorts of autoimmune and control patients. Of note, post-translational modifications of antigens are amenable to detection using our antigen arrays and other proteomics technologies. This is important because such modifications are strongly associated with autoimmune diseases including SLE and RA 303132.

In addition to proteomics monitoring of epitope spreading and isotype usage to gauge need for nonspecific disease-modifying therapies (already described), determination of the specificity of the autoantibody response may enable tailored antigen-specific therapy. Such antigen-specific therapies can be peptide-based or protein-based tolerizing therapies. Alternatively, they can be specific DNA tolerizing vaccines, a strategy we termed 'reverse genomics' 22. We discuss use of the autoantibody response to drive antigen-specific therapy elsewhere 2233.