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There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance. Molecular & Cellular Proteomics 4:1942-1947, 2005.The development of DNA microarrays has had an enormous impact in the research field of functional genomics where it has enabled large scale global analysis of whole genomes and transcriptomes. The developed methodology and technology have now become an established and relatively mature technique in terms of available instrumentation for production and analysis as well as commercially available premade microarrays, robust experimental protocols, and the long range of available tools and software for data analysis. Naturally there have been large efforts to continue the development of the microarray format with similar approaches for global protein analysis where great potential can be envisioned.The microarray technology makes it possible to analyze a large number of proteins within a small sample volume in a single experiment or with a reverse set-up analyze a limited number of proteins in many samples. The basic principles for highly sensitive "microspot" ligand binding assays were described by Ekins (1) and Ekins and Chu (2), who showed that small amounts of capture molecules in microspots can detect low concentrations of the analyte with high accuracy and sensitivity.The protein microarray applications can be divided into two categories: functional protein arrays and protein profiling arrays. Functional protein arrays can effectively screen large quantities of proteins for biochemical activity, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. The arrays have been used to study activity of uncharacterized proteins (3), antibody specificity profiling (4), and immune response profiling. Protein abundance arrays are used to measure protein abundance and/or alterations (5). Lately there has been progress in microarrays printed with antigen and used for detection of circulating antibodies in clinical specimens (6 -8). The protein profiling microarrays have also been used to measure the binding specificity of protein expression libraries (9, 10) and for protein profiling in cancer tissue (11).Currently there are two main types of profiling microarrays: forw...

TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) mutations seem to be associated with autoimmunity and common variable immunodeficiency in humans. Because of its role in immune responses, we investigated the association between TACI mutations and infection proneness/asthma symptoms in children. A total of 2372 children were genotyped for TACI mutations (I87N, C104R, S144X, A181E, R202H and ins204A). Serum IgA, IgG and specific IgE levels were determined in children with mutations. Data on parentally reported allergic diseases and infections were collected. In all, 55 individuals with TACI mutations were identified. Children with TACI mutations had a 2-fold increased risk of wheeze at 2 and 4 years of age and a 2.5-fold increased risk of asthma was seen at 4 years of age. None of the children with mutations suffered from IgA deficiency (o0.07 g l À1 ). No significant differences in serum IgG levels or specific IgE were found. Common variants in asthma susceptibility genes may account for up to 40% of cases of childhood-onset asthma, indicating a high contribution, compared with other common disorders. The role of rare variants/mutations in the pathogenesis of asthma is less clear. We conclude that mutations in TACI are the contributing factors for asthma symptoms in Swedish children, although the mechanisms still remain elusive.

There is a need for neonatal screening tools to improve the long-term clinical outcome of patients with primary immunodeficiency diseases (PID). Recently, a PCR-based screening method for both TRECs and KRECs using Guthrie card samples has been developed. However, the applicability of these excision circle assays is limited to patients with severe T or B cell lymphopenia (SCID, XLA and A-T), whereas the most common forms of PID are not detected. Absence of serum IgA is seen in a major fraction of patients with immunological defects. As serum IgA in newborns is considered to be of fetal origin, eluates from routinely collected dried blood spot samples might thus be suitable for identification of children with PID. To assess the applicability of such screening assays, stored Guthrie card samples were obtained from 47 patients with various forms of primary immunodeficiency diseases (SCID, XLA, A-T, HIGM and IgAD), 20 individuals with normal serum IgA levels born to IgA-deficient mothers and 51 matched healthy newborns. Surprisingly, normal serum IgA levels were found in all SCID, XLA, A-T and HIGM patients and, additionally, in all those IgAD patients born to IgA-sufficient mothers. Conversely, no serum IgA was found in any of the 16 IgAD patients born by IgA-deficient mothers. Moreover, half of the IgA-sufficient individuals born by IgA-deficient mothers also lacked IgA at birth whereas no IgA-deficient individuals were found among the controls. IgA in neonatal dried blood samples thus appears to be of both maternal and fetal origin and precludes its use as a reliable marker for neonatal screening of primary immunodeficiency diseases.

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BackgroundDried blood spot samples (DBSS) from newborns are widely used in neonatal screening for selected metabolic diseases and diagnostic possibilities for additional disorders are continuously being evaluated. Primary immunodeficiency disorders comprise a group of more than one hundred diseases, several of which are fatal early in life. Yet, a majority of the patients are not diagnosed due to lack of high-throughput screening methods.Methodology/Principal FindingsWe have previously developed a system using reverse phase protein microarrays for analysis of IgA levels in serum samples. In this study, we extended the applicability of the method to include determination of complement component C3 levels in eluates from DBSS collected at birth. Normal levels of C3 were readily detected in 269 DBSS from healthy newborns, while no C3 was detected in sera and DBSS from C3 deficient patients.Conclusions/SignificanceThe findings suggest that patients with deficiencies of specific serum proteins can be identified by analysis of DBSS using reverse phase protein microarrays.