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Results for “Protein Array Analysis”

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PubMed2006

G-protein-coupled receptor microarrays for multiplexed compound screening.

Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized "plug-and-play" format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.

Drug Evaluation, Preclinical↗

PubMed2003

Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation.

UNLABELLED: The genomic response to BMP was investigated by ectopic expression of activated BMP type I receptors in C2C12 myoblast using cDNA microarrays. Novel BMP receptor target genes with possible roles in inhibition of myoblast differentiation and stimulation of osteoblast differentiation were identified. INTRODUCTION: Bone morphogenetic proteins (BMPs) have an important role in controlling mesenchymal cell fate and mediate these effects by regulating gene expression. BMPs signal through three distinct specific BMP type I receptors (also termed activin receptor-like kinases) and their downstream nuclear effectors, termed Smads. The critical target genes by which activated BMP receptors mediate change cell fate are poorly characterized. MATERIALS AND METHODS: We performed transcriptional profiling of C2C12 myoblasts differentiation into osteoblast-like cells by ectopic expression of three distinct constitutively active (ca)BMP type I receptors using adenoviral gene transfer. Cells were harvested 48 h after infection, which allowed detection of both early and late response genes. Expression analysis was performed using the mouse GEM1 microarray, which is comprised of approximately 8700 unique sequences. Hybridizations were performed in duplicate with a reverse fluor labeling. Genes were considered to be significantly regulated if the p value for differential expression was less than 0.01 and inverted expression ratios per duplicate successful reciprocal hybridizations differed by less than 25%. RESULTS AND CONCLUSIONS: Each of the three caBMP type I receptors stimulated equal levels of R-Smad phosphorylation and alkaline phosphatase activity, an early marker for osteoblast differentiation. Interestingly, all three type I receptors induced identical transcriptional profiles; 97 genes were significantly upregulated and 103 genes were downregulated. Many extracellular matrix genes were upregulated, muscle-related genes downregulated, and transcription factors/signaling components modulated. In addition to 41 expressed sequence tags without known function and a number of known BMP target genes, including PPAR-gamma and fibromodulin, a large number of novel BMP target genes with an annotated function were identified, including transcription factors HesR1, ITF-2, and ICSBP, apoptosis mediators DRP-1 death kinase and ZIP kinase, IkappaB alpha, Edg-2, ZO-1, and E3 ligase Dactylin. These target genes, some of them unexpected, offer new insights into how BMPs elicit biological effects, in particular into the mechanism of inhibition of myoblast differentiation and stimulation of osteoblast differentiation.

Animals↗

PubMed2005

Discovery and identification of anti-U1-A snRNP antibody in lung cancer.

There are multiple reports of autoimmune response in patients with lung cancer. To investigate whether a novel autoantibody is present in patients with lung cancer and evaluate its clinical diagnostic and prognostic value, sera from 10 patients with lung cancer and 10 normal individuals were analyzed using immunofluorescence and Western blotting. It was found that one serum sample from the patients with squamous carcinoma gave a fine speckled pattern staining in nucleus and had a high titer antinuclear autoantibody which could recognize 31 kD of nuclear protein isolated from both cancer cells and normal cells. The same patient's serum was further used to immunoprecipitate the target antigen. The protein bands were excised from the SDS-PAGE gels and were analyzed with a Qstar Pulser I Quadrupole time-flight mass spectrometer, and the 31 kD target antigen was identified as U1-A snRNP. To test the prevalence of anti-U1-A snRNP antibody, sera from 93 patients including 36 squmaous carcinomas (SCC), 26 adenocarcinomas (Ad), and 31 small cell carcinomas (SCLC) were screened by Western blotting. The results demonstrated that anti-U1-A snRNP antibody was present in 50% of SCC sera, 26.9% of Ad sera and 54.8% of SCLC sera. In this paper, we report for the first time that anti-U1-A snRNP antibody could be detected in the patients with lung cancer.

Adenocarcinoma↗

PubMed2007

Phosphorylation studies using plant protein microarrays.

Identifying protein kinase substrates is one major focus of protein kinase research and supports the elucidation of signal transduction pathways and their complex regulation. In this chapter we describe a protein microarray-based in vitro method, which permits a systematic screening of immobilized proteins for their phosphorylation by specific protein kinases. This high-throughput method allows the identification of potential kinase substrates. The method starts with plant protein microarrays containing hundreds of purified recombinant His-tagged proteins. The microarrays have to be incubated with the soluble and active kinase in the presence of radioactive [gamma33-P]ATP. Only small volumes of active kinase are needed for one microarray experiment. Radioactive signals are then detectable by phosphor imager or X-ray film. The identified potential substrates have to be verified by other in vitro or in vivo methods, as both the kinase and the substrate may not interact with each other under in vivo conditions. This screening method could be generally applicable for direct identification of candidate substrates of various protein kinases.

Arabidopsis↗

PubMed2006

[Mechanism of apoptosis induced by trichostatin A in leukemia Molt-4 cells analyzed by microarray].

BACKGROUND & OBJECTIVE: Histone deacetylase is overexpressed in a variety of cancers and is closely correlated with oncogenic factors. A histone-deacetylase inhibitor, trichostatin A (TSA), has been shown to induce apoptosis in many cancer cells at submicromolar concentrations. However, the mechanism remains unknown. This study was to investigate the underlying mechanism of trichostatin A on apoptosis of Molt-4 cells by characterizing the global gene expression profiles before and after TSA treatment. METHODS: PI single-labeled flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells (PBMC). Microarray and reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the differentially expressed genes of Molt-4 cells after incubation with TSA. RESULTS: TSA could induce apoptosis in Molt-4 cells in a dose and time-dependent manner. Besides, the dose of TSA within the time duration which could induce significant apoptosis in Molt-4 cells did not demonstrate apparent cytotoxicity to PBMCs. After incubation with TSA for 9 hours, 313 genes were detected down-regulated by microarray. Proteins encoded by these genes included signal transduction molecules, transcription factors, enzymes etc., which were involved in the regulation of cell growth, differentiation and survival. STAT5A, MYC and ikaros were down-regulated by 80.4%, 77.3% and 83.1%, respectively. The changes of the three genes were confirmed by RT-PCR and the changes of STAT5A and MYC were further confirmed by Western blot. CONCLUSION: The inhibition of cell growth and induction of apoptosis by TSA in Molt-4 cells may be due to the changes of pro-proliferation genes and anti-apoptosis genes.

Apoptosis↗

PubMedDate not supplied

Plasma proteomic pattern as biomarkers for ovarian cancer.

Early detection of ovarian cancer remains a challenge. Pathologic changes within an organ might be reflected in proteomic patterns in serum or plasma. The objective of this study was to identify new plasma biomarkers in ovarian cancer patients using mass spectrometry (MS) protein profiling and artificial intelligence. The study included 35 women with ovarian cancer and 30 age-matched disease-free controls. For plasma protein signature analysis, the protein chip array surface-enhanced laser desorption/ionization (SELDI) analysis was performed. The strong anion exchange (SAX) and weak cation exchange (WCX) chips were used for analysis. After a training analysis by SAX and WCX protein chips, learning algorithm and clustering analysis was performed to reach a discriminate pattern of protein signature. SELDI mass spectroscopy was highly reproducible in detecting ovarian tumor-specific protein profiles. Four specific protein peaks were identified in plasma of women with ovarian cancer, but not in controls, with relative molecular masses of 6190.48, 5147.06, 11522.6, and 11537.7 d. Two peaks, with Mr 5295.5 and 8780.48 d, were present in plasma of control but not in women with ovarian cancer. A sensitivity of 90-96.3% and specificity of 100% for this studied cases and controls were reached. This study clearly demonstrates that the combined technology of SELDI-MS and artificial intelligence is effective in distinguishing protein expression between normal and ovary cancer plasma. The identified gained and lost protein peaks in plasma may provide as candidate proteins to be used for the detection or monitoring ovarian cancer.

Adolescent↗

PubMedDate not supplied

Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens.

The detection of autoantibodies to tumor antigens has potential utility for the early diagnosis of cancers. In previous studies, we have identified tumor antigens based on Western blot analysis of tumor cell lysates that were incubated with subject sera to identify proteins that elicit specific reactivity in sera from patients with the corresponding tumor type. More recently, we have explored the use of microarrays spotted with tumor proteins as an alternative to Western blots. Microarrays provide a high throughput, high sensitivity alternative to the use of Western blots for tumor antigen profiling. In this study, we have assessed the reproducibility of natural protein microarrays and their ability to distinguish between lung cancer sera and controls. Protein lysates from the A549 human lung adenocarcinoma cell line were separated into 1840 fractions that were spotted in duplicate, along with various controls, on nitrocellulose coated slides. Sera from 18 newly diagnosed patients with lung cancer and from 15 healthy controls were each hybridized to an individual microarray. The reactivity of arrayed proteins with Ig was determined by incubation with biotinylated goat-anti-human-Ig followed by phycoerythrin-conjugated streptavidin. The intensity measures of duplicate spots (within-slide) and duplicate slides (between-slides) were highly reproducible, exhibiting correlation values >0.9. A total of 63 of the 1840 arrayed fractions demonstrated increased reactivity in cancer patients relative to controls as measured by a rank-based statistic (p < 0.008). Microarrays of tumor-derived proteins provide the means for uncovering a repertoire of tumor antigens that have induced an antibody response in patients with specific cancers.

Adenocarcinoma↗

PubMed2006

Identification of protein biomarkers in Dupuytren's contracture using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

BACKGROUND: To study the protein expression profiles associated with Dupuytren's contracture (DC) to identify potential disease protein biomarkers (PBM) using a proteomic technology--Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). METHODS: Normal and disease palmar fascia from DC patients were analyzed using Ciphergen's SELDI-TOF-MS Protein Biological System II (PBSII) ProteinChip reader. Analysis of the resulting SELDI-TOF spectra was carried out using the peak cluster analysis program (BioMarker Wizard, Ciphergen). Common peak clusters were then filtered using a bootstrap algorithm called SAM (Significant Analysis of Microarrays) for increased fidelity in our analysis. RESULTS: Several differentially expressed low molecular weight (<20 kDa) tissue proteins were identified. Spectra generated using both ZipTipC18 aided Au array and WCX2 array based SELDI-TOF-MS were reproducible, with an average peak cluster mass standard deviation for both methods of <1.74 x10(-4). Initial peak cluster analysis of the SELDI spectra identified both up-(14) and down-(3)regulated proteins associated with DC. Further analysis of the peak cluster data using the bootstrap algorithm SAM identified three disease-associated protein features (4600.8 Da, 10254.5 Da, and 11405.1 Da) that were elevated (5.45, 11.7, and 4.28 fold, respectively, with a 0% median false discovery rate). CONCLUSION: SELDI-TOF-MS identified three potential low molecular weight tissue protein markers (p4.6DC, p1ODC, p11.7DC) for DC. The ability of SELDI-TOF-MS to resolve low molecular weight proteins suggests that the method may provide a means of deciphering the biomarker-rich low molecular weight region of the human proteome. Application of such novel technology may help clinicians to focus on specific molecular abnormalities in diseases with no known molecular pathogenesis, and uncover therapeutic and/or diagnostic targets.

Algorithms↗

PubMed2005

[New diagnostics based on the advances of the molecular biology].

Due to the advance of the new technologies of molecular biology, an exposition of the most recent is made (reaction in chain of the polimerasa, sequence and microarrays), and its application is commented in the field of the clinical medicine, of the pharmacology, and mainly, of the centers of diagnostic, especially in microbiology. Above all it, they think about an entire series of challenges and problems to solve, but some new expectations also open up with the creation of new services, introduction of new professionals no doctors and amplification of the diagnostic capacity.

Aged↗

PubMed2003

Protein arrays, an excellent tool in biomedical research.

Although excellent progress has been made in simultaneous analysis of the whole genome, great challenge remains in the highly paralleled analysis of proteome. The development of protein arrays represents a major technology advance in the proteomics field. Advances in protein array technology include chemical surfaces, capture reagents, detection methods and arraying devices. Protein arrays can be classified into different groups. Potential applications of protein arrays include: 1) measurement of protein expression; 2) identification of interactions between protein-protein, protein-DNA and protein-small molecule; 3) analysis of protein modifications; and 4) determination of antibodies. Such protein array technology holds great promise in biomedical research and clinical applications.

Biomedical Research↗

PubMed2005

Layered peptide arrays: high-throughput antibody screening of clinical samples.

High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.

Antibodies↗

PubMed2006

Nuclear and cytoplasmic Maspin expression in primary non-small cell lung cancer.

AIM: To investigate whether nuclear and cytoplasmic Maspin expression is associated with distinct clinicopathological parameters and TP53 expression in a representative series of primary non-small cell lung cancer (NSCLC). METHODS: Tissue microarrays (n=487) were used to immunohistochemically analyse expression of Maspin and TP53. Cytoplasmic and nuclear expression of Maspin was scored on the basis of the percentage of positive tumour cells. Univariate analysis of clinicopathological variables potentially affecting tumour-specific survival was performed. RESULTS: Immunohistochemical Maspin expression (nuclear and cytoplasmic) was informative in 72.3% (352/487) of cases. Cytoplasmic and nuclear Maspin immunoreactivity in >or=10% of tumour cells was detected in 37.8% (133/352) and 65.3% (230/352) of informative cases, respectively. Nuclear and cytoplasmic Maspin staining was observed more frequently in primary squamous cell carcinomas than in other lung cancer types. Only nuclear Maspin immunoreactivity was significantly associated with positive TP53 staining. Cytoplasmic or nuclear Maspin expression was not associated with tumour-specific survival. CONCLUSION: Maspin expression was found both in the nucleus and the cytoplasm of NSCLC, more frequently in squamous cell carcinomas. However, no association with tumour-specific survival could be demonstrated.

Biomarkers, Tumor↗

PubMed2004

DC-SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3.

DC-SIGN is a C-type lectin that binds to endogenous adhesion molecules ICAM-2 and ICAM-3 as well as the viral envelope glycoprotein human immunodeficiency virus, type 1, glycoprotein (gp) 120. We wished to determine whether DC-SIGN binds differently to its endogenous ligands ICAM-2 and ICAM-3 versus HIV-1 gp120. We found that recombinant soluble DC-SIGN bound to gp120-Fc more than 100- and 50-fold better than ICAM-2-Fc and ICAM-3-Fc, respectively. This relative difference was maintained using DC-SIGN expressed on three different CD4-negative cell lines. Although the cell surface affinity for gp120 varied by up to 4-fold on the cell lines examined, the affinity for gp120 was not a correlate of the ability of the cell line to transfer virus. Monosaccharides with equatorial 4-OH groups competed as well as D-mannose for gp120 binding to DC-SIGN, regardless of how the other hydroxyl groups were positioned. Disaccharide competitors and glycan chip analysis showed that DC-SIGN has a preference for oligosaccharides linked in an alpha-anomeric configuration. Alanine-scanning mutagenesis of DC-SIGN revealed that highly conserved residues that coordinate calcium (Asp-366) and/or are involved in both calcium and specific carbohydrate interactions (Glu-347, Asn-349, Glu-354, and Asp-355) significantly compromised binding to all three ligands. Mutating non-conserved residues (Asn-311, Arg-345, Val-351, Gly-352, Glu-353, Ser-360, Gly-361, and Asn-362) minimally affected binding except for the Asp-367 mutant, which enhanced gp120 binding but diminished ICAM-2 and ICAM-3 binding. Conversely, mutating the moderately conserved residue (Gly-346) abrogated gp120 binding but enhanced ICAM-2 and ICAM-3 binding. Thus, DC-SIGN appears to bind in a distinct but overlapping manner to gp120 when compared with ICAM-2 and ICAM-3.

Antigens, CD↗

PubMed2005

Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma.

Follicular lymphoma (FL) is the second most common non-Hodgkin's lymphoma and generally is incurable. Reliable prognostic markers to differentiate patients who progress rapidly from those who survive for years with indolent disease have not been established. Most cases overexpress Bcl-2, but the pathogenesis of FL remains incompletely understood. To determine whether a proteomic approach could help overcome these obstacles, we procured lymphoid follicles from 20 cases of FL and 15 cases of benign follicular hyperplasia (FH) using laser capture microdissection. Lysates were spotted on reverse-phase protein microarrays and probed with 21 antibodies to proteins in the intrinsic apoptotic pathway, including those specific for posttranslational modifications such as phosphorylation. A panel of three antibodies [phospho-Akt(Ser473), Bcl-2, and cleaved poly(ADP-ribose) polymerase] segregated most cases of FL from FH. Phospho-Akt(Ser473) and Bcl-2 were significantly increased in FL (P = 0.001 and P < 0.0001, respectively). Additionally, the Bcl-2/Bak ratio completely segregated FL from FH. High ratios of Bcl-2/Bak and Bcl-2/Bax were associated with early death from disease with differences in median survival times of 7.3 years (P = 0.0085) and 3.8 years (P = 0.018), respectively. Using protein microarrays, we identified candidate proteins that may signify clinically relevant molecular events in FL. This approach showed significant changes at the posttranslational level, including Akt phosphorylation, and suggested new prognostic markers, including the Bcl-2/Bak and Bcl-2/Bax ratios. Proteomic end points should be incorporated in larger, multicenter trials to validate the clinical utility of these protein microarray findings.

Adult↗

PubMed2006

Enhancement of pituitary adenoma cell invasion and adhesion is mediated by discoidin domain receptor-1.

The discoidin domain receptor-1 (DDR1) tyrosine kinases are a family of cell surface receptors that bind to several types of collagen and facilitate cell adhesion that is known association with several cancers. However, no previous study has examined the expression and function of DDR1 in pituitary adenoma. Tissue microarray analysis of DDR1 expression levels in 52 pituitary adenoma tissues revealed that DDR1 expression was significantly related to hormonal background (Kruskal-Wallis test; P < 0.0001). To further elucidate the function of DDR1 in pituitary adenoma, we developed DDR1 over- and under-expressing cell lines using DDR1 clone transfection and short interfering ribonucleic acids (siRNA)-based DDR1 gene silencing, respectively, in a human pituitary adenoma cell line (HP-75). Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting confirmed that expression of both DDR1 isoforms (DDR1a and DDR1b) was elevated by clone transfection and diminished by siRNA. Matrigel invasion assays revealed that cell invasion was increased in HP-75 cells over-expressing DDR1 and decreased in cells under-expressing DDR1. Consistent with this, zymography revealed that the activation levels of matrix metalloproteinase (MMP)-2 and -9 were increased and decreased in cells over- and under-expressing DDR1, respectively. Examination of in vitro cell adhesion to collagen types I, II, III, and IV with respect to MMP-2 and -9 expression revealed that DDR1 regulated cell adhesion to collagen type I, which was responsible for accelerating secretion of MMP-2 and -9 in DDR1 over-expressing cells. Taken together, these results strongly suggest that DDR1 mediates cell invasion-related signaling between collagen type I and MMP-2 and -9 in pituitary adenoma cells.

Adenoma↗

PubMed2004

Mechanism of glutamine-mediated amelioration of lipopolysaccharide-induced IL-8 production in Caco-2 cells.

The mechanism of glutamine (Gln)-mediated down-regulation of inflammation in the intestine is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated IL-8 production in intestinal epithelial cells via transcription factors that counteract the effect of LPS-mediated increase in IL-8. Caco-2 cells were incubated with different doses of Gln with or without methionine sulfoximine (MS), an inhibitor of glutamine synthetase for 24 h before stimulation by LPS (100 microg/ml for 24 h). Inhibitors of the mitogen activated protein kinase (MAPK) family were added to cells for 1.5 h following stimulation by LPS. The p38 inhibitor SB 203580 resulted in a significant decrease in IL-8 peptide production (p < 0.01). However, p38 MAPK activity increased with Gln (p < 0.05), suggesting that this was not involved with Gln-mediated down-regulation of IL-8. Screening of 54 transcription factors demonstrated that STAT-4 was the only inflammation-related transcription factor that was up-regulated by Gln depletion and down-regulated with Gln supplementation (2-fold increase), paralleling IL-8 production. EMSA analysis confirmed these findings (3.5-fold increase). These results indicate that Gln deprivation enhances IL-8 production by Caco-2 cells after LPS stimulation and that down-regulation of IL-8 production with Gln is associated with alterations in STAT-4 transcription factor binding.

Caco-2 Cells↗

PubMed2005

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

Animals↗

PubMed2004

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications.

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.

Antigen-Antibody Complex↗