Array-Based ELISAs for High-Throughput Analysis of Human Cytokines

Moody, Max D.; Arsdell, S W Van; Murphy, Keven; Orencole, Scott F.; Burns, Chris

doi:10.2144/01311dd03

Cited by 181 publications

(123 citation statements)

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“…SearchLight proteome array analyses (Pierce Biotechnology, Woburn, MA) were conducted to measure the levels of IL-4, IL-6, IL-17, and TGF␤ in supernatants from AIF-1-expressing clones, as previously described (36)(37)(38). Briefly, samples were diluted 1:5, 1:50, or 1:1,000 and then incubated for 1 hour on the array plates, which had been prespotted with capture antibodies specific for each protein.…”

Section: Ef070983 and Ef070982])mentioning

confidence: 99%

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Galdo¹,

Jiménez²

2007

Arthritis & Rheumatism

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Objective. Allograft inflammatory factor 1 (AIF-1) was first identified in rat cardiac allografts undergoing chronic rejection. The vasculopathy of chronic allograft rejection is strikingly similar to that seen in patients with systemic sclerosis (SSc). We previously demonstrated AIF-1 expression in inflammatory cells infiltrating skin and lungs from SSc patients, but its role in SSc pathogenesis is unknown. The present study was undertaken to investigate the effects of AIF-1 on T cell migration and production of cytokines capable of modulating normal dermal fibroblast functions.Methods. Stably transfected Jurkat T cells expressing 2 AIF-1 splicing variants were prepared, and their migration toward fibroblast monolayers assayed in Transwell cultures. Cytokine production was assessed by real-time polymerase chain reaction (PCR) and multiplex enzyme-linked immunosorbent assay. Fibroblast gene expression was quantified by real-time PCR, and collagen production by Western blot analysis of culture media.Results. AIF-1 significantly increased Jurkat T cell migration toward fibroblast monolayers. Expression of AIF-1 isoform 2 in Jurkat T cells up-regulated their production of interleukin-4 (IL-4) and IL-17. Conditioned media from AIF-1-expressing clones stimulated synthesis of types I and III collagen and expression of IL-6, transforming growth factor ␤, endothelin receptor, and ␣-smooth muscle actin by normal dermal fibroblasts.Conclusion. These results suggest that AIF-1 may participate in the early pathogenesis of SSc by promoting tissue T cell infiltration and production of cytokines capable of inducing the expression of a fibrotic phenotype in normal fibroblasts.

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Section: Ef070983 and Ef070982])mentioning

confidence: 99%

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Galdo¹,

Jiménez²

2007

Arthritis & Rheumatism

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“…Proliferation was determined by [ 3 H]thymidine uptake. Cytokines were quantitated in 48-h Ag-stimulated supernatants using the Pierce Multiplex SearchLight Arrays technology (͗www.SearchLightOnline.com͘) (37).…”

Section: Determination Of Immunological Responsesmentioning

confidence: 99%

Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance

Bagenstose

Agarwal

Silver

et al. 2005

The Journal of Immunology

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We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.

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“…Polyclonal or monoclonal antibodies for several thousand proteins are available, and are being supplemented with affinity probes generated by phage and ribosomal display, affibodies, and aptamers [8][9][10][11] . Indeed, recent studies have described sensitive 12,13 RCA is a useful alternative for on-chip signal amplification [15][16][17] .It permits sensitive and highly multiplexed assays on microarrays because RCA-amplified signals remain localized at the microarray spot ( Fig. 1) 16,17 .…”

Section: Introductionmentioning

confidence: 99%

Multiplexed protein profiling on microarrays by rolling-circle amplification

Schweitzer¹,

Roberts

Grimwade³

et al. 2002

Nat Biotechnol

542

387

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Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.Several recent reports have established the feasibility of protein arrays for a variety of applications [1][2][3][4][5][6][7] . To meet the emerging needs of expression proteomics, however, such arrays must yield highly multiplexed, sensitive, quantitative, and reproducible measurements of protein levels. It is also desirable that assays on these arrays utilize small sample volumes and be compatible with hardware and software used by the DNA microarray industry. Microarrays of ordered immobilized capture antibodies and attendant sandwich immunoassays are a straightforward, near-term approach for highly parallel measurement of protein levels. Polyclonal or monoclonal antibodies for several thousand proteins are available, and are being supplemented with affinity probes generated by phage and ribosomal display, affibodies, and aptamers [8][9][10][11] . Indeed, recent studies have described sensitive 12,13 RCA is a useful alternative for on-chip signal amplification [15][16][17] .It permits sensitive and highly multiplexed assays on microarrays because RCA-amplified signals remain localized at the microarray spot ( Fig. 1) 16,17 . When utilized on microarrays of printed proteins, RCA has been shown to allow detection of protein analytes with zeptomole sensitivity and broad dynamic range 16,18,19 . In the present study, we establish the utility of RCA for highthroughput analysis of protein expression on microarrays, providing assays that are highly sensitive, quantitative, and reproducible. We describe highly multiplexed, microarray immunoassays with four steps: sample application and...

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Array-Based ELISAs for High-Throughput Analysis of Human Cytokines

Cited by 181 publications

References 10 publications

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance

Multiplexed protein profiling on microarrays by rolling-circle amplification

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