Multiplex Protein Assays Research Articles

Development of open-access mock 510(k) regulatory documents on multiplex proteomic technologies.

99 Background: Proteome-based biomarker science is progressing slowly for a variety of factors, including a lack of dialogue early in the discovery/development process with regulatory agencies such as the Food and Drug Administration (FDA). Although high-throughput multiplex proteomic-based technologies have the potential to accurately measure proteins of clinical significance in complex human matrices, regulatory approval for these analytical technologies have not been explored. Methods: To address this gap, investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) held a forward-thinking workshop, involving clinical laboratories, instrument manufacturers, researchers, and FDA representatives in order to streamline the regulatory approval of protein-based multiplex platforms. Results: Outcomes included a peer-reviewed workshop report published in special proteomics issue of Clinical Chemistry, and first-of-its-kind open access mock 510(k) regulatory documents (based on two most commonly applied quantitative proteomic technologies: multiplex mass spectrometry-based and multiplex affinity-based platforms) as public community resources (also published in Clinical Chemistry). Conclusions: These open-access documents provide a glimpse of the FDA’s perspective of novel approaches to in vitro diagnostics of multiplex protein assays and the process of premarket review. Therefore, these resources should enable others to focus on aspects of the process carefully monitored by the FDA, observe a format that is acceptable to the agency, and understand the iterative nature of review.

Selective release of cytokines, chemokines, and growth factors by minced skin in vitro supports the effectiveness of autologous minced micrografts technique for chronic ulcer repair

A new effective surgical procedure to repair chronic ulcers called minced micrografts technique has been recently reported. The technique consists in spreading a finely minced skin sample upon the wound bed. In this study, we investigate the in vitro release of cytokines (interleukin-6, tumor necrosis factor-α, interleukin-1α, and granulocyte-colony stimulating factor), chemokines (monocyte chemoattractant protein-1 and growth-related oncogene-α), and growth factors (platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor, and nerve growth factor) by minced (referred to as the minced sample) vs. not minced (referred to as the whole sample) human skin biopsy samples from the same donor. Factor release in the culture medium at different time points was detected using a multiplexed protein assay. The minced sample, which could behave like the skin fragments used in vivo in the autologous minced micrografts technique, expressed higher levels of tumor necrosis factor-α, interleukin-1α, platelet-derived growth factor, and basic fibroblast growth factor, and lower levels of interleukin-6, monocyte chemoattractant protein-1, growth related oncogene-α, and vascular endothelial growth factor compared with the whole sample. In conclusion, mincing of healthy skin may allow appropriate regulation of the inflammatory phase of wound healing and could induce overexpression of some growth factors, which facilitates the proliferative phase of healing.

Comparative gene responses to collected ambient particles in vitro: endothelial responses

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 μg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

Angiogenesis Markers Quantification in Breast Cancer and Their Correlation with Clinicopathological Prognostic Variables

Tumoural angiogenesis is essential for the growth and spread of breast cancer cells. Therefore the aim of this study was to assess the diagnostic performance of angiogenesis markers in tumours and there reflecting levels in serum of breast cancer patients. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. We observed that breast cancer tumours exhibited high levels of PDGF-BB, bFGF and VEGF, and extremely high levels of TIMP-1 and Ang-2, whereas in serum we found significantly higher levels of Ang-2, PDGF-BB, bFGF, ICAM-1 and VEGF in patients with breast cancer compared to the benign breast diseases patients. Moreover, some of these angiogenesis markers evaluated in tumour and serum of breast cancer patients exhibited association with standard clinical parameters, ER status as well as MVD of tumours. Angiogenesis markers play important roles in tumour growth, invasion and metastasis. Our results suggest that analysis of angiogenesis markers in tumour and serum of breast cancer patients using multiplex protein assay can improve diagnosis and prognosis in this diseases.

Multiplex protein assay performance/evaluation and the requirement for precision and correlation to clinical assays

Multiplex protein assay performance/evaluation and the requirement for precision and correlation to clinical assays

The definition of the venous ulcer

The definition of the venous ulcer

Comparison of mRNA and Protein Measures of Cytokines following Vaccination with Human Papillomavirus-16 L1 Virus-like Particles

mRNA expression signatures are frequently used as surrogate measures of cellular function and pathway changes. Few studies have directly compared results obtained using gene expression and multiplex protein assays for corresponding gene products. We used data available from a clinical trial of a human papillomavirus-16 vaccine that tracked gene expression and cytokine/chemokine production by peripheral blood mononuclear cells stimulated in culture with various antigens to evaluate the degree to which gene expression levels reflect observed levels of cytokines/chemokines. Twenty-six women enrolled in a phase II clinical trial of a human papillomavirus-16 vaccine were evaluated for gene expression (using the Affymetrix Human Genome Focus Array) and cytokine/chemokine levels (using a bead-based 22-plex cytokine assay developed by Linco Research, Inc.) before and after vaccination. Our results suggest the presence of a wide range of correlations between mRNA expression and secreted protein levels. The strongest correlation was observed for IFN-gamma (R = 0.90 overall levels; R = 0.69 when vaccine induced changes were evaluated). More modest overall correlations ranging from 0.40 to 0.80 were observed for MIP1A, IP10, TNF-alpha, MCP1, IL-2, GM-CSF, IL-5, RANTES, and IL-8. Weaker or no correlation was observed between gene expression and protein levels for the remaining cytokines/chemokines evaluated. The degree of correlation between gene expression and protein levels varied among different cytokines/chemokines. Researchers should be cautious when using mRNA expression array results as a proxy for protein levels using existing technologies.

Measurement and Quality Control Issues in Multiplex Protein Assays: A Case Study

Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays. We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias. Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose-response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms. Multiplex measurements present difficult challenges that require further analytical and statistical developments.

Inflammatory cytokine levels in chronic venous insufficiency ulcer tissue before and after compression therapy

Elevated inflammatory cytokine levels have been implicated in the pathogenesis of non-healing chronic venous insufficiency (CVI) ulcers. The goal of this study was to determine the protein levels of a wide range of inflammatory cytokines in untreated CVI ulcer tissue before and after 4 weeks of high-strength compression therapy. These levels were compared to cytokines present in healthy tissue. Thirty limbs with untreated CVI and leg ulceration received therapy for 4 weeks with sustained high-compression bandaging at an ambulatory wound center. Biopsies were obtained from healthy and ulcerated tissue before and after therapy. A multiplexed protein assay was used to measure multiple cytokines in a single sample. Patients were designated as rapid or delayed healers based on ulcer surface area change. The majority of pro-inflammatory cytokine protein levels were elevated in ulcer tissue compared to healthy tissue, and compression therapy significantly reduced these cytokines. TGF-beta1 was upregulated in ulcer tissue following compression therapy. Rapid healing ulcers had significantly higher levels of IL-1alpha, IL-1beta, IFN-gamma, IL-12p40, and granulocyte macrophage colony stimulating factor (GM-CSF) before compression therapy, and IL-1 Ra after therapy. IFN-gamma levels significantly decreased following therapy in the rapidly healing patients. CVI ulcer healing is associated with a pro-inflammatory environment prior to treatment that reflects metabolically active peri-wound tissue that has the potential to heal. Treatment with compression therapy results in healing that is coupled with reduced pro-inflammatory cytokine levels and higher levels of the anti-inflammatory cytokine IL-1 Ra.

Multiplex protein assays based on real-time magnetic nanotag sensing

Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.

Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy

Elevated matrix metalloproteinases (MMP) levels have been implicated in the pathogenesis of chronic venous insufficiency ulcers. Quantitative measurements of a broad range of MMP proteins in human tissue treated with compression bandaging have not been reported. The goal of this study was to determine the expression of a wide range of proteases in untreated venous leg ulcer tissue and the changes in these levels after 4 weeks of high-strength compression therapy. Twenty-nine limbs with new or untreated chronic venous insufficiency and leg ulceration received therapy for 4 weeks with sustained high compression bandaging. Biopsies were obtained from healthy tissue and from ulcerated tissue before and after therapy. A novel multiplexed protein assay was used to measure multiple MMPs in a single sample. MMP protein activity, TIMP protein levels, and gene expression levels were also addressed. MMP1, 2, 3, 8, 9, 12, and 13 protein levels were elevated in ulcer tissue compared with healthy tissue. MMP8 and 9 were highly expressed in ulcer tissue. MMP3, 8, and 9 significantly decreased following treatment. Reduction in the levels of MMP1, 2, and 3 was associated with significantly higher rates of ulcer healing at 4 weeks. We conclude that compression therapy results in a reduction of the pro-inflammatory environment characterizing chronic venous ulcers, and ulcer healing is associated with resolution of specific elevated levels of protease expression.

A Platform for Ultrasensitive and Selective Multiplexed Marker Protein Assay Toward Early-Stage Cancer Diagnosis

Multiplexed marker protein assay is critical in the diagnosis of complex diseases that cannot be diagnosed by detection of a single marker protein. Gold nanoparticle (Au NP) probes barcoded with reporter DNAs and magnetic microparticles functionalized with a capture antibody were developed for the multiplexed detection of three cancer marker proteins. Three types of Au NP probes were used and each Au NP probe was cofunctionalized with reporter (barcode) DNAs and a specific antibody for each corresponding target protein. Target proteins (antigens) were exposed to magnetic microparticles to form complexes and Au NP probes bound to the resulting target-magnetic microparticle complex through antigen-antibody interaction in a different region of the target protein. After magnetic separation of the complexes, barcode DNAs were released, hybridized with capture DNAs printed on a chip and then identified using a scanometric assay that involved silver amplification. Using this method, Mirkin and colleagues successfully demonstrated, for the first time, a highly selective and sensitive multiplexed protein assay against three cancer marker proteins at low picomolar concentration in a buffer of serum media.

Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy

Remodeling of uterine spiral arteries is critical for the continuation of a successful pregnancy. Uterine natural killer (uNK) cells are the predominant leukocyte population in the early pregnant decidua, and a role for these cells in spiral artery remodeling in pregnancy has been suggested. Angiogenic growth factors were measured in isolated uNK and total (unseparated) decidual cells (8-10 or 12-14 weeks gestation, n=5 each gestational age) after culture for 48 h. Angiopoietin (Ang)1, placental growth factor, transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF)-C were measured by enzyme-linked immunosorbent assay. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. Levels of Ang2, ICAM-1, and KGF, secreted by the total decidual fraction, decreased with increasing gestational age. uNK levels of Ang2 and VEGF-C also decreased with increasing gestational age. At 8-10 weeks gestation, there was no difference in the level of Ang1, Ang2, TGF-beta1, and VEGF-C secreted by uNK cells and the total decidual fraction. At 12-14 weeks, uNK cells secreted significantly lower levels of VEGF-C than the total decidual fraction. Early pregnancy decidua is a major source of angiogenic growth factors whose levels decrease with increasing gestational age, suggesting that they may play a role in spiral artery remodeling. uNK cells appear to be a prominent source of Ang1, Ang2, TGF-beta1, and VEGF-C within the placental bed.

Detection of human cytokines with both chemiluminescent and fluorescent plate‐based protein arrays

We have developed chemiluminescent and fluorescent multiplexed protein assays on the SearchLight® platform for the quantitative analysis of 12 human cytokines that are involved in the Th1/Th2 and inflammatory responses. Twelve different capture antibodies for human cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α) were printed in each well of 96-well microtiter plate. The assay was carried out as a typical sandwich ELISA assay until the last step. After adding streptavidin-horseradish peroxidase (SA-HRP) conjugate and a chemiluminescent substrate, the image of the protein array was captured with a CCD camera. For the fluorescent assay, an infrared dye-labeled streptavidin was applied to the plate, and then the protein array was scanned with a laser imager. Serum samples from normal human controls and sepsis patients were assayed with both the SearchLight chemiluminescent and fluorescent arrays. The levels of several cytokines were elevated in serum from sepsis patients compared to normal controls, and the two signal detection systems produced comparable results. The results showed that the multiplexed protein assay is a powerful tool for quantitative screening of patient samples. We have also developed higher density fluorescent arrays (24 and 37-plex) for profiling cytokines, chemokines, angiogenesis factors, and other protein biomarkers in human serum samples.