Publications by authors named "Paul Rhyne"

19 Publications

  • Page 1 of 1

Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®.

AAPS J 2020 08 3;22(5):104. Epub 2020 Aug 3.

Immunologix Laboratories, 4710 Eisenhower Blvd, Building D, Tampa, Florida, 33634, USA.

The use of biologic-based therapeutics has revolutionized our ability to treat complex diseases such as cancer- and autoimmune-related disorders. Biologic-based therapeutics are known to generate anti-drug immune responses or immunogenicity in clinical patients which can lead to altered pharmacokinetics, decreased drug efficacy, and unwanted adverse clinical events. Assays designed to detect and assess anti-drug immune responses are used to help monitor patients and improve drug safety. Utilizing a tiered approach, screening assays are developed first to identify patients that are potentially positive for anti-drug-specific antibodies. Patients that screen positive are subjected to additional tiers of testing that include a confirmation assay to confirm the presence of expected anti-drug-specific antibodies, a titer assay to assess relative levels of anti-drug-specific antibodies, and, depending on the drug's mechanism of action or concerns of adverse clinical reactions, further characterization such as drug neutralization and anti-drug antibody isotyping. This tiered approach can prove to be detrimental to clinical samples from exposure to multiple cycles of testing, freeze thaws, and repeated handling by lab personnel. Multiplexing some of these assays together may streamline the characterization of anti-drug immune responses and help reduce the repeated usage of clinical samples. In this study, we combined a screening assay and anti-drug isotyping assays into one multiplexed assay using the Luminex® xMAP® Technology. The multiplexed assay was developed and validated to meet the FDA recommended guidelines for immunogenicity assessments. These results show that multiplexed assays perform comparably to industry standards. This study should encourage labs to explore the use of multiplexing immunogenicity assays to characterize anti-drug antibody responses quickly, with less repeat testing and reduced sample handling.
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http://dx.doi.org/10.1208/s12248-020-00487-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399670PMC
August 2020

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

Bioanalysis 2019 Sep 30;11(18s):1-228. Epub 2019 Sep 30.

WuXi Apptec, Shanghai, China.

The 13 GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.
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http://dx.doi.org/10.4155/bio-2019-0207DOI Listing
September 2019

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

Bioanalysis 2019 Jun 19;11(12):1129-1138. Epub 2019 Jul 19.

WuXi Apptec, Plainsboro, NJ 08536, USA.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.
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http://dx.doi.org/10.4155/bio-2019-0131DOI Listing
June 2019

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Bioanalysis 2019 Apr 17;11(7):645-653. Epub 2019 Apr 17.

WuXi Apptec, Plainsboro, NJ, USA.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.
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http://dx.doi.org/10.4155/bio-2019-0072DOI Listing
April 2019

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

Bioanalysis 2018 Dec 29;10(24):1973-2001. Epub 2018 Nov 29.

Amador Bioscience, Pleasanton, CA, USA (formerly of OncoMed, Redwood City, CA, USA).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of , issues 22 and 23 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0287DOI Listing
December 2018

Pharmacokinetic equivalence, comparable safety, and immunogenicity of an adalimumab biosimilar product (M923) to Humira in healthy subjects.

Pharmacol Res Perspect 2018 02;6(1)

Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.

The aims of this randomized, double-blind, three-arm, single-dose study were to demonstrate pharmacokinetic (PK) equivalence of the adalimumab biosimilar M923 (hereafter referred to as "M923") to each of 2 reference products, and to assess M923's safety and immunogenicity. Primary PK endpoints were maximum observed concentration (C ), area under the curve (AUC) from time 0 extrapolated to infinity (AUC ), and AUC from time 0 to 336 hours (AUC ). Secondary endpoints included safety and immunogenicity assessments. Healthy subjects were randomized 1:1:1 to receive a 40-mg dose of M923 (n = 107); adalimumab US Humira (n = 105), hereafter referred to as "US Humira"; or adalimumab EU Humira (n = 103), hereafter referred to as "EU Humira." PK equivalence was demonstrated for all primary PK endpoints. Geometric least squares means ratios (GMRs) for C , AUC , and AUC were 99.4, 100.9, and 100.5, respectively, between the M923 and EU Humira arms and 102.6, 104.2, and 102.9 between the M923 and US Humira arms. The 90% confidence intervals of the GMRs for all PK endpoints were within prespecified confidence bounds of 80%-125%. Adverse event rates were similar across the M923 (47.7%), US Humira (50.9%), and EU Humira (53.3%) arms and were generally mild (73.7%) or moderate (22.0%). The proportion of subjects with a confirmed antidrug antibody (ADA) response was similar across study arms. This study demonstrated bioequivalent PK among M923, US Humira, and EU Humira and demonstrated that the PK parameters were consistent with similar safety and tolerability profile and ADA response rates.
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http://dx.doi.org/10.1002/prp2.380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817835PMC
February 2018

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material.

AAPS J 2017 11 2;19(6):1550-1563. Epub 2017 Oct 2.

Genentech/Roche, 11 Genentech/Roche, 1 DNA Way, South San Francisco, CA, 94080, USA.

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.
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http://dx.doi.org/10.1208/s12248-017-0146-9DOI Listing
November 2017

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

Bioanalysis 2017 Apr 24;9(7):505-516. Epub 2017 Mar 24.

WuXi Apptec, Plainsboro, NJ, USA.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.
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http://dx.doi.org/10.4155/bio-2017-5000DOI Listing
April 2017

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

Bioanalysis 2016 Dec;8(23):2475-2496

OncoMed Pharmaceuticals, Redwood City, CA, USA.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
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http://dx.doi.org/10.4155/bio-2016-4989DOI Listing
December 2016

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

AAPS J 2016 Jan 16;18(1):1-14. Epub 2015 Sep 16.

Ampersand Biosciences, LLC, 3 Main St., Saranac Lake, New York, 12983, USA.

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.
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http://dx.doi.org/10.1208/s12248-015-9820-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706274PMC
January 2016

Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development.

Bioanalysis 2015 ;7(2):229-42

KCAS Bioanalytical Services, 12400 Shawnee Mission Pkwy, Shawnee, KS 66216, USA.

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.
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http://dx.doi.org/10.4155/bio.14.274DOI Listing
September 2015

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

Bioanalysis 2014 ;6(22):2957-63

Covance Laboratories, Chantilly, VA, USA.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
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http://dx.doi.org/10.4155/bio.14.287DOI Listing
July 2015

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

J Pharmacol Exp Ther 2013 Mar 28;344(3):686-95. Epub 2012 Dec 28.

Research and Development, Bristol-Myers Squibb, Wallingford, Connecticut 06492, USA.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.
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http://dx.doi.org/10.1124/jpet.112.199356DOI Listing
March 2013

Characterization and development of a Luminex(®)-based assay for the detection of human IL-23.

Bioanalysis 2010 Sep;2(9):1561-72

Bristol-Myers Squibb Co., Princeton, NJ, USA.

Background: IL-23 is a cytokine produced by dendritic cells, T-cells and macrophages that plays a critical regulatory role in the inflammatory and autoimmune responses. We describe the development and preclinical validation of a highly sensitive Luminex(®) assay specific to IL-23 that is suitable for its measurement in support of early-phase clinical trials.

Results: Intra-assay precision for the BioSource™ ELISA was under 12.3%, and under 5.2% for the eBioscience(®) ELISA. In comparison, the Luminex assays provided an intra-assay precision under 6.2%. The measured inter-assay precision was less than 15.6% for the BioSource ELISA, under 33% for the eBioscience and less than 10% for the Luminex assays.

Conclusions: The Luminex method described provides a way to measure IL-23 in clinical samples either as a single biomarker or as a panel of biomarkers. The assay should prove useful to scientists and clinicians investigating the biology of IL-23 and to those needing to monitor changes in IL-23 as part of a clinical study.
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http://dx.doi.org/10.4155/bio.10.68DOI Listing
September 2010

Electrochemiluminescence in bioanalysis.

Bioanalysis 2009 Aug;1(5):919-35

Bristol-Myers Squibb Company, Pharmaceutical Candidate Optimization, Bioanalytical Sciences, Route 206 and Province Line Rd, Princeton, NJ 08543, USA.

The discovery of electrochemiluminescence (ECL) and its development as a means of detection is truly a success story. Although studies describing ECL were published in the early 1960s, most studies using ECL as a means of detection were not widely published until the mid 1990s. Incorporating ECL into assays provides increased sensitivity, several logs of dynamic range and the ability to electronically control the reaction. These characteristics provide advantages over assays that rely on radioisotopic labels, fluorescence and enzymatic activity. There have been many areas of science that have benefited from the use of ECL, including environmental microbiology, virology, neurobiology, molecular biology and immunology. ECL has improved the understanding and treatment of infectious diseases, cancer, neurodegenerative diseases and even sleep apnea disorders. Drug development has also benefited from ECL via improved assessment of pharmacodynamics, pharmacokinetics and determining immune responses against protein-based therapeutics. This review provides an overview of ECL chemistry and principles with a more detailed emphasis on the applications of ECL-based assays in different areas of science and medicine. The primary purpose of this review is to provide an in-depth discussion of the impact that ECL-based analysis has had on microbiology, immunology, virology, neurodegenerative diseases, molecular biology and drug development. Examples of ECL-based bioanalysis in each of these fields are discussed in conjunction with an overview of ECL principles and instrumentation.
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http://dx.doi.org/10.4155/bio.09.80DOI Listing
August 2009

Immunoassay-based measurement of clinical biomarkers for monitoring changes in nasal cavity.

J Pharm Biomed Anal 2009 Dec 2;50(5):823-30. Epub 2009 Jul 2.

Biomarker and Bioanalytical Sciences, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, Princeton, NJ 08543, USA.

Background: Many drugs for treatment of allergies, migraine headaches, inflammation, and other indications are administered into the nasal cavity providing access to the immune and central nervous systems. One of the concerns for using this route of administration is potential damage to the nasal epithelium and mucosal regions. We assembled a panel of clinical biomarkers that can be used to monitor changes in the nasal epithelium, mucosa, and olfactory regions in preparation for clinical trials involving drugs administered via intranasal route. These biomarkers included albumin, elastase, IL-6, IL-8, lactoferrin, myeloperoxidase and nerve growth factor.

Methods: Immunoassays were developed and used to measure changes in these biomarkers in nasal lavage samples collected twice daily from 30 assumed-healthy volunteers over a 2-day period. Various statistical methods including analysis of variance (ANOVA), paired t-test and Pearson's product-moment correlation were used to evaluate the data.

Results: Although the basal levels of these biomarkers were varied among subjects, the data show that the concentrations of albumin, elastase and IL-8 were significantly higher in samples collected in the morning compared to samples collected later during the day. Pre-washing nasal cavity prior to collecting nasal lavage samples did alter the measurement of elastase and albumin, but did not influence the levels of the other biomarkers.

Conclusions: These data show that this panel of biomarkers can be used to monitor changes in the nasal cavity including those affected by diurnal fluctuations. These results also provide useful baseline values and sources of variability for each biomarker that could be used to help design clinical trials.
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http://dx.doi.org/10.1016/j.jpba.2009.06.043DOI Listing
December 2009

Multiplex analysis of intracellular signaling pathways in lymphoid cells by microbead suspension arrays.

Mol Cell Proteomics 2006 Apr 20;5(4):758-68. Epub 2005 Dec 20.

Center for Comparative Medicine, University of California, Davis, California 95616, USA.

Phosphorylation analysis of signaling proteins is key for examining intracellular signaling pathways. Conventional biochemical approaches, e.g. immunoprecipitation, Western blot, and ELISA, have played a major role in elucidation of individual signaling events. However, these methods are laborious, time-consuming, and difficult to adapt for high throughput analysis. A multiplex approach to measure phosphorylation state of multiple signaling proteins simultaneously would significantly enhance the efficiency and scope of signaling pathway analysis for mechanistic studies and clinical application. This report describes a novel multiplex microbead suspension array approach to examine phosphoproteomic profiles in lymphoid cells. In the Jurkat T-cell leukemia line, the multiplex assay enabled targeted investigation of phosphorylation kinetics of signal transduction from receptor proximal events (tyrosine phosphoproteins CD3, Lck, Zap-70, and linker for T-cell activation) to cytosolic events (serine/threonine phosphoproteins Erk and Akt) to transcription factors (serine/threonine phosphorylated Rsk, cyclic AMP-response element-binding protein, and STAT3). To broaden the application of the multiplex analysis, signaling pathways were also studied in B-cell lymphoid tumor lines that included chronic lymphocytic leukemia lines. In these cell lines, multiplex suspension array enabled phosphoproteomic analysis of signaling cascade mediated by Syk, a homolog of Zap-70. Results obtained by multiplex analysis were confirmed by immunoprecipitation and Western blot methods. The examples of T-cell and B-cell signaling pathway analyses in this report demonstrate the utility of the multiplex suspension arrays to investigate phosphorylation dynamics and kinetics of several signaling proteins simultaneously in signal transduction pathways.
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http://dx.doi.org/10.1074/mcp.T500032-MCP200DOI Listing
April 2006

The functional heterogeneity of type 1 effector T cells in response to infection is related to the potential for IFN-gamma production.

J Immunol 2005 Jun;174(12):7732-9

Trudeau Institute, Saranac Lake, NY 12983, USA.

The expression of IFN-gamma is a hallmark of Th1 cells and CD8(+) effector T cells and is the signature cytokine of type 1 responses. However, it is not known whether T cells are homogeneous in their capacity to produce IFN-gamma, whether this potential varies between tissues, and how it relates to the production of other effector molecules. In the present study we used bicistronic IFN-gamma-enhanced yellow fluorescent protein (IFN-gamma-eYFP) reporter mice (Yeti) and MHC class I tetramers to directly quantify IFN-gamma expression at the single cell level. The eYFP fluorescence of Th1 cells and CD8(+) effector T cells was broadly heterogeneous even before cell division and correlated with both the abundance of IFN-gamma transcripts and the secretion of IFN-gamma upon stimulation. CD4(+) and CD8(+) T cells of influenza-infected mice revealed a similarly heterogeneous IFN-gamma expression, and eYFP(high) cells were only found in the infected lung. Ag-specific T cells were in all examined tissues eYFP(+), but also heterogeneous in their reporter fluorescence, and eYFP(high) cells were also restricted to the infected lung. A similar heterogeneity was observed in Toxoplasma gondii-infected animals, but eYFP(high) cells were restricted to different tissues. Highly eYFP fluorescent cells produced elevated levels of proinflammatory cytokines and chemokines in addition to IFN-gamma, suggesting their coregulated expression as a functional unit in highly differentiated effector T cells.
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http://dx.doi.org/10.4049/jimmunol.174.12.7732DOI Listing
June 2005

Analysis of apoptotic cells using Beadlyte suspension arrays.

Biotechniques 2003 Sep;35(3):624-9

Upstate USA, Lake Placid, NY, USA.

Here we describe a new approach to study apoptosis pathways using multiplex suspension arrays. Apoptosis was induced in Jurkat T cells using the protein synthesis inhibitor, anisomycin. Cells grown in 96-well plates were treated with anisomycin for up to 7 h, washed, and lysed in their respective wells. Samples of each lysate were analyzed using Beadlyte suspension arrays consisting of total Akt/PKB, phosphorylated Akt/PKB, active caspase-3, and single-stranded DNA (ssDNA)-specific Beadmate microspheres and quantified with the X-MAP system. We found that phosphorylated Akt levels dropped dramatically with 2 h or more of anisomycin treatment, whereas active caspase-3 levels rose sharply with 2 h of treatment, signifying the onset of apoptosis. Longer incubation with anisomycin showed increases in ssDNA, which is consistent with the characteristic degradation of DNA that occurs in late-stage apoptosis. This approach demonstrates how apoptosis pathways can be studied from a small amount of sample without the use of more lengthy techniques such as immunoprecipitation or Western blotting. The suspension array is being expanded to measure many other intracellular proteins including posttranslational modifications and should prove to be extremely useful for studying apoptosis and other important cellular pathways.
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http://dx.doi.org/10.2144/03353pf01DOI Listing
September 2003