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James W Jacobson

Publications and source records attributed to James W Jacobson.

6 recordsLinked to original sources
PubMed2006

Analysis of individual data from bead-based assays ("bead arrays").

INTRODUCTION: Typically, bead-based assays ("bead arrays") use the mean or median value of a population of measurements to judge ligand binding or other activity, which results in a change in fluorescence intensity. Individual bead measurements are used here to calculate population parameters integral to the measurement of a bead array. METHODS: Using a commercially-available instrument designed for bead array measurements, a set of beads were labeled with biotin and then titrated with PE-Streptavidin. Data were collected under normal machine conditions as well as variations. RESULTS: The "sensitivity" of the measurements was determined using parametric and nonparametric statistical methods as well as regression analysis over a limited range of the titration (concentration vs. response profile). CONCLUSIONS: Results at low ligand concentrations suggest that precise measurements with bead array systems require a large number of individual bead measurements to be acquired. Individual bead measurements should be used to determine the mean and confidence intervals for the calculated measurements. These results also apply to regression analysis of concentration-response profiles. Furthermore, features of the instrument can be manipulated to achieve increased sensitivity and detection of lower amounts of ligand bound to the bead populations.

Biotin↗
PubMed2005

Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays.

A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data from four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction, and microarray analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples, the cell lines, and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered together regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.

Adenocarcinoma↗
PubMed2005

Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.

A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2005

Cancer diagnostics: decision criteria for marker utilization in the clinic.

A new diagnostic tool must pass three major tests before it is adopted for routine clinical use. First, the tool must be robust and reproducible; second, the clinical value of the tool must be proven, i.e. the tool should reliably trigger a clinical decision that results in patient benefit; and, third, the clinical community has to be convinced of the need for this tool and the benefits it affords. Another factor that can influence the adoption of new tools relates to the cost and the vagaries of insurance reimbursement. The Cancer Diagnosis Program (CDP) of the US National Cancer Institute (NCI) launched the Program for the Assessment of Clinical Cancer Tests (PACCT) in 2000 to develop a process for moving the results of new technologies and new understanding of cancer biology more efficiently and effectively into clinical practice. PACCT has developed an algorithm that incorporates the iterative nature of assay development into an evaluation process that includes developers and end users. The effective introduction of new tests into clinical practice has been hampered by a series of common problems that are best described using examples of successes and failures. The successful application of the PACCT algorithm is described in the discussion of the recent development of the OncotypeDX assay and plan for a prospective trial of this assay by the NCI-supported Clinical Trials Cooperative Groups. The assay uses reverse transcription (RT)-PCR evaluation of a set of 16 genes that were shown to strongly associate with the risk of recurrence of breast cancer in women who presented with early stage disease (hormone responsive, and no involvement of the auxiliary lymph nodes). The test is highly reproducible. It provides information to aid the physician and patient in making important clinical decisions, including the aggressiveness of the therapy that should be recommended. A trial is planned to test whether OncotypeDX can be used as a standalone trigger for specific treatment decisions. The problems that have been encountered and have delayed the development of other diagnostic tools are exemplified in the development of tests for human epidermal growth factor receptor (HER2) overexpression, for predictors of response to epidermal growth factor receptor inhibitors, and for the detection of residual disease following chemotherapy.

Algorithms↗
PubMed2003

Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.

Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.

Antigens, Bacterial↗