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Biomedical subjects

Kerry G Oliver

Publications and source records attributed to Kerry G Oliver.

5 recordsLinked to original sources
PubMed2006

High-throughput fluorescent multiplex array for indoor allergen exposure assessment.

BACKGROUND: Current enzyme immunoassay methods for detection of common indoor allergens in environmental dust samples are labor-intensive and time consuming. OBJECTIVE: To develop and validate a fluorescent multiplex array to measure 6 (Der p 1, Der f 1, Der p 2, Der f 2, Fel d 1, and Can f 1) indoor allergen levels simultaneously. METHODS: A multiplex array for 6 allergens, using mAbs covalently coupled to fluorescent microspheres, was developed using a single universal standard composed of purified natural allergens. The multiplex array was validated by comparing the measured dust mite, cat, and dog allergen levels in household dust samples to those obtained by standard ELISA methods. RESULTS: Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for dust mite, cat, and dog allergens: R(2) values ranging from 0.90 to 0.99 (P < .001). In addition, the sensitivity, limit of detection (<0.1 ng/mL), reproducibility, intra-assay coefficient of variance (<5%), and interassay coefficient of variance (<25%) of the fluorescent multiplex array were shown to be equal to or better than the ELISA method. CONCLUSION: A multiplex array has been developed to measure simultaneously 6 indoor allergens from a single sample. The array will facilitate epidemiologic studies and indoor air quality assessments and can, in principle, be expanded to include other allergens and biologics. CLINICAL IMPLICATIONS: The multiplex array lends itself to clinical studies, population-based environmental surveys, and allergen avoidance studies comparing allergen exposure in large populations over several time points.

Air Pollution, Indoor↗
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

Design of a microsphere-based high-throughput gene expression assay to determine estrogenic potential.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

Animals↗
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↗