Rapid, sensitive results when timing is critical

PlexMark™ 3 Renal Biomarker Panel

Clinical Diagnostics

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Figure 1. Quantification of MIG, IP-10, and OPG in urine samples. Biomarker proteins were added to normal human urine or to buffer, and the samples were serially diluted and assayed in triplicate using the PlexMark™ 3 Renal Biomarker Panel.

Potential early detection of renal dysfunctionPlexMark™ 3 Renal Biomarker Panel

g Sensitive and specific—accurate detection of IP-10, MIG, and OPG

g Rapid—results in less than 5 hours from urine samples

g Easy and cost-effective—no blood draws required, reduced labor and material costs

g Noninvasive—no biopsy required

Monitoring the profile of cytokines, chemokines, and receptor levels in the urine of re-

nal transplant patients may be a noninvasive way to predict potential renal damage. Recent

studies have profiled levels of three analytes: monokine induced by interferon gamma (MIG),

interferon-inducible protein 10 (IP-10), and osteoprotegerin (OPG) in patient urine samples as

predictors of renal dysfunction.1 The IP-10, MIG, and OPG triplex assay system may aid in the

evaluation of the cryptic occurrence of acute kidney injury.

The PlexMark™ 3 Renal Biomarker Panel Assay uses the Luminex® xMAP® technology in

a standard sandwich immunoassay format to offer ease of use, sensitivity, and rapid results.

This assay has the advantage of assessing whole kidney function by measuring biomarker

levels in urine. As a biological medium, urine is harsh and variable, making it difficult to use

in biochemical and molecular analysis. The PlexMark™ 3 Renal Biomarker Panel Assay uses a

patented buffering system that allows quantitation of urinary proteins with unprecedented

sensitivity, specificity, and reproducibility (Figure 1). One potential research application in-

cludes the detection of post-transplant kidney injury.

Clinical Diagnostics

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www.invitrogen.com

The Luminex® xMAP® technology The Luminex® xMAP® technology combines the efficiencies of multi-

plex analysis with reproducibility similar to ELISA. The technology uses 5.6

µm polystyrene beads that are internally dyed with both a red and an infra-

red flurorophore. Beads are manufactured to contain any one of 100 differ-

ent spectrally distinct red:infrared fluorescence intensity ratios (called bead

regions). As beads pass in front of the laser in the Luminex® instrument, the

bead region is detected, and this serves to identify the bead and distinguish

it from others in the sample.

Beads of known bead region are conjugated to protein-specific cap-

ture antibodies. The beads together with test samples are added to wells of

a filter-bottom microplate. Standards (of known protein concentration) and

control samples (if applicable) are processed in parallel wells using aliquots

of the same capture beads. During a 2-hour incubation, proteins are specifi-

cally captured to the bead surface.

After the incubation period, the beads are washed, and protein-spe-

cific biotinylated detector antibodies are added and allowed to bind to the

captured proteins during a 1-hour incubation. After removal of excess bi-

otinylated detector antibodies, streptavidin conjugated to the fluorescent

protein, R-Phycoerythrin (streptavidin R-PE), is added and allowed to incu-

bate for 30 minutes. The streptavidin R-PE binds to the biotinylated detector

antibodies associated with the immune complexes on the beads, forming a

four-member, solid-phase sandwich.

After washing to remove unbound streptavidin-RPE, the beads are an-

alyzed with the Luminex® detection system. By simultaneously monitoring

both the bead region of the beads and the amount of associated R-Phyco-

erythrin (R-PE) fluorescence, the concentration of one or more proteins can

be determined (Figure 2).

Quantitating MIG, IP-10, and OPG from urine using the PlexMark™ 3 Panel

MIG, IP-10, and OPG levels in urine were quantified using the Plex-

Mark™ 3 Renal Biomarker Panel. Assays were performed on samples of

pooled urine or buffer, both containing known amounts of MIG, IP-10,

or OPG. The assay produced accurate quantitation results, regardless of

whether the spiked sample was buffer or human urine (Figure 1). These

results demonstrate that the PlexMark™ 3 Renal Biomarker Panel is re-

liable and sensitive, even in the harsh, variable background of human

urine. In a separate experiment, samples of human urine were spiked

with MIG, IP-10 or OPG (each at a concentration of 5 ng/ mL) and assayed

using the PlexMark™ 3 Renal Biomarker Panel. Specific quantitation was

obtained for each biomarker with little cross-reactivity (Table 1).

For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

© 2009 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. B-083787 0509

The PlexMark™ 3 Renal Biomarker Panel provides sensitive, reproducible

quantification of three biomarkers implicated in renal dysfunction. Per-

formed on the Luminex® instrument, this assay allows you to take ad-

vantage of powerful multiplexing capabilities and delivers rapid results.

To learn more about the PlexMark™ 3 Renal Biomarker Panel Assay, visit

www.invitrogen.com/plexmark3.

www.invitrogen.com

Ordering informationProduct Quantity Cat. no.

PlexMark™ 3 Renal Biomarker Panel 1 kit LHC6007

Table 1—The PlexMark™ 3 Renal Biomarker Panel provides a highly specific assay with extremely low cross-reactivity. Samples were as-sayed in duplicate; results are expressed in terms of mean fluorescence intensity (MFI).

OPG IP-10 MIG

OPG (recombinant, 5 ng/mL)6,584 7 1016,940 11 98

IP-10 (recombinant, 5ng/mL)12 10,831 511 11,464 5

MIG (recombinant, 5ng/mL)16 25 9,63413 28 9,591

Reference1. Hu, H. et al. (2004) Elevation of CXCR3-binding chemokines in urine indicates acute renal-allograft dysfunction. Am J Transplant 4:432-437.Am J Transplant 4:432-437.Am J Transplant