Cayman Chemical Company
Currents | Issue 18 • Winter 2006
Advanced Platforms for Eicosanoid Measurements
Article By Daniel J. Tew, Adam Uzieblo, Margaret L. Collins, Elizabeth A. Meade, Nisha T. Palackal, Jeff K. Johnson, and Kirk M. Maxey
Immunoassay technology has advanced considerably since the era when all assays were performed in glass test tubes with dangerous radioactive tracers over a period of several days. The microwell plate is ubiquitous, and its standard 96 well version is now routinely subdivided into over 1,500 assays that can fit in the palm of your hand. Enzymatic and fluorescent tracers make it possible to perform assay work quickly and in a relatively uncontrolled environment. The solid-phase format of immunoassays can often be replaced with no-wash, homogenous assays utilizing time-resolved fluorescence (TRF), fluorescence resonance energy transfer (FRET, TR-FRET), and fluorescence polarization (FP) based methods. One of the more clever and productive of the recent assay innovations is the multiplexed assay, pioneered by Luminex as well as other vendors. Multiplexed assays allow one to run two, three, four, or even a dozen assays simultaneously on one single sample of as little as 100 microliters. Even so, nearly all of the commercial multiplexed assays are 2-site, immunometric sandwich assays that only work with large analytes (for oligopeptides, about 28-30 amino acid residues is a critical minimum size). So while this has worked well for large substances like interleukins, the small molecule analytes like steroids, prostaglandins, and leukotrienes have not been developed to the same extent. This article briefly describes Cayman Chemical’s development of assays for prostaglandins (PGE2 and PGD2), thromboxanes (TXB2), and leukotrienes (LTC4 and LTB4) utilizing Luminex® Meso Scale Discovery®, and FP technologies.
Fluorescence Polarization
Fluorescence polarization (FP) assays are homogeneous, single-step assays ideally suited for high-throughput screening (HTS) of large numbers of samples. All FP assays employ a large molecular species, or binding partner (BP), in conjunction with a small, low molecular weight fluorophore-labeled analyte (FA). When the large BP molecule is an antibody, the assay is referred to as a fluorescence polarization immunoassay (FPIA). FP is typically assessed by measuring fluorescence emission though a polarized filter in both parallel and perpendicular orientations compared to the polarized excitation filter.
FPIAs are based on the competition of FA with free (i.e., unlabeled) analyte in the samples or standards for the high affinity binding site of an antibody. A microplate well filled with the FA:antibody complex will give a high FP reading (measured in milli-polarization units or mP) due to the slow rotation of the larger complex compared to that of a smaller molecule. As competition with the free analyte happens, some of the FA will be released from the antibody, and will resume its intrinsic, rapid rate of rotation. This will cause a detectable loss of FP in the well. The addition of increasing amounts of analyte will result in increased reduction in mP. Plotting mP versus analyte concentration allows the construction of a standard curve with a broad dynamic range. This is similar to, but not strictly analagous to, the sigmoidal dose-response curve in a traditional solid phase EIA. Figure 1 depicts the standard curve and Z′-factor determination for our recently developed FPIA for PGD2. Duplex measurement of PGE2 and PGD2 can be accomplished when fluorescein or rhodamine tracers are utilized for separate assays as depicted in Figure 2.
Figure 1 • PGD2 FPIA Red
FP tracers were prepared by labeling PGD2 with Texas Red. Measurements were performed using excitation and emission filters at 560 and 645 nm, respectively. (A) PGD2 standard curve: The standard curve is an average of 4 replicates with an IC50 of approximately 12 ng/ml and a limit of detection of 250 pg/ml. (B) Z′-Factor Determination: Z′-Factor for the PGD2-FPIA was determined by measuring mP in two 96 well plates. Each plate had 48 negative control wells containing only FPIA buffer and 48 positive control wells containing 40 ng/ml PGD2. The Z′-Factor was determined to be 0.63.
Figure 2 • Simultaneous Measurement of PGE2 and PGD2
PGD2 and PGE2 standard curves were prepared separately (control) and in the same well (duplex). Polarization was measured with the appropriate filters (PGD2-fluorescein – ex: 485 nm, em: 530 nm, dichroic: 505 nm · PGE2-rhodamine – ex: 560 nm, em: 645 nm, dichroic: 595 nm).
Meso Scale Discovery®
The MSD® assay platform utilizes ruthenium (II) tris-bipyridine-(4-methylsulfone) [Ru(bpy)3] that, once conjugated to the analyte, serves as the tracer in competitive assays. The Ru(bpy)3-based tag undergoes a rapid redox reaction that emits light in the presence of an applied voltage. Only label bound in proximity of the electrode is detected (see Figure 3). Assays for several eicosanoids have been adapted to the MSD® platform. Figure 4 shows the standard curves for both PGE2 and TXB2. The standard curves are characterized by low variability and a dynamic range of approximately 3 logs.
Figure 4 • Measurement of PGE2 and TxB2 on the MSD® Platform
PGE2 (A) and TXB2 (B) standard curves were quantified by Ru(bpy)3 electrochemi-luminescence using 384 well MULTI-ARRAY plates on the MSD® platform (A) The PGE2 curve is characterized by a 50% B/B0 of 300 pg/ml and an 80% B/B0 of 65 pg/ml (B) The TXB2 assay exhibited a 50% B/B0 of 2.6 ng/ml and an 80% B/B0 of 500 pg/ml, with further optimization still to be performed.
Luminex®
Luminex® xMAP technology is synonymous with multiplexing and is now being utilized in labs worldwide in both research and clinical settings. The technology is based on microsphere beads that have been color-coded to create 100 distinct sets. Each microsphere set carries a different assay similar to the wells on an ELISA plate or different spots on a microarray. The xMAP® microspheres are readily suspendable in liquid and exhibit very favorable kinetics during an assay. This ability to suspend the microspheres in liquid combined with unique spectral addressing (i.e., color-coding) allows assays to be multiplexed.
For application to the measurement of eicosanoids in a competitive format, microspheres are coated with antibodies against a specific analyte of interest. Individual analytes are conjugated to phycoerytherin and serve as tracers in the assay. The tracer and analyte (whether in a sample or standard) compete for a limited number of antibody binding sites on the bead within each well of the plate. After sufficient equilibration time, typically 4 hours, the assays are read using the Luminex analyzer.
Once inside the instrument, fluidics cause the microspheres to line up single file as they pass by two lasers. A red laser classifies each color-coded microsphere, determining which assay is carried on that particle, and the green laser measures the amount of bound tracer on its surface. Since thousands of each microsphere set are used for each separate assay, many readings are made of each individual assay, yielding robust data. In a competitive assay, the amount of bound tracer is inversely proportional to the concentration of analyte in the sample. Figures 5 and 6, respectively, show a simplified picture and data from a duplex assay of PGE2 and LTC4.
Figure 6 • PGE2 and LTC4 standard curves on the Luminex xMAP® Assay Platform
PGE2 (A) and LTC4 (B) standard curves were measured on the Luminex® 100 separately (i.e., as individual assays) and as a duplex assay. The PGE2 and LTC4 assays exhibit nearly identical sigmoidal-shaped curves when acquired in either modality with 50% B/B0 values of 60 pg/ml and 7 ng/ml, respectively.
In summary, FPIA is a very rapid, high-throughput system ideally suited for screening libraries. MSD® is a more sensitive platform that enables multiplexing as well as rapid, homogenous screening. The Luminex® platform has by far the most versatile multiplex capabilities, is quite sensitive, but is not as rapid as the other platforms. The standard 96 well EIA kits developed decades ago by Cayman for lipid signaling molecules will always be the alternative simple and cost effective option to advanced platforms.