Fertility-eli title.pdf

A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
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2004 Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC. All rights reserved.
We have developed simultaneous, multiplexed assays for Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), and Progesterone on the MSD MULTI-ARRAY™ platform for measurement in serum. LH and FSH are measured in sandwich immunoassays, whereas Progesterone is measured in a competition format. An electrochemiluminescent tag is used to label the detection antibodies and the progesterone competitor. The assay protocol involves a single 1-hour incubation followed by a wash step. Assays are performed in MSD 4-Spot 96-well MULTI-ARRAY Plates, and are read on MSD’s SECTOR™ Imager 6000, with a throughput of 80 samples per minute.
Assay ranges are 0.02 – 200 mIU/ml for LH, 0.1 – 200 mIU/ml for FSH and 0.1 – 70 ng/ml (0.3 – 225 nM) for Progesterone.
All assays are highly specific, and cover the expected clinical ranges.
LH and FSH are formatted as sandwich immunoassays using monoclonal antibodies, and progesterone is formatted as a competitive assay using a polyclonal capture antibody and a labeled progesterone competitor. A 4-spot 96-well MULTI-ARRAY plate is used which is precoated with the three capture antibodies on separate spots within each well. The progesterone competitor and the detection antibodies for FSH and LH are labeled with a Ru(bpy) 2+ compound, and electrochemiluminescence is used as the detection technology.
A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
• Plate: 4-spot 96-well MULTI-ARRAY plate, pre-coated with all three capture antibodies. • Calibration: 8-level multi-analyte calibrator set (serum based matrix).
• Detection Antibodies: labeled antibody solution containing labeled reagents (two labeled antibodies and labeled progesterone) and • Add 25 µl of labeled antibody solution to each well. • Add 25 µl of serum sample or calibrators to each well. • Incubate with shaking for 60 minutes. • Wash 3X with PBS.
• Add MSD-T Read Buffer. • Read plate on SECTOR Imager 6000 Reader (1 minute read time). 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
Three female serum samples were diluted with calibrator matrix and measured three times. The average measured concentration is compared to Dilutions are linear, and average recoveries are 109% for FSH and 100% for LH over all three samples and three dilutions.
Concentration (mIU/ml) Concentration (mIU/ml) Concentration (mIU/ml) Concentration (mIU/ml) Three female serum samples were Spiked with known quantities of LH and FSH and measured three times. The measured average concentration is compared to the expected concentration.
Average recovery is 102% for LH and 96% for FSH over all three samples and 3 spike levels.
Concentration (mIU/ml) Concentration (mIU/ml) Concentration (mIU/ml) Concentration (mIU/ml) A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
Inter-assay cross reactivity was evaluated by testing calibrators spiked with FSH (42 mIU/ml), LH (25 mIU/ml) and/or progesterone (12 ng/ml).
Only a slight cross-reactivity between LH and FSH was noted at the tested concentrations.
The progesterone assay was unaffected by the tested levels of FSH and LH, and elevated progesterone levels had no measurable effect on the other Images of ECL from multiplexed fertility marker assays in MSD 4-Spot Plates.
Note: Progesterone is formatted as a competitive assay, therefore high ECL signal on the progesterone spot indicates low progesterone concentration. LH and FSH are sandwich immunoassays: increased concentration leads to increased ECL emission from their respective spots.
A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
Individual female serum samples were measured on both the MSD plate-based assay and the Roche Elecsys 1010 clinical Good correlations were obtained for all three assays.
10 Performance: Comparison to a Clinical Laboratory Analyzer Analytical sensitivity = 2.5 SD above background A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .
1. Multiplexed FSH, LH, and Progesterone immunoassays have been demonstrated on the MSD platform.
2. Assays are rapid (1 hour incubation), simple (one addition/one wash), use small sample volumes (25 µl), are very sensitive and have 3. Performance is comparable to clinical laboratory analyzers.
A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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