Mosaic ELISAs are 96-well microplates spotted with up to 8 antibodies per well for simultaneous detection of multiple analytes. To calculate sample concentrations, Mosaic ELISA kits include purified analyte standards.
Introduction
Mosaic ELISA kits employ a two-site sandwich ELISA technique to simultaneously detect up to 8 analytes in cell culture supernates, serum, and plasma samples. Multiple capture antibodies that specifically recognize the target analytes have been pre-spotted into each well of a 96-well microplate. Standards and samples are added, and analytes present in the samples are bound by the immobilized antibodies. After washing away unbound material, biotinylated detection antibodies are used to detect the specific analytes. Unbound detection antibodies are washed away and streptavidin-HRP is added. Following an additional wash, chemiluminescent substrate reagents are added to the wells, and a signal proportional to the amount of each analyte bound in the initial step is produced. Plates are read using a digital camera imaging system, and pixel intensity is measured using an analytical software package.
Available Kits
Product Summary
| Format |
Pre-spotted 96-well microplate
Each well is spotted with up to 8 capture antibodies
Each well is spotted with 1 reference spot to allow easy visualization of well location for template alignment during data analysis |
| Number of Tests |
80 |
| Number of Data Points |
320 data points per microplate (40 samples in duplicate plus 2 standard curves) |
| Number of Analytes |
Up to 8 |
| Sample Type |
Cell culture supernates, serum, and plasma |
| Sample Size |
50 µL (serum/plasma/platelet-poor plasma samples require a 2-fold dilution) |
| Assay Length |
4.5 hours |
| Detection System |
Chemiluminescence |
| Data Acquisition |
Requires a Digital Imaging System and Acquisition Software |
| Amenable to Automation |
Yes |
Kit Contents
- 96-well Microplate Antibody Array
- Cocktail of Purified Protein Standards
- Detection Antibodies
- Diluents & Wash Buffers
- Substrate Reagents*
- Plate Covers
*Substrates 1 and 2 are comprised of TMA-6, a product of Lumigen, Inc., Southfield, Michigan, USA, and are covered by the following US Patent Numbers: 5,922,558 and 6,858,733 and International Patent Numbers: 733,086, 1,019,525, 2,300,071, 1,015,461, 2,002,352,881, ZL02805225.0, and 1,456,716
Step-by-step procedures and instructional videos for imaging Mosaic ELISAs.
Data examples
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| Antibody spotting and data examples for Mosaic ELISA kits. Columns 1 to 5 depict the arrangement of spot locations in microplate arrays that simultaneously detect different numbers of analytes. Columns 6 to 12 show how actual data samples might appear in an 8 antibody spotted microplate. In addition to the 8 spotted capture antibodies, data shown in columns 6 to 12 also depict one Reference Spot (lower rightmost spot in each well). Reference Spots provide a strong positive signal to allow easy visualization of well location for template alignment during data analysis. |
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| Simultaneous detection of multiple analytes in PHA-treated PBMCs. Human peripheral blood mononuclear cells were treated with 10 µg/mL PHA for 0 - 72 hours. Following treatment, analytes were detected using R&D Systems Mosaic ELISA Human Cytokine Panel 1 (Catalog # MEA001). A. Upper panel shows the well map for Mosaic ELISA Human Cytokine Panel 1 and representative images of individual wells for the standard curve and data shown in the histograms below. In addition to the 8 spotted capture antibodies, well images show a Reference Spot (lower rightmost spot in each well). PBMC supernatants were diluted 1:64 to ensure that the values for all of the analytes fell within the standard curve. Reference Spots provide a strong positive signal to allow easy visualization of well location for template alignment during data analysis. B. Lower panel shows examples of analyte concentration quantification. |