This work was funded by Luminex Corporation.

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This work was done at Luminex Corporation with equipment manufactured at Luminex Corporation.
R&amp;D Systems &amp; EMD Millipore are strategic partners of Luminex Corporation; licensed to develop and commercialize multiplex xMAP based assays.

The conversion of an ELISA assay to the Luminex xMAP platform can be as simple as substituting streptavidin horseradish peroxidase (SA-HRP) in a typical ELISA kit with streptavidin phycoerythrin (SA-PE), and optimizing for performance. For those who want to create an xMAP immunoassay from the ground up, this can be accomplished with a straightforward protocol that also enables the fast, multiplex evaluation of the antibody pairs. The reagents for the xMAP assay were easily prepared using the xMAP Antibody Coupling kit to couple the designated capture antibodies to MagPlex low concentration microspheres. The use of low concentration microspheres reduces the cost of assay development while providing the same assay performance of higher concentration microspheres. The amount of time required to prepare coupled MagPlex microspheres is approximately 3 hours, which is much faster than the 22 - 24 hours required to coat the well of an ELISA plate, followed by treatment of the coated wells. The performance of the xMAP assay is also superior to the ELISA in terms of limit of detection (&lt;4 pg/mL vs. &gt;31 pg/mL) and dynamic range (&lt;4 pg/mL to &gt;8000 pg/mL vs. 16 pg/mL to 1000 pg/mL). Plate readers have a limited OD range which is either 3 or 4 OD, restricting the upper limit of the dynamic range for an assay.
Undoubtedly, not all antibodies will work in an ELISA format and not all antibodies that work well in an ELISA are readily transferable to the xMAP assay format. However, since xMAP assays can be multiplexed (i.e., run simultaneously), it is possible to evaluate several capture and detection antibody combinations concurrently to identify the best pair to use for an assay. This process saves significant time and reagents compared to the ELISA development procedure, which is limited to the evaluation of one pair at a time. Should two or more antibody pairs perform equivalently; other parameters of the assay can be considered to determine suitability of the pair (e.g., availability, cost, etc.).
In addition to improved assay performance and flexibility with the xMAP assay, there are also significant cost savings. The recommended quantity of antibody required to coat a single well of an ELISA plate is 400ng, whereas the quantity required for the beads used in one well of an xMAP assay is approximately 7.5 ng. Thus, the amount of antibody required for one ELISA well will provide more than 50 test results, if used in an xMAP assay. For applications involving precious samples, xMAP also has a significant advantage. The volume of sample recommended for the ELISA is 100 &mu;L whereas the volume required for the xMAP assay can be half of that, or less.
In summary, conversion of an ELISA assay to the Luminex xMAP platform is uncomplicated, efficient and cost-saving, while producing an assay with superior dynamic range and sensitivity.

<table border="1">
 <tbody>
 <tr>
 <td>Description</td>
 <td>Vendor</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Human TNF-&alpha;/TNFSF1A DuoSet ELISA kit</td>
 <td>R &amp; D Systems</td>
 <td>DY210</td>
 <td>Includes monoclonal and biotin coupled polyclonal antibodies and recombinant TNF-&alpha; protein standard</td>
 </tr>
 <tr>
 <td>Monoclonal Antibody to TNF-&alpha;</td>
 <td>Abcam</td>
 <td>Ab18696</td>
 <td>Capture Antibody, clone CH8820</td>
 </tr>
 <tr>
 <td>Monoclonal Antibody to TNF-&alpha;</td>
 <td>Abcam</td>
 <td>Ab16166</td>
 <td>Biotin coupled Detection Antibody, clone AS1</td>
 </tr>
 <tr>
 <td>TNF alpha protein</td>
 <td>Abcam</td>
 <td>Ab9642</td>
 <td>Recombinant TNF-&alpha; protein standard</td>
 </tr>
 <tr>
 <td>Monoclonal Antibody to TNF-&alpha;</td>
 <td>Novus</td>
 <td>NBP1-50115</td>
 <td>Capture Antibody, clone 4H31</td>
 </tr>
 <tr>
 <td>Monoclonal Antibody to TNF-&alpha;</td>
 <td>Novus</td>
 <td>NB100-78162</td>
 <td>Biotin coupled Detection Antibody, clone MAb11</td>
 </tr>
 <tr>
 <td>TNF alpha protein</td>
 <td>Novus</td>
 <td>NBC1-18460</td>
 <td>Recombinant TNF-&alpha; protein standard</td>
 </tr>
 <tr>
 <td>Monoclonal Antibody to TNF-&alpha;</td>
 <td>EMD Millipore</td>
 <td>MAB1141</td>
 <td>Capture Antibody, clone 3C7.2</td>
 </tr>
 <tr>
 <td>Polyclonal Antibody to TNF-&alpha;</td>
 <td>EMD Millipore</td>
 <td>654250</td>
 <td>Rabbit polyclonal Detection Antibody</td>
 </tr>
 <tr>
 <td>Streptavidin-Phycoerythrin</td>
 <td>Moss</td>
 <td>SAPE-001</td>
 <td>Fluorescent reporter reagent for Luminex xMAP Assay</td>
 </tr>
 <tr>
 <td>MAGPIX w/ xPONENT Software</td>
 <td>Luminex Corporation</td>
 <td>MAGPIX-XPONENT</td>
 <td>Luminex Instrument</td>
 </tr>
 <tr>
 <td>xMAP Antibody Coupling (AbC) Kit </td>
 <td>Luminex Corporation</td>
 <td>40-50016</td>
 <td>Includes EDC reagent, Sulfo-NHS reagent, activation buffer, wash buffer, 1.5 mL reaction tubes, and disposable pipettes</td>
 </tr>
 <tr>
 <td>MagPlex Microspheres, Low Concentration</td>
 <td>Luminex Corporation</td>
 <td>MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID</td>
 <td>Low concentration beads
 (@ 2.5 x 106 bead/mL)</td>
 </tr>
 <tr>
 <td>EZ-Link Sulfo-NHS-LC-Biotin</td>
 <td>Thermo Fisher</td>
 <td>PI-21335</td>
 <td>Biotinylation kit for unmodified detection antibody</td>
 </tr>
 <tr>
 <td>Tecan Infinite F200 Reader</td>
 <td>Tecan</td>
 <td>&nbsp;</td>
 <td>ELISA plate reader</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline</td>
 <td>Sigma-Aldrich</td>
 <td>P-3688</td>
 <td>1% PBS-BSA, Assay Buffer</td>
 </tr>
 <tr>
 <td>One-Pint Compact Ultrasonic Cleaner, 115 VAC</td>
 <td>Cole-Parmer</td>
 <td>WU-08849-00</td>
 <td>Produce an effective operating frequency of 55 kHz</td>
 </tr>
 <tr>
 <td>Magnetic Tube Separator</td>
 <td>Luminex Corporation</td>
 <td>CN-0288-01</td>
 <td>For single 1.5mL tube magnetic separation in coupling wash steps</td>
 </tr>
 <tr>
 <td>Magnetic Plate Separator</td>
 <td>Luminex Corporation</td>
 <td>CN-0269-01</td>
 <td>For 96-well plate magnetic separation in assay wash steps</td>
 </tr>
 </tbody>
</table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

conversion-capture-elisa-to-luminex-xmap-assay-using-multiplex

Research

JoVE Journal

Biology

43.3K visualizações.  Luminex Corporation. Um ELISA pode ser facilmente convertido para um ensaio de xMAP Luminex e, através dos benefícios da multiplexação, vários anticorpos podem ser rastreados simultaneamente para identificar um par de anticorpos óptima, resultando em aumento da sensibilidade e gama dinâmica, enquanto reduz o custo do ensaio.