ProQuantum high-sensitivity immunoassays

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1 ProQuantum high-sensitivity immunoassays The next generation in protein quantitation

2 Measure protein analytes with high specificity and sensitivity Invitrogen ProQuantum high-sensitivity immunoassays are a new line of ready-to-use kits that provide an easy method of measuring protein analytes with high specificity and sensitivity using a very small amount of sample. The assay workflow is simple, with only one antibody incubation step and no washing prior to placing the plate in any qpr instrument for signal detection. The data analysis is performed on newly designed, free cloud-based software. This is an affordable, scalable open platform that provides easy accessibility for every researcher who wants to measure protein concentrations. Fast, easy workflow no wash steps, typically 2 hours from sample to results road dynamic range 5 logarithmic units, minimizing the need to dilute samples to fall within the range No expensive, proprietary instrument to purchase runs on any qpr instrument Includes intuitive, free cloud-based software for easy, robust data analysis and statistical groupwise comparison enefits include: High sensitivity detect low levels of proteins with greater sensitivity than other traditional methods like ELIS Low sample consumption use 2 5 µl of sample (e.g., 2 µl vs. 75 µl for triplicate wells with other methods) ProQuantum immunoassays can answer your protein detection needs o protein levels correlate with my mrn data? Is there a faster way to measure protein? ProQuantum high-sensitivity immunoassays I have limited samples. o I have enough to measure my proteins of interest? I m trying to measure miniscule amounts of protein. How can I do that? 2

3 How does it work? ProQuantum immunoassays utilize proximity ligation assay (PL ) technology to combine antigen antibody binding for analyte detection with qpr signal amplification and readout (Figure 1). The assay is a two-step process:. nalyte binding by paired antibodies conjugated to oligonucleotides Two antibody conjugates are provided in each kit: a 3 end oligonucleotide and a 5 end oligonucleotide, each conjugated to a target-specific antibody. When the antibody pair binds to two different epitopes of the protein, the 3 and 5 oligos come into close proximity.. Ligation of the oligonucleotides by N ligase and amplification by pplied iosystems TaqMan qpr ssay Only when the pair of antibodies binds to the analyte () can the associated oligos become bound to the complementary splint oligo and subseqently joined to each other with N ligase (). Following the oligo ligation, 95 heat inactivation denatures the ligase, antibodies, and other proteins, leaving 1-base strands in concentrations proportional to the level of antibody analyte binding in the first stage. This 1- base N strand serves as the amplification template for 4 cycles of qpr using TaqMan ssays. 3 3 Splint oligo 5PL regression 3 oligo nalyte 5 oligo 5 5 Fluorescence t nalyte nalyte ycle number nalyte conc. Figure 1. How ProQuantum immunoassays work. 3

4 What comes in each kit? Each kit provides all of the reagents needed, in sufficient quantities to run 96 assay reactions (Figure 2). nalyte binding Ligation and qpr Each kit contains: 2 protein standards (lyophilized) ssay dilution buffer ntibody-conjugate dilution buffer ntibody-conjugate ntibody-conjugate Master mix Ligase ssay workflow The assay workflow is simple. ompared to traditional methods like ELIS, there are fewer incubation steps and no wash steps, and timeto-results is faster (Figures 3 and 4). working plate is recommended in order to do all the preparation steps, including mixing the antibody conjugates, 1-fold dilution of unknown samples, and serial dilutions of the protein standards. The assay run is performed in 2 hours. The first incubation step allows the antibodies to bind to the target analyte for 1 hour. The next step is to add the qpr reaction mixture and place the plate in the qpr instrument for ligation and qpr readout. Protein standard ssay dilution buffer ntibodyconjugate ntibodyconjugate ombine ntibody-conjugate dilution buffer Figure 2. ProQuantum kit contents. omponents are color-coded for ease of use E F G H ntibody conjugates dd 4 μl of qpr reaction mixture to all assay wells. 5 ml ELIS ProQuantum assay Working plate Unknown samples Sample binding to plate: 2 hr Standards 3 µl nalyte binding 5 µl Ligation and qpr Transfer 5 µl of antibody-conjugate mixture to all wells. Then add 5 µl of the standard or the unknown samples to appropriate wells. PR plate ntibody conjugates 3x wash etection antibody binding: 1.5 hr 4 µl PR plate PR plate Master mix 3x wash Time (hours) Figure 3. ssay run time compared to ELIS. ind analyte Perform qpr E F G H E F G H ombine dd substrate, stop, read Standards Unknown samples Total volume per well is 1 µl. 6 minutes Incubate at room temperature E F G H Ligase Master mix Ligase onical tube Reagent reservoir Total volume per well is 5 µl. Place in real-time PR instrument. Figure 4. Schematic of assay run steps. This is only an example of running 5 µl sample volumes in triplicate on an entire 96-well plate. Volumes can be scaled down to 2 µl samples. 4

5 nalyte LO (pg/ml) How has the assay been validated?* With over 32 years of immunoassay experience, we have developed a high-performance assay with the best quality testing in mind. The ProQuantum platform measures the target of interest with great sensitivity, dynamic range, and precision (Figures 5 8). 1, , IL-17E (IL-25) IL-27 IP-1 MIP-1β MP-1 MP-2 MP-3 VEGF EGF G-SF Eotaxin TNF-α IL-6 IL-1β IL-8 IL-2 IFN-Υ IFN-β IL-1 IL-4 IL-12 (p4) IL-12 (p7) IL-9 IL-5 IL-1α IL-1ra IL-17 IL-17F IL-21 IL-22 Flt-3 ligand Fractalkine IGF-1 IL-3 IL-7 IL-13 IL-15 IL-2R ProQuantum immunoassay ELIS ProQuantum immunoassay ELIS Figure 5. nalytical sensitivity. Limit of detection (LO) is defined as 2 standard deviations above background. omparison of LOs for a number of human cytokine targets demonstrates that the ProQuantum platform has 1 1,x greater sensitivity than ELIS. *The use or any variation of the word validation refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses. t ELIS ProQuantum immunoassay IFN-γ (pg/ml) , 1, Figure 6. road dynamic range. The standard curve for the ProQuantum assay overlaid with that of an ELIS demonstrates the larger dynamic range as well as low-end separation ProQuantum (pg/ml) R 2 = ELIS (pg/ml) Figure 7. orrelation of human IL-8 ProQuantum vs. ELIS assays. Serum samples were tested by ELIS and show correlative data between the two platforms. Intra-assay precision Inter-assay precision Parameters Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Mean measured conc. (pg/ml) V (%) Figure 8. Precision. Samples of known human IL-6 concentration were determined in replicates of 1 within an assay (intra-assay precision). Samples of known human IL-6 concentration were determined in replicates of 1 in 3 different assays (inter-assay precision). 5

6 Each kit has been tested on natural samples such as serum, and evaluated using tests such as expected protein levels, spike and recovery, linearity of dilution, and parallelism (Figures 9 and 1). The standards are calibrated to NIS proteins, if available. t Sera , MP-1 (pg/ml) Standard curve Serum 1 Serum 2 Serum 3 Serum 4 3/28 PM + ionomycin Measured MP-1 (pg/ml) Human sera R 2 =.999 R 2 =.953 R 2 =.999 R 2 = Expected MP-1 (pg/ml) 5 PM supernatants PM + ionomycin R 2 = Expected MP-1 (pg/ml) PM supernatants 3/28 R 2 = Expected MP-1 (pg/ml) Figure 9. Parallelism and linearity of dilution. () ssays of a recombinant protein and the corresponding natural protein found in native samples show parallel results. Serum and supernatant samples were tested against the MP-1 standard curve to show parallelism. () Linearity indicates that the observed signals in the standard curve match the general rate of dilution stoichiometry principles. Serum and supernatant samples show that the measured vs. expected results are the same in the linear range of the curve. Endogenous protein measurement (pg/ml) IL-6 IL-2 IFN-γ IL-1 IL-4 IL-12 p4 IL-5 IL-tra MP-3 EGF IL-15 Figure 1. etectable protein levels in normal human serum from 4 donors, measured using various ProQuantum immunoassays. ll samples showed quantifiable protein levels within the range of their respective standard curves. These values often are not quantifiable on other platforms like ELIS. 6

7 ata analysis using cloud-based ProQuantum software The intuitive Invitrogen ProQuantum cloud-based software is available to support your data analysis. This step-by-step software helps you set up your assay, including standard curve dilution calculation, plate layout design, and customized lab bench instructions (Figure 11). Once the assay has been performed at the lab bench, simply upload your run files from the qpr instrument to the ProQuantum cloud app and analyze the data. nalysis features include standard curve qualification, outlier detection, 4 or 5PL regression fit, results for unknown samples in pg/ml, and groupwise statistics. The modern laboratory is undergoing a technological revolution. Easily access and securely share results with colleagues anywhere, anytime, when connected to Thermo Fisher loud. ccess free ProQuantum cloud-enable software at apps.thermofisher.com/apps/proquantum Figure 11. ssay setup and analysis using ProQuantum software. () Standard curve setup. () Plate setup. () ustomized lab instructions. () ata and results. 7

8 Need a new qpr instrument? The pplied iosystems QuantStudio 3 and 5 Real-Time PR Systems provide our latest advancements in touch screen usability, allowing you to stay connected to your data easily. They re designed for both new and experienced users who need simple and affordable real-time PR systems without compromising on performance or quality. For more information and other system options, go to thermofisher.com/ quantstudioqpcrfamily ccessibility* ccess experiment runs from any location, anytime, with remote monitoring Wi-Fi enabled connectivity Utilize portable devices to quickly analyze data when you need to Interactivity Interactive touch screen Run and edit directly from touch screen Easy and intuitive interface Quickly share data and protocols online Send large files securely around campus or around the world nalyze and integrate multiple data sets and data types into one project ollaboration* * With internet access and Thermo Fisher onnect For any questions or to submit future protein target requests, contact us at invitrogenimmunoassays@thermofisher.com New target-specific assays are being developed on a monthly basis. For the most up-to-date product list and to learn more, go to thermofisher.com/proquantum For Research Use Only. Not for use in diagnostic procedures. 217 Thermo Fisher Scientific Inc. ll rights reserved. ll trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. OL