Multiplexed ELISA Kits
A:Q-Plex technology is a multiplex ELISA, an assay platform that can simultaneously quantify the concentrations of multiple analytes by utilizing enzymes and adsorbed antibodies and/or antigens. Each fully-quantitative Q-Plex kit consists of arrays of up to 20 distinct capture antibody nano spots deposited into each well of a microtitre plate, meaning that in a 96-well plate, you can obtain up to 96 X 20 = 1920 data points. This is different from a traditional ELISA, where an entire well is devoted to a single type of antibody population, such that only 96 data points are acquired in one plate. Q-Plex kits can be imaged and analyzed using our high-quality, low-cost Q-View imager, or other imagers, and our Q-View Software. More about Q-Plex.
A: Yes, Quansys specializes in assay development. Customizing with us is easy and fast. We can help you measure specific analytes or biomarker panels. Please call or email for details and pricing. Read more on the customization webpage.
A:Q-Plex plates are stable for at least two years when stored unopened at 4°C (unless stated otherwise on the kit). The SHRP and Substrate are only stable for 1 year and 18 months, respectively. Contact us if your unused kits are more than a year old, and we can arrange to send replacement reagents to you.
A: The Q-Plex kits consist of a printed array of capture antibodies for a variety of biological markers. The user adds a calibrator and samples to individual wells of a 96-well plate. After incubation and washing, a mixture of biotinylated antibodies is added to the plate. This is followed by incubating with streptavidin-HRP or -dye and is completed by capturing the light produced from adding chemiluminescent substrate with an imaging system. Pixel intensity values from the images are used to construct a standard curve and calculate the concentrations of the unknown sample for each system. Please visit this webpage for a detailed description of our technology.
A: Yes, the array uses a standard curve to accurately quantify samples and the curve has been designed to encompass the biological ranges for individual proteins.
A: The Quansys Multiplex ELISA assay has <15% CV between kit lots. Samples are usually linear out to a 1 to 64 dilution.
A: The Q-Plex Array has been tested with serum, plasma, urine, and cell and tissue lysate. We have experienced no interference from FBS, 1% NP-40, 1% Tween, 1% Triton X-100, heparin (30 mg/ml), 1M urea, 20 mM EDTA, 20% citrate. SDS has been shown to interfere with the assay. Human samples containing human antibodies must be diluted 1:2 in the provided sample dilution buffer to remove potential false postives. If tissue homogenates or cell lysates are used, centrifuging is recommended to clarify samples prior to using in the assay. When using tissue homogenates, we recommend preparation in a protein-free buffer so that samples can be tested for total protein to normalize cytokine responses.
A: Most Q-Plex kits work best with 30 µl—50 µl (total volume per well, sample plus diluent). Smaller sample volumes may be used when high levels of detectable proteins are anticipated. Samples containing antibodies MUST be diluted 1:2 in sample dilution buffer prior to running the assay.
A: Most protocols require as little as two and a half hours for completion including analysis with the Q-View Software.
A: No, for best results it is recommended that the assay be completed once started. After reading the results with a camera, the user has the option to delay data analysis to a convenient time. The IR plate may be read at any time after completion of the assay.
A: Once the lyophilized reagents are reconstituted they should be used immediately. If the user desires to run several assays using the 96 well plate, we recommend the user purchase the Q-Plex Stripwell kits. These kits come with additional reagents for multiple testing periods. Customers can purchase additional reagents as needed.
Q: HOW SHOULD I DILUTE MY SAMPLE IF I EXPECT A HIGH RESPONSE FOR ONE BIO MARKER AND A LOW RESPONSE FOR ANOTHER?
A: We recommend testing each sample in step dilutions. For example, when testing cell culture supernatant for unknown cytokine concentrations; a 1:2, a 1:10, and a 1:50 dilution are typically used.
Software & Analysis
A: The LLD is the lowest concentration of an analyte where its signal is distinguishable from the background (2 times the standard deviation of the mean of the background). This is different than the lower limit of quantification (LLOQ) which is the lowest concentration of an analyte that can be accurately measured. In many cases the typical standard curve does not reach the LLD nor the LLOQ for a given protein. The user has an option of using additional dilutions of the calibrator mix to achieve the lower limit of quantification (LLOQ) for a given protein. A quick determination of LLOQ is to examine the backfit of your regression analysis and determine where the model no longer predicts within 80-120% of the actual standard value. I.E. the LLOQ is the lowest point where the backfit is within 80-120% of its corresponding value on the standard curve.
A: The Q-View Software allows the user to open the plate image, mark the spots, input well assignements and select the product type. The software then automatically analyzes the pixel intensity of individual spots on the plate and process the data. All customers can download a free trial of the software and have full access for 90 days. After 90 days, the software becomes the trial version which will still process images and calculate raw data but will not perform regression analysis. Please contact us for more information on Q-View Software or visit this webpage.
Imaging & Equipment
A: The user will need to provide a pipette and imager. For convenient processing we also recommend an automatic plate washer and multichannel pipette for best results.
Q: WILL DIFFERENT CCD AND CMOS CAMERAS GIVE DIFFERENT PIXEL INTENSITIES AND HOW WILL THIS AFFECT MY ASSAY?
A: Yes, different CCD and CMOS cameras give different pixel intensities based on the sensitivity of the camera. However, this will not affect assay performance or standard curve generation as the pixel intensities are relative values.
A: Yes, your samples can be mailed to Quansys where they will be tested using the Quansys Multiplex ELISA platform. Data, along with statistical analyses will be provided to the user within two weeks. Please contact us for details and pricing.