High background | Bead concentration is too high | We recommended using 40 μg/mL Donor beads and 10 μ/mL Acceptor beads (final concentration). |
One of the antibodies can bridge both the Donor and Acceptor beads simultaneously | Re-evaluate bead selection. Example: Use of a biotinylated rabbit IgG antibody in an assay using Streptavidin Donor beads and Protein A Acceptor beads. Protein A can bind strongly to rabbit IgG antibodies. Because of this, one single biotinylated rabbit IgG antibody can bring together both a Streptavidin Donor bead and a Protein A Acceptor bead in the absence of analyte. |
Buffer selection | We recommend using AlphaLISA Immunoassay Buffer (Cat. No. AL000) for dilution of your beads and antibodies. If this buffer still gives high background, you may need to try AlphaLISA HiBlock Buffer (Cat. No. AL004) or AlphaLISA NaCl Buffer (Cat. No. AL007). |
Bead selection | Certain bead pairs can associate with themselves in the absence of other assay components. For more information, refer to our description and table. |
Pre-mixture of components | Pre-mixing reagents that may cross-react could result in high background |
Contamination | You may be inadvertently introducing target analyte from your skin, saliva, etc. while preparing your reagents. If you are measuring a human analyte, take appropriate measures to prevent contamination. |
Interference by diluent used | Change or evaluate other buffers |
Assay configuration | Switch antibody configuration and evaluate alternate protocols |
Low maximal signal | Significant exposure of Donor beads to light | Alpha Donor beads are somewhat light-sensitive. If your beads have been exposed to light for a prolonged period, you may need to use fresh beads. |
Matrix interference | Some components of cell culture media may interfere with the AlphaLISA signal. If possible, try switching to a different medium. |
Order-of-addition | Some binding partners may interfere with the association of other binding partners if allowed to interact in the wrong order. Try an alternate order-of-addition protocol. |
Incubation time | You may need to extend the incubation time of your protocol steps to allow more time for antibody to bind target. |
Hook effect | The signal should increase with increasing concentrations of analyte, until you reach saturation (the Hook Point). At analyte concentrations above the Hook point, signal will begin to decrease. Make sure you are in the right range of your assay. The easiest way is to perform a full titration curve of the analyte. |
Instrument settings | Verify instrument settings and ensure instrument is working properly |
Verify concentrations of your reagents | If possible, take analyte OD measurements, and double-check calculations for preparation of working solutions |
Antibody selection | You may need to test other antibodies in your assay |
Poor sensitivity | Order-of-addition | Try an alternate order-of-addition protocol (3-step protocol) – order-of-addition can influence assay sensitivity. |
Matrix interference | Some complex matrices may contain components that can interfere with the assay. For example, biotin present in culture media may interfere with binding of a biotinylated antibody to streptavidin Donor beads. You may need to dilute your samples in a buffer that will not interfere with the assay. |
Antibody selection | Try a different assay configuration (switch biotinylated and bead-conjugated antibody), or try a different antibody. |
Analyte selection | Make sure you have chosen an appropriate standard analyte for your antibodies and your assay. Recombinant versions of your analyte may be available in full-length or cleaved form, in active or inactive form, etc. |
Cannot fit a standard curve | Trying to fit sigmoidal data to a linear curve | AlphaLISA data should be plotted to fit a dose response (or sigmoidal, 4PL) curve. If you would prefer to use just the linear portion of the curve, you will need to omit some of the higher and lower concentrations of analyte from your data. |
Hook effect | The signal should increase with increasing concentrations of analyte, until you reach saturation (the Hook Point). At analyte concentrations above the Hook point, signal will begin to decrease (resulting in a bell-shaped/parabolic curve). Omit higher concentrations of standard and re-fit the data to a sigmoidal curve. |
Shifted curve (curve shifts right or left) | Order-of-addition | Your order-of-addition must stay consistent from assay-to-assay. Changing the order in which assay components are added can shift your standard curve. |
The diluent used for the standard curve was changed | The diluent used for your standard curve must closely match your sample type, and must be consistent from assay-to-assay. If your diluent was changed, this would likely cause a shift in your standard curve. If you are measuring different sample types, you need to use different diluents for each of your standard curves. |
Change in analyte | If your analyte has changed (for example, you changed lots and the new lot has a different level of glycosylation) or become degraded, your curve may shift. |
Sticky analyte | If your analyte is sticky and can stick to the walls of your tubes or pipette tips, you will see a curve shift reflecting the lower soluble concentration of analyte. You may need to use low-bind tubes and tips. |
Change in antibody lot | If you are working with a polyclonal antibody, a change in antibody lot may result in a curve shift due to differences in affinity. |
% recovery too low or too high | Spike-in concentration is not within the dynamic range of the assay | Try using a different spike-in concentration for assessing % recovery. |
Selection of diluent for standard curve. | Try using an alternate diluent for your standard curve. Your diluent for the standard curve must match your sample matrix as closely as possible. You might also need to dilute your samples to decrease matrix interference. |
AlphaLISA results do not match ELISA results | *It is important to determine which assay is giving accurate results. If possible, use a third method to assess which assay is accurate. |
Standard curve is not run in a diluent similar to the sample type | The standard curve should be run in a diluent that closely resembles your sample type. For example, if you are working with cell culture supernatant samples, you should run your standard curve in the same culture media used to treat your cells. |
Change in antibody | If you use different antibodies, you may see different selectivity. Different results may be obtained. |
Change in analyte | If you use a different standard for your standard curve, different results may be obtained. |
Standard curve is not being fit properly (e.g., sigmoidal data is being fit to a linear curve) | AlphaLISA data should be plotted to fit a sigmoidal curve. Data weighting is recommended (1/Y2) to get the best curve fitting for lower concentrations. If you would prefer to use just the linear portion of the curve, you may need to omit some of the higher and lower concentrations of analyte from your data. |
Buffer selection | Your target analyte may exist in a state bound to another molecule in your sample and cannot be recognized by the antibodies. If this is a possibility, we recommend using AlphaLISA Dissociation Buffer (Cat. No. AL006) to disrupt the interaction within your sample. |
Concentration of analyte in your samples is too high or too low (beyond the dynamic range of the assay) | Sample dilution may be required to adjust your samples to be within the dynamic range of the assay. Refer to Hook effect – concentrations of analyte above the Hook point can give low signal. |