Alpha Troubleshooting Tables

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General Alpha troubleshooting
AlphaLISA immunoassay troubleshooting

General Alpha Troubleshooting

Problem	Possible Cause	Solution
No signal	Reagent	Donor beads have been exposed to light/photobleach. Use another lot of beads.
	Reagent	Binding partners do not interact. Check for potential steric hindrance. Re-optimize the assay by changing the order of addition.
	Buffer	Inhibitor or quenching component in buffer. Avoid the use of components that quench singlet oxygen (azide, transition metals). Avoid the use of components that absorb light strongly in the 520-680 nm range for AlphaScreen^®, or the 615 nm range for AlphaLISA^®.
	Instrument/plates	Incompatible microplate choice (for example, use of black plates). Use standard solid opaque white plates.
	Instrument/plates	Plate reader error or failure. Consult the instrument manufacturer.
Lower signal than expected	Reagent	Concentration of Donor or Acceptor beads is too low. We recommend using bead concentrations from 10-40 µg/mL.
		Wrong concentration of binding partners. Titrate the binding partners to determine the optimal concentration.
		Degradation of bead conjugates due to incorrect or prolonged storage. Beads should be stored at 4°C, in the dark.
	Assay	Non-optimal order of addition of binding partners or beads. You may need to play with order of addition.
		Inappropriate assay buffer composition. Check for the correct pH, buffering capacity, and salt concentrations, as well as for requirements for educing reagents, detergents, chelators, metal cofactors, blocking reagents, or enzyme inhibitors.
		Incubation time too short (for example, the enzyme reaction time, cell stimulation time, or pre-incubation time of binding partners). Try adjusting your treatment time, your reaction time, or your incubation times.
	Instrument/plates	Incompatible microplate choice (for example, use of black, clear-bottom plates, or plates made of polypropylene. Use standard opaque white plates).
	Instrument/plates	Plate reader error or failure. Consult the instrument manufacturer.
	Temperature	Abnormally low temperature prevailing in the room where the reader is located will result in decreased signal. Ensure that the temperature is kept consistent, around 23°C.
Signal inconsistency	Plates	Warped or distorted plates. Avoid storage of microplates under heavy objects or next to sources of heat.
		Uneven plate molding or tissue culture treatment. Check your plates for defects.
		Light penetrating edges of plate. Ensure that your plates are covered completely and protected from light during incubations.
		Poorly-fitted plate seal (especially in shallow well 384- or 1536-well plate types).
	Assay	Differential evaporation from wells around plate edges. Use a plate seal cover to minimize evaporation of sample; avoid incubation of plates at elevated temperatures.
	Assay	Degradation of buffer components, especially BSA blocking reagent. Prepare fresh buffer; store the buffer without BSA at 4°C and use it within 3-4 days.
	Temperature	If a plate is not equilibrated to the same temperature as the instrument prior to reading, the plate may warm or cool as it is being read, leading to left-right trends. Incubate the plate for at least 30 minutes next to the instrument or in the plate stacker, or use incubators to control the plate temperature during the final incubation.
High background	Assay	Non-specific interaction between assay components. Use blocking agents, such as BSA at higher concentration (>0.1% w/v), or detergent such as Tween-20.
	Plates	Inappropriate dark adaptation. Incubate the plates in dark.
	Plates	Use of white top plate cover. Use a black top cover plate or foil instead.
	Detection	Unsuitable laboratory lighting conditions; avoid use of 'simulated daylight' type fluorescent tubes. Use an alternative location, dim the lighting, or cover the light source with green filter.
		Accidental exposure of beads to light just prior to reading: Acceptor beads will auto-fluoresce for 2-3 minutes. Re-dark adapt for at least 5 minutes prior to reading of plate.
		Air bubbles trapped in some wells. Using electronic multipipettes or automated liquid handling dispensers, and ensure sufficient dead volume in tips to minimize bubbling.
	Temperature	Abnormally high temperature prevailing in the room where the reader is located will lead to higher background.
High degree of signal variability	Assay	Differences due to transfer from assay development to HTS lab. Often there are different operators from assay development to HTS. Ensure that the operators are trained adequately.
		Differential liquid evaporation from wells. Use a plate seal such as TopSeal™-A.
		Mixing problems. For 96-well plates, use a shaker during incubations. For higher density plates, try to add aliquots of no lower than 5 µL to ensure that the liquid expulsion speed from the tip is sufficient to promote adequate mixing.
	Day-to-day variability	Check standard operating procedures. Ensure that the experimental procedure is the same from day-to-day. Prepare the beads in the same area, and ensure that incubation times and temperature do not fluctuate. You may need to use an incubator to control the ambient temperature.
	Instrumentation	Pipetting or dispensing errors. Ensure all pipettes (manual and automated) and liquid handling systems are calibrated. Use suitable tips, and optimize the dispense height and programming of automated dispensers.
	Instrumentation	Temperature difference between the plate and the detector chamber within plate reader. Incubate the plate next to the instrument or in the plate stacker. Check for correct operation of instrument temperature control devices.
Unexpected gradient of signal across entire plate	Instrument/plates	Uneven microplates. Check the microplates.
		Plate is kept at a too low temperature prior to reading. The chemistry is designed to give best results at room temperature. Do not chill the plates or incubate on ice before reading.
		Temperature of plate not equilibrated to instrument ambient temperature. Incubate the plate for at least 30 minutes next to the instrument or in plate stacker. A large number of stacked plates may require longer to reach the instrument ambient temperature.
	Robotic liquid dispensing	This can be caused by inconsistent placing of aliquots in wells, clogged liquid head dispenser, uneven placing of plate on dispenser platform, incorrect tip choice, automated dispenser program error/inaccuracy. Check your robotics/liquid handling system.

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AlphaLISA Immunoassay Troubleshooting

Please note the information in this table refers to troubleshooting in the development of your own AlphaLISA immunoasay, though many of the tips are also applicable if you are using one of our AlphaLISA immunoassay kits.

Problem	Cause	Solution
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.
Cannot fit a standard curve	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.
% recovery too low or too high	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.

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