Picokine ELISA Troubleshooting

Weak or No Signal

Weak or no color development in an ELISA assay can indicate that the target protein is present in minute quantities in the sample, if at all. It can also mean that there is something wrong with the assay or the reagents that prevent efficient detection. If your control reactions indicate that an error is causing your poor results, use this troubleshooting guide to diagnose and resolve your ELISA weak signal.

There are three main causes of weak signals in ELISA

• Antibody/epitope reaction problems
• Insufficient reporter enzyme activity
• Plate related errors

S.No.	Possible Cause	Solution
1	Blocking protein in coating solution	Eliminate blocking protein from coating solution
2	Capture antibody (or antigen) does not bind to plate	Use ELISA plate, not tissue culture plate Try longer coating time Increase concentration of coating components Use Boster pre-coated ELISA kits
3	Problem with the standard	Use new sample Check that the standard is appropriately handled
4	Incubation time too short	Follow the manufacturer guideline (If the problem persists, try incubating samples at 4°C overnight)
5	Incubation temperature too low	Ensure incubations are done at correct temperature Before proceeding, all reagents, including plate, should be at room temperature or as recommended by the manufacturer
6	Incompatible sample type	Use sample that the assay is known to detect a positive control (Include such control in your experiment)
7	Incompatible assay buffer	Ensure assay buffer is compatible with the target of interest
8	Target present below detection limit	Decrease dilution factor or concentrate samples
10	Incorrect/Insufficient/No substrate	Check the substrate identity Increase concentration or amount of substrate Follow manufacturer guidelines
11	Incorrect/Insufficient/No antibody	Check the antibody identity Repeat the assay with higher antibody concentrations to find the optimal one for your experiment
12	Loss of binding activity because antibody was stored at 4°C for several weeks or subjected to repeated freeze-thaw cycles	Use fresh aliquot of antibody that has been stored at -20°C or below Avoid repeated freeze-thaw cycles
13	Incorrect reagents added/ prepared; Missing reagents	Check protocol, ensure correct reagents are added in proper order and prepared to correct concentrations (e.g. TMB for HRP-labeled antibodies)
14	Expired/Contaminated reagents	Make and use fresh/uncontaminated reagents
15	Enzyme inhibitor present	Avoid sodium azide in HRP reactions Avoid phosphate in AP reactions
16	Incorrect storage of components	Double check storage conditions on kit level (Most kits need to be stored at 4°C)
17	Excessive plate washing	Gently pipette wash buffer (manual method) Ensure correct pressure (automatic wash system)
18	Wells dry out	Cover the plate using sealing film or tape for all incubations
19	Wells scratched with pipette or pipette tips	Carefully dispense/aspirate solutions into and out of wells
20	Plate read at incorrect detection wavelength	Use recommended wavelength/filter Ensure plate reader is set correctly for type of substrate used
21	Slow color development	Prepare substrate immediately before use Allow longer incubation Ensure stock solution is unexpired and uncontaminated
22	Epitope recognition impeded by adsorption to plate	Conjugate peptide to large carrier protein before coating onto plate
23	Primary antibody concentration too low	Increase primary antibody concentration Incubate for longer
24	Detection reagent old, contaminated, or wrong pH	Use fresh substrate at the correct pH

Saturated Signal

If your ELISA signal is too high, the results of the experiment can become unusable. Saturated signal can cause wells to appear uniformly reactive, or cause the standard curve to become unusable. Before you repeat your ELISA experiment, we've provided some troubleshooting tips to identify possible sources of the saturated signal error as well as solutions to solve it.

S.No.	Possible Cause	Solution
1	High sample concentration	Use higher sample dilutions. Determine the optimal dilutions by titration assay.
2	Excessive substrate	Decrease concentration or amount of substrate. Follow manufacturer guidelines (e.g. the substrate provided with the ELISA kit might require further dilution).
3	Substrate color changed before use	Make substrate immediately before use.
4	Nonspecific antibody binding	Try different formulations of coating solutions. Ensure wells are pre-processed to prevent nonspecific binding. Use affinity-purified antibody and preferably one that is pre-adsorbed. Use Boster antibodies guaranteed to only react with their targets. Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended).
5	Incubation time too long	Reduce incubation time. Follow the manufacturer guidelines. If the problem persists, try incubating samples at 4°C overnight.
6	Excessive antibody concentration	Repeat the assay with lower antibody concentrations to find the optimal one for your experiment.
7	Contaminated buffers with metals or HRP	Make and use fresh buffers.
8	Insufficient washing	Follow the manufacturer guidelines. At the end of each washing step, thoroughly drain the plate by flicking the plate over a sink and patting the plate on a paper towel.
9	Plate sealers not used or re-used	During incubations, cover plates with plate sealers. Use a fresh sealer every time the used sealer is removed from the plate.
10	Plate read at incorrect detection wavelength	Use recommended wavelength/filter. Ensure plate reader is set up correctly for type of substrate used.
11	Excess time before plate reading	Read your plate within 30 minutes after adding the substrate. If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate.

High Background

S.No.	Possible Cause	Solution
1	Insufficient washing	Follow the manufacturer guidelines At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel
2	Ineffective/Contaminated blocking buffer	Try higher blocking protein concentration Increase blocking time Use fresh buffer
3	Excess antibody	Repeat the assay with lower antibody concentrations to find the optimal one for your experiment
4	Excess substrate	Decrease concentration or amount of substrate Follow manufacturer guidelines (Note: The substrate provided with the ELISA kit might require further dilution)
5	Cross-reactivity (Detection antibody reacts with coating antibody)	Run appropriate controls. Run negative control to determine cross-reactivity. Cross-adsorb your antibody against cross-reactive targets Use Boster antibodies guaranteed to bind only their specified targets
6	Non-specific antibody binding	Try different formulations of coating solutions Ensure wells are pre-processed to prevent non-specific binding Use affinity-purified antibody and preferably one that is pre-adsorbed Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended)
7	Insufficient Tween in buffers	Use PBS containing 0.05% Tween
8	Suboptimal salt concentration in washing buffer	Optimize salt concentration as high concentration can reduce non-specific interactions
9	Incubation temperature too high	Optimize incubation temperature for your assay (antibodies bind optimally at very specific temperature)
10	Reagents were not mixed properly	Thoroughly mix all reagents and samples before pipetting solutions into wells
11	Blanks contaminated with samples	Change pipette tips when switching between blanks and samples Put a lid on pates to avoid any spilling between wells
12	Sample contaminated with enzymes	Test samples with substrate alone to check for contaminating enzymes
13	Contaminated TMB substrate	Use a clean container to check that the substrate in not contaminated (TMB substrate should be clear and colorless before adding to wells)
14	Substrate incubation in light	Carry out substrate incubation in dark or follow recommendation from manufacturer
15	Uneven evaporation of solution from wells during incubation	Always incubate with a lid on the plate
16	Precipitate created in wells upon substrate addition	Increase dilution factor of sample or decrease concentration of substrate
17	Incubation time too long	Follow the manufacturer guidelines (If the problem persists, try incubating samples at 4°C overnight)
18	Incorrect standard curve dilutions	Check pipetting techniques Double check calculations
19	Plates stacked during incubations, leading to uneven temperature distribution	Avoid stacking plates
20	Dirty or defective plates	Wash plates before coating Clean the plate bottom Use Boster pre-coated ELISA plates
21	Unstopped color development	Use Stopping solution to prevent over-development
22	Excess time before plate reading	Read your plate within 30 minutes after adding the substrate (If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate) Note: Color continues to develop even after adding the stopping solution (although at a slower rate)
23	Incorrect plate reading setting	Use recommended wavelength/filter Ensure plate reader is set correctly for type of substrate used

Low Sensitivity

S.No.	Possible Cause	Solution
1	Assay format not sensitive enough	Switch to a more sensitive detection system (e.g. colorimetric to chemiluminescence). Switch to a more sensitive assay type (e.g. direct ELISA to sandwich ELISA). Use Boster's high-sensitivity ELISA kits. Increase incubation time and/or temperature.
2	Improper storage of ELISA kit	Store all reagents as recommended by the manufacturer. Note: All reagents may not have identical storage requirements.
3	Insufficient target	Reduce sample dilution or concentrate sample
4	Inactive substrate	Ensure reporter enzyme has the expected activity. You can run a control to verify reporter enzyme reacts with the substrate.
5	Poor target adsorption to wells	Covalently link target to wells.
6	Insufficient substrate	Increase concentration or amount of substrate.
7	Incompatible sample type	Include positive control in your experiment. Use sample types (e.g. serum vs. cell extract) that have been verified to work as positive controls.
8	Interfering ingredients in buffers and sample	Check reagents for any interfering chemicals (e.g. sodium azide in antibodies inhibit HRP enzyme; EDTA used as anti-coagulant for plasma collection inhibits enzymatic reactions).
9	Mixing or substituting reagents from different kits	Avoid mixing components from different kits.
10	Incorrect plate reading setting	Use recommended wavelength/filter. Ensure plate reader is set correctly for the type of substrate used.

Poor Standard Curve

S.No.	Possible Cause	Solution
1	Improper standard solution	Confirm dilutions are done correctly Make new standard curve as appropriate
2	Standard improperly reconstituted	Briefly spin vial before opening Inspect for undissolved material after reconstituting
3	Standard degraded	Store and handle standard as recommended Prepare standards no more than two hours before use
4	Improper curve fitting	Try plotting using different scales, e.g. log-log, 5-parameter logistic curve fit
5	Pipetting error	Use calibrated pipettes and proper pipetting technique
6	Insufficient washing	Follow the manufacturer guidelines At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel
7	Poorly mixed reagents	Thoroughly mix reagents
8	Poor/variable adsorption of reagents to plate	Extend incubation time Check coating buffer Use a different plate as appropriate Check homogeneity of samples
9	Plates stacked during incubation	Keep plates separated if not using rotating plates
10	Dirty or defective plates	Clean the plate bottom

Poor Replicate Data

S.No.	Possible Cause	Solution
1	Bubble in wells	Ensure no bubbles are present prior to reading plate
2	Insufficient washing of wells	Carefully wash wells Follow recommended protocols Check that all ports of the plate washer are unobstructed
3	Incomplete reagent mixing	Ensure all reagents are mixed thoroughly
4	Inconsistent pipetting	Use calibrated pipettes and proper pipetting techniques Use a fresh sealer every time the used sealer is removed from the plate
5	Inconsistent sample prep or storage	Ensure consistent sample prep and optimal sample storage conditions (e.g. minimize freeze/thaw cycles)
6	Particulates in samples	Remove the particulates by centrifugation
7	Plate sealers not used or re-used	During incubations, cover plates with plate sealers Use a fresh sealer every time the used sealer is removed from the plate
8	Cross-well contamination	Ensure plate sealers and pipette tips are not contaminated with reagents
9	Edge effect (higher or lower OD in peripheral wells than in central wells)	Ensure plates and reagents are kept at room temperature before pipetting into wells unless otherwise instructed During incubation, seal the plate completely with a plate sealer and avoid stacking plates

Inconsistent Assay-to-Assay Results

S.No.	Possible Cause	Solution
1	Insufficient washing of wells	Carefully wash wells Follow recommended protocols Check that all ports of the plate washer are unobstructed
2	Variation in incubation temperature	Adhere to recommended incubation temperature Avoid incubating plates in area where environmental conditions vary
3	Variation in protocol	Adhere to the same protocol from run to run
4	Plate sealers not used or re-used	During incubations, cover plates with plate sealers Use a fresh sealer every time the used sealer is removed from the plate
5	Incorrect dilutions	Confirm dilutions are done correctly for standard solutions, etc Make new standard curve as appropriate
6	Contaminated buffers	Make and use fresh buffers
7	Plates stacked during incubation	Keep plates separated if not using rotating plates

Slow Color Development

S.No.	Possible Cause	Solution
1	Substrates too old, contaminated or used at incorrect pH	Make and use fresh substrates at correct pH: they should be prepared immediately before use
2	Expired/Contaminated solutions	Make and use fresh reagents
3	Incorrect incubation temperature	Ensure plates and reagents are kept at room temperature before pipetting into wells unless otherwise instructed During incubation, seal the plate completely with a plate sealer and avoid stacking plates
4	Low antibody concentration	Repeat the assay with higher antibody concentrations to find the optimal one for your experiment
5	Low substrate concentration	Add more substrate to the wells Make substrate no more than one hour before use Note: Typical ELISA sensitivity is ~0.1 pg/mL with exact value depends on antibody used.

Plate Imaging Problem

S.No.	Possible Cause	Solution
1	Oversaturated image after acquisition	Use full resolution image to analyze results (Do not use jpeg or other compressed formats)
2	Blurry spots in images	Re-focus your camera before taking a new image
3	Repeated pixel values or rectangular spots	Use lower bin size, higher image resolution and/or lossless file type
4	Flat standard in images	Reduce acquisition time

High Well-to-Well Variation

S.No.	Possible Cause	Solution
1	Plates stacked during incubation	Do not stack plates to ensure even temperature distribution.
2	Plates warmed unevenly before use	Allow plate temperature to stabilize before use to avoid edge effects.
3	Bubbles in wells	Check for bubbles before incubations.
4	Uneven washing	Wash all wells uniformly. Check plate washer for obstructed parts.
5	Incomplete reagent mixing	Make sure all reagents are homogenous before use.
6	Inconsistent storage.	Make sure all samples and reagents are stored under uniform conditions.

Matrix Effect

The matrix effect occurs when the target antigen interacts with matrix components in plasma or serum samples. These matrix components can be endogenous biological components such as phospholipids, carbohydrates, and metabolites. Matrix components can reduce the binding of the antibody to the target protein, or non-specifically bind the antibody and generate weak or noisy results.

S.No.	Possible Cause	Solution
1	Matrix effect	Centrifuge the sample. Centrifugation can separate matrix components from soluble antigens, reducing the concentration of the components and the matrix effect on results. Increase dilution. Increasing the dilution factor 2-5 fold will reduce matrix component binding and mitigate the matrix effect. Note: when diluting the samples remember to use the same diluents as used for the standard curve.