Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: Matrix effect

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, generating weak or noisy results.
Here are some tips to reduce matrix effect:

  • Centrifuge the sample. Centrifugation can separate matrix components from soluble antigens, reducing the concentration of matrix components and their 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.

Keywords:- Matrix Effect, ELISA Troubleshooting

Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: Low Sensitivity

When using small amounts of precious protein sample, the sensitivity of your ELISA assay can be its most important feature. Boster pre-coated ELISA kits are guaranteed to have consistently high sensitivity, but other ELISA formats can encounter errors that reduce their sensitivity. Use this troubleshooting guide to avoid common sources of reduced ELISA sensitivity:

Incorrect assay typeAssay format inherently not sensitive enoughSwitch to a more sensitive detection system
Switch to a more sensitive assay type
Use Boster high-sensitivity ELISA kits
Incompatible sample typeUse sample types (e.g. serum vs. cell extract) verified to work as positive control
Include a positive control in your experiment
Reagent/sample problemsInsufficient targetReduce dilution factor or concentrate sample
Inactive substrateRun a control to verify reporter enzyme reacts with substrate
Insufficient substrateIncrease concentration of substrate
Mixing or substituting reagents from different kitsAvoid mixing components from different kits
Plate problemsImproper storage of ELISA kitFollow manufacturer storage recommendations
Poor target adsorption to wellsCovalently link target to wells

Keywords:- ELISA, troubleshooting, sensitivity, low sensitivity, low detection limit, sample not detected, no color development

Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: High Variation

High well-to-well variation can be a symptom of a flawed experiment, or it can be a simple error that’s easy to fix. Errors in plate handling or loading, or mistakes in the usage of reagents are all common causes of well variance in ELISA. Use this troubleshooting guide to help solve some of the common sources of error that drive high well-to-well variability.

Plate handling problemsPlates stacked during incubationDo not stack plates to ensure even temperature distribution
Plates warmed unevenly before useAllow plate temperature to stabilize before use to avoid edge effects
Bubbles in wellsCheck for bubbles before incubations
Uneven washingWash all wells uniformly
Check plate washer for obstructed ports
Reagent problemsIncomplete reagent mixingMake sure all reagents are homogenous before use
Inconsistent storageMake sure all samples and reagents are stored under uniform conditions

Keywords:- elisa, troubleshooting, well-to-well, variation, high variation, intra-assay variation

Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: High Background

The signal-to-noise ratio of your ELSIA assay is critical for the proper interpretation of results. High background signal can ruin the data of an entire assay. Before you redo your experiment, consult this guide for tips to help resolve the source of your high background results.

Antibody problemsExcess antibodyLower antibody concentration
Run an optimization trial to determine most effective concentration
Cross reactivityRun negative control to determine cross reactivity
Cross-adsorb your antibody against cross-reactive targets
Use Boster antibodies guaranteed to bind only their specified targets
Non-specific antibody bindingUse a different formulation of coating solution
Ensure wells are pre-processed
Use affinity purified antibody
Block with serum from same species as secondary antibody
Buffer/substrate problemsIneffective/contaminated blocking bufferUse higher blocking protein concentration
Increase blocking time
Mix fresh buffer
Excess substrateReduce concentration of substrate
Optimize substrate concentration
Insufficient Tween 20 in buffersUse PBS containing 0.05% Tween
Suboptimal salt concentration in wash bufferOptimize salt concentration to reduce nonspecific interactions
Plate errorDirty or defective platesWash plates before coating
Use Boster pre-coated ELISA plates
Unstopped color developmentQuench the color development reaction to avoid over-development

Keywords:- elisa, troubleshooting, high background, saturated background, signal-to-noise, high signal to noise ratio

Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: 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, read these troubleshooting tips to identify possible sources of your saturated signal error, and solutions to solve it.

Substrate problemsExcessive substrateDecrease substrate concentration
Incubation time too longReduce incubation time
Reduce incubation temperature
Substrate activation before useMake substrates immediately before use
Plate errorInsufficient washingFollow manufacturer’s washing instructions
Thoroughly drain plate after washes
Plate sealer not used or reusedCover plate with plate sealer during incubations
Use fresh sealer every time
Plate read at incorrect wavelengthUse recommended wavelength/filter
Check that plate reader is set up for the type of substrate used
Antibody problemsExcessive antibody concentrationReduce antibody concentration
Nonspecific antibody bindingUse a different formulation of coating solution
Use Boster antibodies guaranteed to only react with their targets

Keywords:- ELISA, Troubleshooting, Saturated Signal, O.D., Off Scale

Tip: For personalized troubleshooting assistance, contact us at support@bosterbio.com.

ELISA Troubleshooting: Weak 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 prevents 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.

Antibody/epitope reaction problemsCapture antibody failed to absorb to plateCoat the plate for longer
Use more concentrated coating components
Use Boster pre-coated ELISA Kits
Epitope recognition impeded by absorption to the plateConjugate target protein to carrier peptide before coating to plate
Primary antibody concentration too lowIncrease primary antibody concentration
Incubate for longer
Loss of binding activity due to improper storageStore antibodies ant -20C or below
Avoid repeated freeze-thaw cycles
Insufficient reporter enzyme activityEnzyme inhibitor presentAvoid sodium azide in HRP reactions
Avoid phosphate in AP reactions
Detection reagent old, contaminated, or wrong PHUse fresh substrate at the correct pH
Detection substrate too diluteIncrease concentration of detection substrate
Incorrect incubation temperatureOptimize incubation temperature
Make sure all reagents are at room temperature before beginning
Plate errorExcessive washingCalibrate automatic washer to the correct pressure
Wash gently with a manual pipette
Wells dried outCover the plate with sealing film during incubations
Well bottoms scratched by pipette tipsUse caution when dispensing reagents

Keywords:- ELISA, Troubleshooting, Weak Signal, Low Signal, Weak Staining, No Results, No signal