ELISA Tips and TricksOla H. Elgaddar

MD, PhD, MBA, CPHQ

Lecturer of Chemical Pathology

Medical Research Institute – Alexandria University

ola.elgaddar@alexu.edu.eg

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3Wash

ELISA Tips - WashingØ Follow procedures for the preparation of wash buffer.Ø Check washers before use to determine they areworking properly. Perform routine maintenance.

Ø Completely fill the wells. (Fluid dome)ØWhen washing do not allow wells to overflow.Ø Be certain to wash the specified number of times.Ø Examine the wells for completeaspiration of contents.Ø Upon completion of wash cycle, blot to remove residualfluid.

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5Pipetting

ELISA Tips - PipettingØ Calibrate pipettes regularly according to manufacturer'sinstructions.

Ø Avoid touching side wall of well with tips.Ø Avoid splashing of sample and reagents.Ø Use a new tip for each sample/control/reagentaddition.

Ø New tips should be used on the multichannel pipettesfor each reagent to be added.

Ø Forward Vs Reverse pipetting!

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ELISA Tips - PipettingØ Check pipette tips are long enough to provide air spacebetween top of tip and pipette barrel.

Ø Check pipette barrel for residual fluid of dried material,remove if present.

Ø Ensure pipettes tips are fitted tightly.

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8Microplate

ELISA Tips - MicroplateØ Bring microplate pouches to room temperature beforeopening.

Ø Level microwells evenly in microplate frame as theindividual breakaway wells have very flexible plateframes leading to bowing off wells and yield poorwashes.

Ø Place plates in dark immediately after addition ofsubstrate solution, provided the substrate is sensitive tolight.

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ELISA Tips - MicroplateØ Seal unusedwells in purchase along with the desiccant.Ø Date the poucheswhen first opened.Ø Clean bottom surface of plates with wash buffer toremove fingerprints.

ØMake sure microwells are at level during washing,reagent addition and plate/strip reading.

Ø Do not allow microwells to become dry once the assayhas begun.

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11Temperature

ELISA Tips - TemperatureØ Bring test reagents to room temperature (22-28 C)approximately 30minutes prior to use.

ØMaintain proper incubation temperature:-Lower temperaturecan decreaseOD values.-Higher temperatures can increase OD values.-Evaporation in wells can cause edging effect.

Ø Check temperature against calibrated thermometer.

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ELISA Tips - TemperatureØ Sometimes incubation may be@37ºCØ Strict adherence to timemustbemaintained.Ø Check calibration of timers.Ø Record time of incubation.Ø Read plate with specified time limits of adding stopsolution

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ELISA Tips - IncubationØ In certain ELISA systems, the plates are rotated duringincubation for better antigen-antibody reaction.

Ø The effect of rotating plates is to mix the reactantscompletely during the incubation step.

Ø Since the solid-phase limits the surface area of theabsorbed reactant, the mixing ensures that, potentiallyreactive molecules are continuously coming intocontactwith the solid-phase.

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ELISA Tips - Incubation

Ø During stationary incubation, mixing only takes placebecause of diffusion of reagents.

Ø Thus, to allow maximum reaction from reagents instationary conditions, greater times of incubation maybe required, than if they are rotated.

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ELISA Tips - Incubation

Ø Rotation also allow ELISA to be performed independentof temperature conditions.

Ø The interaction of antigen & antibodies relies on theircloseness, and the kinetic energy provided to thesystem, which is encouraged with the mixing duringrotation.

Ø Stationary incubation relies on the diffusion ofmolecules & thus is dependent on temperature.

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ELISA Tips - OthersSubstrate Preparation:qUse freshly prepared substrateqDo not hold substrate solution longer than 1 hour.qFollow procedure of working substrate solution.qThe temperature of solution is important because iteffects the rate of color development.

qDo not add fresh substrate to reagent bottle containingold substrate.

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ELISA Tips - OthersConjugate:qStore at recommended temperature.qNever store excessively diluted conjugate for use atsome later time.

qAlways make up the working dilution of conjugate justbefore you need it.

qNever leave conjugateon the bench for excessive time

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ELISA Tips - OthersAddition of Samples:Problems are usually caused by failure to put sample intobuffer in well, leaving it on the side of the plate. Payattention to the proper addition of samples.

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ELISA Tips - OthersStopping Reagents:qStopping reagents are added to prevent further enzymereaction in ELISA.

qThe stopping is usually made at a time when therelationship among the enzyme-substrate product is inthe linear phase.

qMolar concentration of strong acids or strong basesstopsenzyme activity by quickly denaturing enzymes.

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ELISA Troubleshooting – High O.DqExcessive color development or high optical densityeven when the color development is not dark.

q Mostly due to insufficientwashing or contamination

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ELISA Troubleshooting – High O.DCauses:qPoor-quality water was used to wash plates or toprepare wash solution.

qDeteriorated substrate (Colored)q Insufficientwashing or poor washer performance.qWasher systemhad microbial contamination.qWash systemcontained an alternatewash formulation.qReagents were intermixed, contaminated or preparedincorrectly.

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ELISA Troubleshooting – Low O.DCauses:qLaboratory temperature was too low or reagents /plates were too cold.

qWasher system had microbial contamination orcontained an alternate wash formulation.

qToomany wash cyclesq Incubation periodswere too shortqWrong conjugate was used, conjugate was preparedincorrectly or has deteriorated.

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ELISA Troubleshooting – Low O.DCauses:qAssay plate read was at wrong wavelength, or readerwas malfunctioning.

qAssay plates were compromised or previously used.q Insufficient amount of antigen was coated to microtiterplate.

qNot enough antibody used, or too much dilutedconjugate.

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30No Color Development!!

ELISA Troubleshooting – No Color DevelopmentCauses: (carelessness!Ormalfunctioning kit)qReagents were used in the wrong order or an assay stepwas omitted.

qWrong conjugate was used, conjugate was preparedincorrectly or has deteriorated.

q Incorrector no detection antibodywas added.qSubstrate solutionwas not added.

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ELISA Troubleshooting – Poor ReproducibilityCauses:qExcessive time was taken to add samples controls orreagents to the assay plate.

qMultichannel pipettewas not functioning properly.qThere was inconsistent washing or washer systemmalfunctioning.

qTherewas poor distribution of antibody in the sample.

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