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ELISA tips and tricks - March 2016
This document provides comprehensive tips for performing ELISA (Enzyme-Linked Immunosorbent Assay) effectively, covering crucial aspects such as washing procedures, pipetting techniques, microplate handling, incubation practices, and troubleshooting common issues related to optical density readings. Proper preparation, temperature control, and accurate timing are emphasized to ensure reliable results. Additionally, specific guidelines are offered to address potential problems such as high or low optical density and no color development.
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ELISA tips and tricks - March 2016
- 1. ELISA Tips andTricks Ola H. Elgaddar MD, PhD, MBA, CPHQ Lecturer of Chemical Pathology Medical Research Institute – Alexandria University [email protected]
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- 4. ELISA Tips -Washing Ø Follow procedures for the preparation of wash buffer. Ø Check washers before use to determine they are working properly. Perform routine maintenance. Ø Completely fill the wells. (Fluid dome) Ø When 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 residual fluid. March2016 4
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- 6. ELISA Tips -Pipetting Ø Calibrate pipettes regularly according to manufacturer's instructions. 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/reagent addition. Ø New tips should be used on the multichannel pipettes for each reagent to be added. Ø Forward Vs Reverse pipetting! March2016 6
- 7. ELISA Tips -Pipetting Ø Check pipette tips are long enough to provide air space between top of tip and pipette barrel. Ø Check pipette barrel for residual fluid of dried material, remove if present. Ø Ensure pipettestips are fitted tightly. March2016 7
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- 9. ELISA Tips -Microplate Ø Bring microplate pouches to room temperature before opening. Ø Level microwells evenly in microplate frame as the individual breakaway wells have very flexible plate frames leading to bowing off wells and yield poor washes. Ø Place plates in dark immediately after addition of substrate solution, provided the substrate is sensitive to light. March2016 9
- 10. ELISA Tips -Microplate Ø Seal unused wells in purchase along with the desiccant. Ø Date the pouches when first opened. Ø Clean bottom surface of plates with wash buffer to remove fingerprints. Ø Make sure microwells are at level during washing, reagent addition and plate/strip reading. Ø Do not allow microwells to become dry once the assay has begun. March2016 10
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- 12. ELISA Tips -Temperature Ø Bring test reagents to room temperature (22-28 C) approximately 30 minutes prior to use. Ø Maintain proper incubation temperature: -Lower temperaturecan decrease OD values. -Higher temperaturescan increase OD values. -Evaporation in wells can cause edging effect. Ø Check temperature against calibrated thermometer. March2016 12
- 13. ELISA Tips -Temperature Ø Sometimesincubation may be @37ºC Ø Strict adherence to time mustbe maintained. Ø Check calibration of timers. Ø Record time of incubation. Ø Read plate with specified time limits of adding stop solution March2016 13
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- 15. ELISA Tips -Incubation Ø In certain ELISA systems, the plates are rotated during incubation for betterantigen-antibody reaction. Ø The effect of rotating plates is to mix the reactants completelyduring the incubation step. Ø Since the solid-phase limits the surface area of the absorbed reactant, the mixing ensures that, potentially reactive molecules are continuously coming into contactwith the solid-phase. March2016 15
- 16. ELISA Tips -Incubation Ø During stationary incubation, mixing only takes place because of diffusion of reagents. Ø Thus, to allow maximum reaction from reagents in stationary conditions, greater times of incubation may be required, than if they are rotated. March2016 16
- 17. ELISA Tips -Incubation Ø Rotation also allow ELISA to be performed independent of temperature conditions. Ø The interaction of antigen & antibodies relies on their closeness, and the kinetic energy provided to the system, which is encouraged with the mixing during rotation. Ø Stationary incubation relies on the diffusion of molecules & thus is dependent on temperature. March2016 17
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- 19. ELISA Tips -Others Substrate Preparation: qUse freshly prepared substrate qDo not hold substrate solution longer than 1 hour. qFollow procedure of working substrate solution. qThe temperature of solution is important because it effectsthe rate of color development. qDo not add fresh substrate to reagent bottle containing old substrate. March2016 19
- 20. ELISA Tips -Others Conjugate: qStore at recommendedtemperature. qNever store excessively diluted conjugate for use at some later time. qAlways make up the working dilution of conjugate just before you need it. qNever leave conjugateon the bench for excessive time March2016 20
- 21. ELISA Tips -Others Addition of Samples: Problems are usually caused by failure to put sample into buffer in well, leaving it on the side of the plate. Pay attention to the proper addition of samples. March2016 21
- 22. ELISA Tips -Others Stopping Reagents: qStopping reagents are added to prevent further enzyme reaction in ELISA. qThe stopping is usually made at a time when the relationship among the enzyme-substrate product is in the linear phase. qMolar concentration of strong acids or strong bases stopsenzyme activity by quickly denaturing enzymes. March2016 22
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- 25. ELISA Troubleshooting –High O.D qExcessive color development or high optical density even when the color development is not dark. q Mostly due to insufficient washing or contamination March2016 25
- 26. ELISA Troubleshooting –High O.D Causes: qPoor-quality water was used to wash plates or to prepare wash solution. qDeteriorated substrate (Colored) qInsufficientwashing or poor washer performance. qWasher systemhad microbial contamination. qWash systemcontained an alternate wash formulation. qReagents were intermixed, contaminated or prepared incorrectly. March2016 26
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- 28. ELISA Troubleshooting –Low O.D Causes: qLaboratory temperature was too low or reagents / plates were too cold. qWasher system had microbial contamination or contained an alternate wash formulation. qToo many wash cycles qIncubation periods were too short qWrong conjugate was used, conjugate was prepared incorrectly or has deteriorated. March2016 28
- 29. ELISA Troubleshooting –Low O.D Causes: qAssay plate read was at wrong wavelength, or reader was malfunctioning. qAssay plates were compromised or previously used. qInsufficient amount of antigen was coated to microtiter plate. qNot enough antibody used, or too much diluted conjugate. March2016 29
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- 31. ELISA Troubleshooting –No Color Development Causes:(carelessness!Or malfunctioning kit) qReagents were used in the wrong order or an assay step was omitted. qWrong conjugate was used, conjugate was prepared incorrectly or has deteriorated. qIncorrector no detection antibody was added. qSubstrate solution was not added. March2016 31
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- 33. ELISA Troubleshooting –Poor Reproducibility Causes: qExcessive time was taken to add samples controls or reagents to the assay plate. qMultichannel pipette was not functioning properly. qThere was inconsistent washing or washer system malfunctioning. qThere was poor distribution of antibody in the sample. March2016 33
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