Technical Guide
Troubleshooting for common lab applications.

Troubleshooting guide: ELISA

Many factors can affect the performance of an ELISA. Reading the instructions carefully prior to starting will help avoid unnecessary errors. Using good laboratory practices as well as calibrated equipment will all assist in a successful assay. Use clean reservoirs and a clean lab space and change pipette tips between each reagent. Running all standards and samples in dulicate will improve accuracy.

Washing Techniques:

Multi-channel Pipette or squirt bottle		Ensure that each prong of the pipette is dispensing properly or that the squirt bottle has enough pressure. Empty the contents of the well and fill well with volumes of wash buffer indicated. Decant the wells and invert and blot on a clean paper towelling. Repeat this process as indicated in the insert. After the last decanting, remove any remaining wash buffer by inverting and blot it on a clean paper towelling. Do not allow wells to sit dry, move to the next step as indicated in the insert. Note: numbering the strips prior to washing and decanting in a clean sink is useful in the event that strips become loose while decanting.
Manifold dispensor or autowasher		Ensure that there is an appropriate vacuum supply. Check all cannulae or prongs for proper dispensing and aspirating.Empty the wells by aspirating or decanting. Fill each well the appropriate volume indicated in the insert. Aspirate the wells completely. Do not over aspirate the wells by allowing the aspiration aparatus to sit in the wells after the buffer has been removed. Repeat this process as indicated in the insert, not allowing the wells to sit dry after the last aspiration.

Pipetting Techniques:
Ensure that the pipette that you are using is calibrated and that the pipette tip is seated properly and securely on the pipette. Set the pipette to the desired volume and pre-rinse with sample or reagent. Draw the reagent up slowly, allowing the pipette to return to the up position slowly. Wait a moment to allow the reagent to reach volumetric equilibrium in the tip. Dispense the reagent slowly to the first stop and then gently to the second stop. Hold the pipette at this position until it is removed from the reservoir or well to avoid drawing the reagent back up. As you remove the pipette, draw the tip up the side of the reservoir or well to release any liquid that may be on the outside of the tip.

Troubleshooting

Poor Pecision		Incomplete washing: ensure that apparatus is working correctly and that wells appear dry after aspiration. Unequal mixing of reagents: Ensure that reagents are mixed adequately. Contamination: Be sure to use clean reservoirs and change pipette tips between each reagent. Pipetting error: use good lab techniques and ensure pipette is calibrated properly.
Standard Curve		Improper standard preparation: Ensure that standards are reconstitued appropriately and that dilution instructions are adhered too. Incomplete washing: ensure that apparatus is working correctly and that wells appear dry after aspiration. Unequal volumes added to well: check that pipette is calibrated and that pipette tips are seated properly. Ensure that blank and NSB values have been substracted from the net ODs.
High Background		Ensure that the plate was washed correctly. Review washing techniques above. Adhere to all incubation times and temperatures. High backgrounds can result from extended incubation periods or incubation at higher than recommended temperatures.
Drift		Bring all reagents to room temperature prior to use.  Prepare reagents prior to running the assay or as directed in the kit insert. Avoid interuppted assay set-up.
Edge Effect		Be aware of temperature variations or drafty areas when incubating the plate in ambient conditions. Placing the plate under an air conditioning or heating vent may cause uneven color development.
Color Development		Ensure all reagents are added at the correct volumes and at the correct times in the assay. Adhere to correct incubation times. Read plate immediately after stop solution is added unless otherwise indicated in the kit insert. Was the substrate prepared correctly and added