Washing of ELISA Plates

ELISA wash steps are used for a variety of reasons.
The washing steps are an important tool to minimize background signal by removing the loose, attached antibody. This helps in improving the signal-to-noise ratio of the analysis. Washing in between steps ensures that only specific (high-affinity) binding events are preserved; result in a signal in the final step. WASHING IS THE MOST IMPORTANT STEP FOR YOU. DON’T UNDERSTIMATE IT. IT HELPS IN IMPORVING THE SENSITIVITY AND ALSO MINIMZE THE VARIABILITY BETWEEN YOUR RESULTS.

Volumes and parameters for ELISA washing
The number of wash depends on the manufacturer’s instructions for the ELISA reagent. After an incubation step, the higher the wash volume, the less excess antibody or antigen is left over. The number of wash cycles is the second key factor that influences wash efficacy.

Cycles of ELISA plate wash
Filling the wells entirely with buffer, usually PBS with some concentration of a non-ionic detergent such as Tween-20 is an essential procedure. To effectively remove unbound material, washing is normally performed 3-5 times between each step in the ELISA.

ELISA plate washing – manual aspiration of buffers
During an ELISA process, aspiration of buffers is essential to extract all excess buffer, inappropriate complexes, or antibody. Establish new pipettes tips on the multichannel pipette between steps when using a multichannel pipette. When aspirating buffer, place the multichannel into the well at a titled angle, being careful not to touch the wells’ sides or bottoms. After removing the buffer, flip the plate upside down and tap it on paper towels to remove any remaining buffer. Between the wash and buffer steps, do not allow the plate to dry.

ELISA PLATE WASHING – TIPS & TRICK.

TIP#1: Whether you are doing an automated wash or a manual wash, the flipping of the strips/plate onto an absorbent paper towel is the most important part. Hit hard on to the towel to remove any residual wash buffer or liquid reagent left in the wells. This helps to improve the accuracy of your results !!!

TIP#2: When manually washing plates with removable strips, number the strips with a permanent marker. If strips fall-out from the holder when decanting the reagents, they are easily replaced in the proper sequence, ensuring proper data reporting.