ELISA Plate Washing

ELISA assays are antibody based experiment that allows researchers to determine the amount of their analyte present in a sample. ELISA assays are commonly used in immunology, neuroscience and cancer research to analyze a range of targets focussed on inflammation, growth factors and signaling molecules. Key targets included IL6, IL8, BDNF & VEGF.

Common types of ELISA

The two common types of ELISA assays that researchers predominantly used to measure analytes are the Sandwich ELISA and competitive ELISA techniques. Sandwich (antibody is bound to the plate) and competitive (antigen is bound to the plate) ELISA assays involve two main antibody binding steps which are seperated with key wash steps that allow for the removal of contimating factors, unbound antibody & low-affinity bound complexes.

TMB reaction

In order to measure output of an ELISA experiment numerous substrates can be used to detect the final antibody-antigen bound complex. Colorimetric, chemiluminescent & fluorescent based assays are the three main technologies. Commonly ELISA refers to the colorimetric based measurement of samples, whereby TMB (3,3’,5,5’-Tetramethylbenzidine) acts as a hydrogen donor for the reduction hydrogen peroxide to water in the presence of horse radish peroxidase. This reaction results in the production of a diimine which changes the colour of the solution to blue. This colour change to blue can be read on a spectrophotometer at 370 & 650 nm. To stop this reaction sulphuric acids is added to the sample and results in a colour change to yellow. The ELISA plate can then be analyzed using a spectrophotometer and read at 450 nm.

Reasons for ELISA wash steps

A key step in the ELISA protocol is the wash steps. Washing steps are critical in order to reduce background signal, which can be due to unbound, conjugated antibody resulting in the increase in ratio of signal to noise. Therefore washing steps ensure that only high fidelity binding interactions occur between antigen and antibody. When inefficient washing occurs, this can result in high background levels, which can obstruct the acquisition of data, result in high variation between samples, ultimately resulting in poor results.

ELISA washing volumes & parameters

The volume of wash buffer added to the ELISA plate is key to ensure effective washing of your plate. Depending on whether you use an ELISA plate washer or multichannel pipette, pay attention to the volume required when washing your plate. Washing buffer volume should be higher than the coating buffer volume added to each well. For instance, if your plate was coated with 100µL of coating buffer, you will need to added a higher volume of wash buffer to ensure complete washing of the well such as 200 – 350 µL.

ELISA plate wash cycles

After wash volume, the number of wash cycles are important to increase the removal of background but also to prevent the unneccessary washing of bound antigen-analyte. If samples are over-washed, this may reduce the signal strength and make it difficult to measure and analyze your data. Depending on your postion in the protocol 1-3-5 wash steps maybe required. In general, pre-coated plates require fewer wash steps than DIY coated plates, however, this may depend on the manufacturer and the technology used to read the ELISA plate.

Aspiration of buffers during ELISA plate washing – manual

Aspiration of buffers during an ELISA protocol is key to remove residual buffer, unwanted complexes or antibody. When using a multichannel pipettte, place fresh pipettes tips on the multichannel between steps. When aspirating buffer, place the multichannel at a titled angle into the well, while being careful not to touch the side or the bottom of the wells. Following removal of buffer, you can turn the plate upside and tap the plate on paper towels to remove residual buffer. Do not let the plate dry between wash steps and buffer steps.