ELISA assay spectrophotometry

The ELISA reader is used for measuring and quantification of the colour differences in the wells of the plate.

These readers do spectrophotometry; they emit a beam of light at one wavelength and measure the amount of light absorbed and reflected by the target analyte and are able to detect both ultraviolet and visible light.

Furthermore, ELISA plate readers can also be used for measuring fluorescence and luminescence.

Chemical dyes emit one colour or wavelength when excited by a specific wavelength. The amount of reflection, absorption and the colour detected by the ELISA reader are used for measuring the amount of a substance.

The other notable advantage of ELISA readers compared to conventional spectrophotometers is that the first require much less sample volume to get a reading and can measure up to 96 samples (wells) at the same time compared to around 6 that spectrophotomers achieve. 

The electromagnetic spectrum is a range of spectrum frequencies ranging from Gamma rays up to radio- and microwaves. 

In spectrometry, light from the visible spectre is commonly used and ELISA plate readers are usually set up to analyse TMB substrate at 450nm, which is blue in colour.

Using 450 nm for measuring TMB substrate after the reaction has been terminated by the addition of an acid is preferred because its peak absorbance is at that wavelength, giving us the best reading and increase the sensitivity of the assay. 

When the substrate reacts with the immobilised HRP-conjugated secondary antibodies, the solution in the wells turn blue in colour. Its intensity is directly proportional to the amount of target analyte in the well in most ELISA variations and it is inversely proportional in sandwich ELISA.

When the desired colour intensity is reached, the reaction is terminated by the addition of sulfuric acid. This leads to oxidation of the substrate and a colour change from blue to yellow. 

These readers are able to detect light signals that are produced by a sample, converted by it or transmitted through it. 

The signal is then measured by a photomultiplier tube (PMT), which convert photons into electrical signal that is quantified by the machine.