🧪🧪 ELISA | ENZYME LINKED IMMUNOSORBENT ASSAY | Enzyme ImmunoAssay

If we wanted to see if an individual has been infected with COVID-19 for example in the past ( they are well, so not acute infection where we could detect with RT-PCR, what technique could we use?

We could try and detect whether the individual has produced antibodies to covid19 virus or components of the virus such as the spike protein. The antibody would be in  the blood of the individual. The blood is collected and presented as serum (blood with coaggulation factors removed) or plasma (blood with anti-coaggulant such as heparin or acid-citrate-dextrose plasma).

This works because individuals have an immune system and when they are in touch with a virus or bacteria, they respond. In the case of infection, evidence of past infection can be found in the blood using a technique called Enzyme Linked ImmunoSorbent Assay (ELISA). ELISA is an Enzyme ImmunoAssay (EIA).
The history of the development of this technique begins in 1960 when Rosalyn S YALOW  and Solomon A Berson develop RadioImmuno Assay where antibodies and radioisotopes are used to detect insulin in plasma.
1969 Stratis Avrameas couples enzymes to antibodies or antigens for use as probes in  detecting antigen-antibody interactions. Researchers prefer this approach and adopt it  due to concerns with safety of radioisotopes.
By 1971 Engvall and Perlman introduces us to the form of ELISA's as we know it with the antibody or antigen immobilised on a microtitre plate.
How does coupling enzymes to antibodies help us in ELISA? Enzymes such as Horse Radish Peroxidase, Alkaline Phosphatase and B-galactosidase can react with specific chromogenic substrates to produce colour when they are part of an antibody-antigen complex. The enzymes coupled to antibodies could also react with chemiluminescent substrates or fluorogenic substrates to produce luminescence or fluorescenct signals. Fluorescence and luminescent signals are much more sensitive than chromogen-based detection. 
To increase the sensitivity of the detection , indirect methods for detection are often used to detect the antibody-antigen complex. In such a setup, rather than coupling the antibody used for primary detection to the enzyme, a secondary antibody is introduced to detect the initial antibody-antigen complex. It is the secondary antibody that has the enzyme

The appropriate substrate - either chromogenic, fluorescent or chemiluminescent is then introduced to visualise the interaction.

For chromogenic detection, light absorbance of the visibly coloured substrate is detected using a spectrophotometer. The principle here is that every compound – coloured or not – absorb light characteristically. Hence by measuring how much light passes through the coloured product produced by the enzyme in response to the underlying antigen-antibody interaction, you can analyse the sample/specimen.
The ELISA (aka Enzyme Immuno Assay) technique involves first coating component of the pathogen we wish to detect on a micro titre plate:
for example for covid19 we can coat the surface with the spike protein from the virus

Then we can take blood from the individual, incubate the blood with the spike protein on the surface and see if there are antibodies in the individuals blood that recognise the spike protein we have coated on the surface.

We can use ELISA/EIA as a simple – yes/no for the presence of an antibody. for example, it may be any biomolecule that has a specific affinity for another biomolecule such as hormones or cytokines.

In the yes/no scenario, we have a positive control and a negative control. We then specify what is positive or negative based on absorbance values when the colour produced by the action of the antibody-coupled enzyme reacting with its substrate.
For example, we my say that any well (representing an individual) with a signal that is 0.2 optical density above the negative control well is a positive.

We can also set up the ELISA/EIA technique to tell us relative amounts of the pathogen that we want to assess.
If this is the scenario we want, we have to first place different known amounts of the pathogen/antigen/antibody on the plate in decreasing amounts, to act as what we call a standard curve.
Based on the reading of the known amounts, we can infer the amount of pathogen in the individuals we are measuring.
For some pathogens, amounts are important so we may want to know not just yes/no, but how much is there.
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//MUSIC
Music: Shine by Joakim Karud
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// ADWOA
I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. I’ve worked in medical research for years and want to be useful to people new to the lab life. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. Hopefully it helps if you're new to the topic/technique.

 This video is about Enzyme linked immunosorbernt assay, enzyme immunoassay, ELISA, immunoassay, enzyme-linked immunosorbent assay