how do we tell if two individuals that look very healthy have been exposed to a particular pathogen
in the past? given the context that this recording is being made let's say we have two individuals
that look very healthy and they are very healthy nothing's wrong they're not sick but we want to
know if they've been exposed to the virus that causes covid19 in the past how would we do it
you may say we could do a reverse transcription realtime PCR to see if they've had the virus but
don't forget they look healthy and so this is the case where they're not likely to feel sick and
go and get tested and the reverse transcription, real time PCR may also not detect the virus if it's a long
time since they were in touch with the virus so if it's been several weeks and in this case these
individuals look very healthy there's they don't feel any symptoms it may have been a
very long time that they got exposed so reverse transcription PCR would not pick up the virus
what's another technique that we could use we could use if you said enzyme-linked immunosorbent
assay or ELISA then you are correct. so ELISAs can be used to detect antibodies we could try and use
analyzer to detect antibodies to the component of the virus such as the spike protein in the
blood of the individual. the technique works or this idea works because individuals - living
systems - have an immune system that helps them protect themselves against invading other living
organisms so in the case of virus or bacteria that enters any human body unless the individual has
an immuno-compromising disease they will defend themselves and the output of that defense
are antibodies ultimately so antibodies are a marker that an individual has been
in contact or has been exposed to a particular pathogen in the past because it does take time
to produce these antibodies if the individual is exposed immediately acutely there will there isn't
going to be any antibodies in the individual's blood but if it's been they've been exposed in
the past there's going to be antibodies so we can use that concept to see if the individual
has been exposed to any pathogen in this case let's say the spike protein of covid19 virus.
so how did we come up with this technique how did we get this to this point with the technique? so
back in the 1960s Rosalyn S Yalow and Solomon A Bersen developed an assay that's very similar
to the ELISA and it was a radio immunoassay and this was a scenario where antibodies and radio
isotopes were used to detect insulin in plasma plasma being a component of blood in 1969 Stratis
Avraemeas coupled enzymes to antibodies rather than using radioisotopes and this turned out to
be a modification that researchers preferred because it's just there's some safety there
are some safety concerns with radioisotopes the format of ELISA that you know now where
an antigen or an antibody is coated on a plastic surface a micro teeter plate 96 well plate is the
it's a format that was introduced by Engvall and Pelman in 1971. how does this coupling of enzymes
to antibodies or antigens work it turns out that enzymes such as horseradish peroxidase alkaline
phosphatase and beta galactosidase can react with specific colored compounds they're called
chromogenic substrates they can react with colored or chromogenic substrates to produce color when
they are part of an antibody antigen complex now the enzymes could also react with other substrates
that give us color or give us give off light such as chemiluminescent or fluorogenic substrate
chemiluminescent and fluorogenic substrate tend to give off tend to be more sensitive readouts for us
so it would be preferred if you know you don't have a lot of the antibody that you're trying to
detect or the antigen that you're trying to detect and chemiluminescence is where you use a chemical
reaction to result in a luminescent signal so you could also use these enzymes and then introduce
substrates that are chemiluminescent or fluorescent in order to detect your
antigen-antibody interactions. the process for ELISAs involves you first coating the surface
with a viral or bacterial component that you want to detect generally and then you go ahead
and take blood from the individual who that you want to determine if they've been exposed to that
particular pathogen and then you would incubate it in this case if you're looking at SARS-CoV-2 you
incubate the blood with the spike protein that's been created to the surface of the 96 well plate
see if there are antibodies in the blood that recognizes the spike protein
the initial antibody antigen detection can be the sensitivity of it as well as the specificity of
it can be increased by then introducing an additional detector so a secondary antibody
which allows you to detect the initial interaction indirectly so it's called that indirect ELISA and
this tends to be much more sensitive and less likely to give you false positives
secondary antibody that that you introduce the fluorescent can be luminescent substrate later
on so the enzyme will just be coupled where you introduce a chromogenic detection system
light absorbance of the visibly colored substrate is used to detect the interaction and you use
something called a spectrophotometer the principle here is that every compound colored or not
absorbs light characteristically so they have a specific way that they absorb light and so
by measuring how much light passes through the colored product produced by the enzyme
in response to the underlying antigen antibody interaction you can analyze the sample or specimen
at this point i've introduced the idea of using light to detect
the interaction ultimately as a readout so light visible light is actually part of a
wider spectrum of electromagnetic radiation big word visible light is usually defined as having
wavelengths in the range of 400 to 700 nanometers and so you're used to the scale of meters,
millimeters even, we don't tend to use nanometers that's the wavelengths of light they're very small
nanometers so the visible light that we see is in the wavelength of 400 to 700 nanometers so
other non-visible light sources are referred to as infrared and these are longer wavelengths they're
around 700 to 1000 to fifteen thousand nanometers much longer so fifteen thousand nanometers is
fifteen micrometers human eye cannot detect this wavelength of light there are also smaller
wavelengths of light which are around 185 to 400 nanometers and they are ultraviolet light so
these spectrum is going to be used to detect the antibody interaction antibody antigen interaction
thanks to the color that the enzyme has produced happens is when light is shown
through the compound dissolved in a solution so the substrate that introduced the enzyme catalyzed
a reaction and it gave of light well when light is shown through it a section of it is absorbed
as the concentration of the dissolved compound increases which correlates with more antibodies
and therefore more antibody antigen interactions so as that concentration increases so will the
absorbance as that concentration decreases so too the absorbance will decrease so you can use this
principle to decide if there has been an antibody produced against an antigen just as a yes or no or
you can even if you fine tune it and put standard so a known amount of the antigen in there against
which you can read your unknown concentrations then you can even decide how much of the antibody
antigen interactions are there in a sample of amongst individuals okay in the next video we're
going to look at the data analysis there are two main ways that you can analyze data from
ELISA so we'll look at it in the next video and if you are interested in joining the study group
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