how do we tell if two individuals that look very healthy have been exposed to a particular pathogen
in the past we could try and use analyzer to detect antibodies 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 immunocompromising disease they will defend themselves and the output of that defense
are antibodies ultimately the technique is not just for detecting antibodies though it can be
used to detect any secondary metabolite that has an affinity with another molecule can be used
to detect proteins even peptides nucleic acids hormones herbicides a huge range of applications
the process for elisa's 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 in
incubate the blood with the spike protein that's been coated 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
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 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 in the scenario where you just wanted to know is the antibody interaction there
but you don't necessarily want to measure how much of it there is then you can use a simple
cut off analysis the simple cut of analysis you're going to have a positive control and a
negative control on the well and the positive control checks that the assay... the ELISA
worked as it should the negative control that's provided in the kit or if you prepared it yourself
is going to be used to determine the cutoff and the cutoff is the value that will determine
that an interaction has occurred so anything that is below the negative control value or at the same
as the negative control value will be considered that there is no interaction between your
what you know your your ligand and your antibody or epitope that you're interacting with and then
the anything above that a particular value will be calculated to show that anything
above it it could be like a standard deviation above the average of the negative control it
could be two standard deviations above or three standard deviations or it could be a customized
value that's based on the actual assay parameters or conditions that you're measuring
and so that value will be worked out and then you have to use that to in addition to the negative
value absorbance reading the average of it because usually you do this in replicates so
that you can determine the cut off value for those that are to be considered as a proper interaction
you will be giving a value that will represent a cut-off value and the cut-off value would usually
be your reading of the negatives whatever absorbance - your negative- where there's
no antibody antigen interaction whatever that absorbance region is plus a particular
amount so you're measuring... because with the spectrophotometer you're measuring optical
densities so you say plus 0.4 for instance and that's going to be your cutoff so it's
your negative read plus a value - a particular value - and anything above that is going to
be considered positive, anything below that is going to be considered negative. these scenarios
when you get your cutoff value you actually work out a ratio so whatever the reading was
you would divide your reading by the cut-off value and it is the ratio of that that will
tell you whether it's a 'yes' or 'no' for your antibody/antigen that you're trying to detect.
More commonly you may want to actually know how much of the antigen you have between samples or
between individuals where you're detecting a particular antigen or antibody and this is
because sometimes the amount of something you have actually determines whether the person is
sick or you know what the outcome is going to be for the person so typically people... researchers
or your experiments you want to know how much and compare how much you have between samples.
this scenario you would need a standard curve. you would take a known amount of the antibody or
antigen that you're trying to detect you do serial dilutions: i have a video up here on how to desire
dilution is a critical technique for bioscientists for anyone working in the laboratory in biological
sciences so take a look at that. so you take a known amount you do a serial dilution and
the serial dilution allows you to just to incrementally decide how much protein is
in each well that's going to act as something called a standard curve a standard curve in the
sense is going to be a reference point for all the samples which you are going to analyze from
sources that you don't know how much there is. so the standard curve will be used to reference that
and you read off the standard curve... compare the standard curve with your reading from the unknown
patients or samples or experimental groups to see how they compare from that... you know estimate
how much protein or how much antibody or antigen you have in the unknown samples or specimens
in a spectrophotometer the absorption of light through that solution and depending on how much
color there is there will be different absorption or optical densities as is reported and your
optical densities is then the y variable because of the response and then your independent variable
is the known concentration so you put on the x-axis and then from that you'll be able to infer
the unknown concentrations and so you draw a line and the equation of the line is just
turns out to be y equals mx plus b (y= mx+b) but you know you can't use a straight line for
biological responses it turns out it's actually a sigmoidal response in a case where there's
sort of the binding is different at different concentrations so it's not a linear scale so
that's why you can't use a straight line when you draw the graph you have the concentrations
and then you have their responses and so if the points are close to a straight line that's when
you can use a linear regression that's when you can use the equation y =mx+b and y equals mx plus
b (y=mx+b) means the response equals or depends on m times x plus b (m x X + b) and m it turns
out is the slope so the angle of the line... it's the angle of the line times your concentration
which is x plus your y-intercept and your y-intercept is given the symbol b and it just
means the intercept of the line on y which is your response and so it turns out that unless you're
using very limited range of concentrations you're not really going to have your responses being
fitting around a straight line they're more likely to fit on in a shape that's called the sigmoidal
shape the that s shape for like a dose response and so that s shape is analyzed in a different way
because you don't just have these two constants they're m and b the slope and the y-intercept
being the critical factor there are other factors and it turns out there are four so
in the straight line you just have two constants or two parameters that describe that slope or that
y is exactly y equals so the y is the response but when is the sigmoidal shape you actually have four
constants that describe it and so that's the four p (4P) or the four parameter logistics.
It turns out that the four parameters (4P) you need are really just to fit the shape so when it
was linear you only needed two you just needed the slope and you needed the intercept of the
of b which is the y-intercept so you just need a slope and the y-intercept when it's a straight
line but when it's sigmoidal you need to know the minimum signal they have maximum signal and
then you just have this inflection point where it sort of turns and starts to go up to plateau
and then you also need to need a maximum and so those are the four parameters and that's
why you assess these sigmoidal shaped fits with a four parameter fit rather than a straight line
which is just two parameters it just captures more accurately so that you can read the unknown values
the minimum signal is the point where there isn't much of a signal it's very close to your negative
control the maximum signal is the point where even though you have a lot of interactions between your
ligand and your antibody or your antigen and your antibody you just won't be able to detect any more
signal it's saturated it's the maximum output you're going to get from the system from the
assay that you set up then there's your inflection points your inflection point is given the alphabet
b or the symbol b it's halfway off your slope halfway up the slope and it's called another name
is called the heel coefficient and finally there's the fourth parameter which is the concentration
where you get half of your maximum signal so there's the maximum signal way saturated and so
halfway of that is your l50 so that's your ligand concentration when your ligand concentration
is at half of its maximal saturation and if you are interested in joining the study group go ahead
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