• If we consider how you'd want to best approach a potential exam answer these are the kind of things you want to keep in mind when you are structuring your essay.
• Why do we use biomarkers, what are they used for? They are used for diagnosis specifically they are used to manage a particular...
Today we today we're going to look at conditions of the heart, specifically muscle damage.
If we consider how you'd want to best approach a potential exam answer these are the kind
of things you want to keep in mind when you are structuring your essay.
Why do we use biomarkers, what are they used for? They are used for diagnosis specifically
they are used to manage a particular condition so you can use biomarkers to monitor the
progression of the disease if a particular protein increases as a disease gets more aggressive,
then you can measure the severity of the disease. Equally once you start administering a
treatment you can measure to see if the levels of that biomarker decrease that would be
indicative of how well this patient is responding to treatment and in the worst-case scenario
once you have administered a therapeutic intervention, if the biomarker reappears in the
blood, then you can have an idea whether the disease is going into remission.
On a broader scale of things biomarkers are used for screening and we looked at new-born
screening as a particular example, when they found the FTO gene which identified in obese
mice they look for that particular gene in the population as well to look for patients who
might have a genetic predisposition for developing obesity as well.
If these criteria are difficult to achieve always use a battery of tests, never just go off one
test back it up with two or three others and they could be biochemical so you may look at
three or four different proteins in the blood or it could even be non-biochemical tests as
well.
The heart is a muscle therefore the best type of biomarker you could use to analyse the
condition of the muscle tissue is to run an assay for a serum enzyme or protein or even an
enzyme activity.
Serum is just a fancy word for component in the blood, if you centrifuge a blood sample it
separates into whole blood or red blood cells and serum and finding a protein in the serum
is a useful way to diagnose a particular condition. The easiest way to do this of course is
using a colorimetric assay in which you are looking for a particular protein and as you release
an antibody for that protein it binds that particular protein any causes a colour change, and
the intensity of that colour is in direct proportion to the concentration of that protein.
It's not always possible to have a reaction which produces a distinct colour change so as you
can see on the slide here there was an example of how you would measure aspartate, a
reaction takes place and at the end of the reaction it produces oxaloacetate which is
colourless as is malate, so you have a problem as there is not going to be a colour change.
This is an example of quantifying something based on structures.
When you do an assay which looks at aspartate and you want to quantify how much
oxaloacetate or malate you can look at the concentration of NADH to NAD+. NADH and
NAD+ absorb light at different wavelengths so you get different readings based on whether
you have more NADH or NAD+.
Of course, we'd opt for colorimetric assay as they're very easy to read and to a certain
extent you could eyeball and tell if there's a difference in concentration but that's not always
the case and sometimes reactions are colourless so in these instances, we look for changes
in structure.
, 2. Cardiac Function Tests
How do we go about quantifying protein or an enzyme in the serum? One way is to run an
ELISA. There are four different types.
Direct ELISA – green little circle is the antigen, so this is a protein that you're after or an
enzyme, you do is you generate an antibody which binds to that antigen, you attach a
particular protein to the antibody that when it meets its chromogenic substrate it causes a
colour change and then the concentration of the colour is in direct proportion to the
concentration of that antigen.
In direct ELISA – this is a bit more specific; we are releasing an antibody for an antigen and
then we're using a secondary antibody to bind that primary antibody so it's just an additional
level of security really, we are making sure that the antibodies are definitely binding the
antigen, the end stage is the same you have a chromogenic substrate which changes colour.
Sandwich ELISA – is considerably more accurate because you first coat the plate with the
capture antibody and this this kind of coating is commonly done overnight so you'd coat
your plate with capture antibody and you leave it for more than 12 hours, then when you
introduce your sample to the ELISA plate the antigen is already being bound by specific
capture antibodies which are only binding that antigen, you would then wash the plate
numerous times to make sure that any antigen that hasn't bound an antibody gets washed
off. This is very similar to an indirect ELISA where you use a secondary antibody and a
detection antibody. It's a lot more specific, it's a lot more sensitive and requires more
antibodies and therefore you can imagine from a technical standpoint that these kind of
ELISAs are a lot more expensive to run.
Competitive ELISA – these aren't used that often, there are specific tests which may use
them but generally speaking we would be using sandwich ELISAs, in a competitive ELISA you
release two antibodies which compete for the same antigen and then you use a substrate
which will cause a colour change, the colour change might be different in antibody one
compared to antibody two and you would quantify which colour is more prevalent.
When we do get a colorimetric assay the higher the concentration the higher the
absorbance. So, if your protein is in the serum in a very high concentration let's say it's there
in the milligrams per mil, you're going to get a big colour change, a lot more absorbance and
you can use that to quantify what the actual concentration is.
This line which is intercepting through zero and it's got these dots on them is normally used
to determine the concentration of the unknown, you will be given a set of standards so let's
say if we're looking for leptin which is a hormone produced by the adipocytes in a blood
sample when you order an ELISA kit for leptin it comes with standards or premade solutions
of a known concentration, hypothetically if we look at these particular points you can
imagine the first point is maybe 10 milligrams per mil then you have 5 milligrams per mil, 2.5
milligrams per mil, 1.25 milligrams per mil then you have 0 which is no absorbance (going
from right to left). You will put these samples onto your plate, you will run them and get a
straight line like this because as the concentration is increasing so is that absorbance. Once
you've established this standard curve when you measure your unknown samples they will
dot around somewhere in and around these lines and you will simply draw a line from your
unknown straight down and you can figure out your concentration from that.
Now we will look at the conditions which can affect cardiovascular function.
The heart consists of four chambers: atria at the top and your ventricles at the bottom.
The blood supply from the heart consists of two circulatory loops; one will be the systemic
circulation which will provide blood to the entire body and then you have the pulmonary
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