hi my name is Adwoa and in this video we're going to cover the kinds of controls you need to
consider when doing your assay these controls are in relation to multi-well assays such as elisas
and other plate plate based assays but the principles may apply in other instances. now
when you're doing an assay you want to have a sense of the data that you collect is related to
the biology that you're studying and not related to the instrument or the equipment or the assay
that you're doing - the technique itself and so there's this idea of normalization and in
the elisa setting you need a blank well. these blank wells allow you to subtract information
that is irrelevant to the actual assay that you're studying so information that is due to the buffer
that you're using to do the enzyme reactions in. in other words your blank measures the background
values and the blanks are... how they handled is that they are averaged and then subtracted
from each value, each well on the microplate so that you can subtract out information that is
irrelevant to the study, the target, the biology. a necessary control is a negative control.
negative controls allow you to confirm scenarios where you do not expect a result and in that
case it confirms that your setup is working. so it involves you either omitting... omitting the
antibody that you're using to capture the particular target or analyte or you may omit
the actual sample so the sample is not there it doesn't contain the analyte so there's nothing to
give a result or you may just omit the standard now this negative control when you are doing a
sandwich elisa, yes it's a negative control but when you're doing a competitive elisa keep in mind
that it's going to be called a non-specific binding well so they may designate it as an
nsb well nsa: non-specific activity well. in this well you're omitting the antibody and so
you don't really expect a result and if you do get a result then you know something went wrong
in your assay. in the vein of negative controls there is a B nought (B0) designation that you may
encounter and this is specifically in competitive ELISAs the B nought (B0) is specific... is a
specific denomination of the zero standard. so you would have standards on your plate
ranging from high to low or low to high that you serially diluted and the B nought (B0) will
refer to that well where you did not actually include any of the standard its equivalent in a
regular in a sandwich elisa is the s nought (S0). They're given different designations on a sandwich
elisa versus a competitive elisa because on in a competitive elisa, because there is no
standard or sample analyte, it will be the well that leads to maximum color development:
on a sandwich elisa when you don't have a standard or sample analyte in the s nought (S0) you will
actually have the least color development. so that's why it really has the different
designations so in a competitive elisa it's b nought (B0): you don't have a standard in that
well and so you get the maximum color development owing to the fact that the conjugated analyte
does not face binding competition and so maximal conjugate binding is achieved. B
nought (B0) in this way communicates the maximum color development in the competitive elisa and
is used to control for full functionality of all assay components. it is expected that it
could generate results that are above the maximum detection range of the assay. how you handle this
particular value is that the B0 value is used to reference the relative binding in a sample
or standard to the maximum possible binding and it's used to analyze the assay readout.
The ratio that result is commonly referred to as percentage of binding percent binding or b/B0 and
is therefore recommended to include B0 controls in every assay... in every competitive elisa.
Again in competitive elisa this is your hundred percent (100%) bound analyte as opposed to
competitor because there's no competitor in the system. In contrast the nsb which is the S0
in a competitive elisa is your zero percent (0%) binding.
so that's it for negative control. You also want to include a positive control. now positive
controls confirm that the assay is working as it should or as expected and so when you're trying
to measure a particular analyte you will find a situation where you know the analyte is present
and use that in a couple of the wells so that you can track that the response is as expected.
Now standards are not typically thought of as a positive control but you have known quantities
of the analyte that you add to the buffer in a standard and so it's probably the purest form
of a positive control. so standards while they are their own particular category in your assay could
be looked at like a positive control where you've got known quantities of the analyte that you've
added to the assay and if the response is as it should be in terms of the dilutions that you've
performed, that gives you an indication that the setup is working also, but it is common place to
also include a positive control well. I'm going to mention that in a competitive elisa you would also
have a total activity (TA) well and so this sort of activity was actually controlling and
allowing to ensure that you have enzyme activity throughout the length of the incubation. so the
total activity (TA) serves to ensure enzymatic activity of the enzyme conjugated antibody,
biotin or analyte. In this well only the substrate and enzyme conjugate are incubated together during
the assays development. they are used to monitor the enzyme activity over a long assay incubation
procedure for instance. so if you know that in that well the enzyme and its substrate
are reacting and doing as they should over a long period of time, then your responses can be
validated in that... in that way. so you know that it's a genuine response and not maybe due to the
substrate and its enzyme conjugate decaying over time. You're probably familiar with the format
of these sorts of micro well assays, if not i do have a previous video here that goes through
a regular sandwich elisa and so in here i'll talk more about like a competitive elisa where
the analyte is in your sample or in your standard and then the analyte is incubated
with the analyte specific antibody; it's allowed to react for a given amount of time - an
hour or so - or even overnight at four degrees celsius. Then the analyte that is coupled to
an enzyme being a competitive elisa is then introduced to the sample so that it competes for
antibody where antibody has not bound. well we say compete but it just essentially also goes to bind
to the antibody and where there was an analyte in the sample it's already bound to the antibodies
so it has no opportunity to bind and the analyte allows you... analyte coupled to the enzyme allows
you to visualize the assay and so your plate layout would look something like this where you
have your standards ranging from high to low, and then you have control wells and the control wells
will get... give you a sense of what's happening that things are progressing as it should.
Thanks for watching :)