Diagnostic and detection method
There are many types  of application
used in diagnostic and detection method used in immunology
such as antibody generation, immunoprecipitation,
agglutination reactions,
antibody assays based on molecules binding to
solid phase support, affinity of antigen-antibody reactions
microscopic visualization of cell and subcellular structure
immunofluorescence-based imaging technique
and lastly, flow cytometry
In this video, we will be focusing on antibody assays
based on molecule binding to solid phase support
The most commonly used method is ELISA
ELISA is used to measure antibodies, antigens, proteins and glycoproteins
Generally carried out in 96 well microtiter plates
and these plates are special absorbent plates
(for example, NUNC Immuno plates)
to ensure the antibody or antigen sticks to the surface
Each ELISA measures a specific antigen hence
there are kits available for a variety of antigens
How ELISA works?
Firstly, ELISA plate is coated with capture antibody.
which in the excess, unbound antibody will be washed from the plate
Next, the sample (e.g. urine, serum, or cell supernatant) will be added.
Any antigen found in the sample will bind
to the capture antibody already coating the plate
Again, any excess sample is washed from the plate
Next, detection antibody will be added.
This antibody is labelled with an enzyme, usually horse radish peroxidase or
alkaline phosphatase. Detection antibody binds to
any target antigen already bound to the plate
Finally, a substrate is added to the plate. ELISA assays are usually chromogenic,
using a reaction that converts the substrate (e.g. TMB or ABTS)
into a coloured product which
can be measured using a plate reader.
There are three types of ELISA formats
which are direct assay, indirect assay and capture assay
There are three advantages which are quick
because only one antibody and fewer steps are required
Secondly, they are versatile because
many primary antibodies can be made in one species and the same labelled
secondary antibody can be used for detection
Thirdly, the sensitivity is increased because each primary antibody contains
several epitopes that can be bound by the
labelled secondary antibody, allowing for signal amplification
Disadvantages of ELISA are
time-consuming and expensive. Secondly,
there are minimal signal amplification and no flexibility
in choice of primary antibody label from one experiment to another
Second, immunoblot (Western Blot)
The HIV immunoblot is used for confirmation of HIV infection
The steps involved in HIV immunoblot are
the blood sample taken from patient is loaded
and they are separated on SDS-PAGE
 
The viral proteins will then be
transferred into
a nitrocellulose membrane and
the membrane
will be blocked with neutral protein
such as BSA or casein
The next step will be incubating the membrane with primary antibody
specified to the target protein. Next, it will be incubated
with HRP-labelled
secondary antibody which is specified to
the primary antibody
and finally, it will be incubated
with chemiluminescent HRP substrate and exposed to the film
Finally, it is the rapid test
Rapid test is used to detect HIV antibodies in the blood
mostly HIV-1 and HIV-2 Abs in the blood
They work by capturing antibodies (or antigen) on a solid surface
and then attaching molecules to them that allow detection
by the naked eye
There are two types of HIV rapid test present, they are immunoconcentration and immunochromatography
Immunoconcentration involved dot test
whereas immunochromatography involved line test
In immunoconcentration, flow-through devices are used
which are usually cartridges, with HIV antigen attached to a membrane.
HIV antibody links to bound HIV peptide
antigens forming the colour spot
For interpretting the result
HIV negative produces all colourless dots except for the control
Next, for HIV positive
which involves dual reaction will
show red in all the three dots HIV 1 positive
red will be present at the control dot and
HIV 1 dot and finally if the result shows
HIV 2 positive, red will be present
at the control and the HIV 2 dot
Immunochromatography involved line test which
uses lateral flow devices
HIV negative shows non-reactive result where
only control area shows a line
Next HIV positive will show reactive result in both lines
of the control and test area
If there is no any line showing, it shows that the result is invalid
and required test repetation
The other implications of immunochromatography are
HIV ½ triline rapid test
which the test is based on immunochromatography
Secondly is the INSTI™ HIV-1/HIV-2
Antibody Test It involves
the single-use, rapid, in vitro qualitative
immunoassay for the detection of antibodies to HIV-1 and HIV-2
in human
Thank you for watching and hope you enjoy the video.
