Enzyme wells are coated in an antigen-specific antibody first,

Enzyme linked
immunosorbent assay (ELISA) is a well-established solid-phase, biochemical test
that uses antibody-antigen interactions together with a conjugated enzyme,
which reacts with a complimentary substrate to produce a product that is
detectable (usually in the form of colour change in the solution) and
measurable by a specialized spectrophotometer that can measure ultra-violet
light, fluorescence and luminescence .There are different ways in which to
perform ELISA, such as the direct method, indirect method, competitive method
and the sandwich method. The direct method involves coating the microtitre
plate in the antigen of interest and blocking of the remaining binding sites.
The analyte is detected by using a primary-labelled antibody which binds to the
target antigen and can be visualized after the addition of the correct
substrate causes a colour change in the solution. This makes it the simplest
and quickest ELISA technique to perform. The indirect method detects the
analyte similarly to the direct method, but includes an extra step that
involves the addition of a secondary labelled antihuman antibody (cultured in a
different species) bound to the primary antibody. The secondary antibody is
usually polyclonal allowing it to bind to multiple epitopes on the antigen,
increasing the signal and giving the assay a high sensitivity. It produces a
colour change that can be analyzed in the spectrophotometer. The sandwich ELISA
differs from the direct and indirect methods in the first step. The microplate
wells are coated in an antigen-specific antibody first, and then after the
blocking step, the antigen is added. After this it follows the same step as the
direct method and requires a second, region-specific antibody to bind to the
antigen. Competitive ELISA uses a 96 well microplate with bound antibodies.
After blocking any remaining binding sites it involves the use of two ligands;
one being the antigen of interest and one being a similar molecule or the same
molecule with an enzyme conjugate that is capable of binding to the antibodies
attached to the microplate well. The ligands will compete with each for the limited
number of antibody binding sites. The signal or colour change produced is
inversely proportional to the amount of analyte in the sample. In other words,
high colour intensity means the level of analyte is low, whereas low colour
intensity means the level of analyte is high.

The procedure in
this practical involved the use of microtitre plates with 96 wells. This is so
that a dilution series can be prepared easier. These wells are coated in the
antigen of interest, which is typically done on the manufacturer of the plate’s
part. In the case of this practical, the plates were coated in Myelin Basic
Protein. The use of the indirect ELISA technique, it was determined whether or
not the given patient serums are positive for an autoimmune disease relating to
demyelination (e.g. multiple sclerosis).

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Multiple Sclerosis (MS) is a complex, inflammatory, demyelinating,
autoimmune disorder that affects the central nervous system (CNS). The disease
affects 0.24% of young adults in the United States and Canada, and around 0.19%
in some European countries (Miljkovi?,
2013). The mechanism or pathophysiology of MS is not precisely understood; that
is to say, whether the disease is caused by the immune system or whether it’s
caused by failure of the cells that produce the myelin (Farez, 2014). Clinical
expressions of MS include sensory loss, limb weakness or numbness, issues with
the bladder and bowel, sexual dysfunction, vision problems and fatigue (Miljkovi?,


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