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ELISA (Enzyme­Linked ImmunoSorbant

Assay)
The purpose of an ELISA is to determine if a particular protein is present in a 
sample and if so, how much. There are two main variations on this method: you
can determine how much antibody is in a sample, or you can determine how 
much protein is bound by an antibody. The distinction is whether you are trying
to quantify an antibody or some other protein. In this example, we will use an 
ELISA to determine how much of a particular antibody is present in an 
individuals blood.
ELISAs are performed in 96­well plates which permits high throughput results. 
The bottom of each well is coated with a protein to which will bind the 
antibody you want to measure. Whole blood is allowed to clot and the cells are 
centrifuged out to obtain the clear serum with antibodies (called primary 
antibodies). The serum is incubated in a well, and each well contains a different
serum (see figure below). A positive control serum and a negative control 
serum would be included among the 96 samples being tested.

After some time, the serum is removed and weakly adherent antibodies are 
washed off with a series of buffer rinses. To detect the bound antibodies, a 
secondary antibody is added to each well. The secondary antibody would bind 
to all human antibodies and is typically produced in a rodent. Attached to the 
secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. 
These enzymes can metabolize colorless substrates (sometimes called 

chromagens) into colored products. After an incubation period, the secondary 
antibody solution is removed and loosely adherent ones are washed off as 
before. The final step is the addition the enzyme substrate and the production of
colored product in wells with secondary antibodies bound.
When the enzyme reaction is complete, the entire plate is placed into a plate 
reader and the optical density (i.e. the amount of colored product) is determined
for each well. The amount of color produced is proportional to the amount of 
primary antibody bound to the proteins on the bottom of the wells.

Enzyme-Linked Immunosorbent Assay
(ELISA)
There are a number of ways to determine whether an antibody has bound to its
target antigen.
One simple method is an ELISA.
Generally, an ELISA has the following steps (each seperated by extensive
washes):

Antigen Binding:A solid support surface is initially coated with an
appropriate amountof the antigen of interest. The whole surface must be
blocked to ensurebiologically specific binding of the subsequent
components. Having theantigen bound to the solid surfaced makes it

simple to seperate boundfrom unbound molecules. Nonspecifically bound
materials are washed awayduring the process of the assay.
Blocking: All unbound sites on the solid support are blocked to prevent

nonspecific binding of the antibodies.
Primary Antibody: The primary antibody ia added and will be bound if

there is a recognized epitope within sample antigen.
Secondary Antibody:An enzyme-linked secondary antibody is added which
will bind to anyavailable primary antibody. Primary antibody will only be

available onthe surface if it has bound to the antigen.
Detection:After adding an enzyme substrate, a colored product will
develop ifthis binding has occurred. The color intensity reflects the
amount ofprimary antibody bound to the target antigen and can be
measured.

There are many different applications of ELISA and the assay may be modified in
a number of ways.
Direct versus Indirect ELISA
 Indirect ELISA: Essentially the method described above.

Direct ELISA:This is similar to the process described above, except that the
primaryantibody is linked to the detection enzyme. Therefore, no
secondaryantibody is required. This method is very quick, but is typically
notas sensitive or flexible as the indirect method.

Sandwich ELISA
 A less-common variant of this technique, called "sandwich" ELISA, is used

to detect sample antigen.
Asoild support is coated with a capture antibody. A solution containingthe
antigen is added followed by washing. Detection and quantitation ofbound
antigen is then accomplished by the direct or indirectmethodology as
above.

Competitive ELISA

The steps for this ELISA are somewhat different.
Theantigen is bound to the solid support. Unlabeled antibody is
incubatedin a solution containing the antigen. These bound
antibody/antigencomplexes are then added to an antigen coated well
followed byextensive washes to remove unbound complexes. (The
moreantigen in thesolution, the less antibody will be available to bind to

theantigen inthe well, hence "competition."). This is then processed
similar to theabove methods.
For competitive ELISA, the higher the original antigen concentration, the
weaker the eventual signal.

Microtiter plates were coated with the ANTIGEN of interest. Then filled
with the patient’s dilutions of serum. If antibodies against the antigen
are present, they will bind to the antigen fixed at the bottom of the
well. But only antigen-specific antibodies will bind to the well. The wells
are then washed out to remove the unbound antibodies. Next, a
solution of animal antibody against human antibodies is added. This
second antibody is covalently conjugated to an enzyme. The wells are
washed again this time to remove unbound enzyme conjugated
antibody. Finally, a solution of colorigenic enzyme substrate is added.
The interaction of the substrate with the enzyme on the second
antibody generate visible color. The devt of color in the wells, with the

specific antibody can be seen with the naked eye or quantified with the
reader.
Antigen – antibody – enzyme – substrate – color reaction