m Enzyme Linked Immunosorbent Assay (ELISA)

m Term Was Coined By Engvall and Pearlmann in

1971
m Different Types
î Sandwich
î Indirect
î Competitive
m Similar To RIA
m Can Be Used To Detect Both Antibody and
Antigen
m Very Sensitive
m ˜sandwich˜ ELISA, is used to
detect sample antigen.
m The steps are followed
m The wells of microtitre plate are coated
with specific antibody against the
antigen to be detected
m Specimens to be tested are added in
coated wells, If antigen present are bind
with coated antibody
m Wash the plate, so that unbound antigen is
removed
m Apply enzyme linked primary antibodies as
detection antibodies which also bind specifically
to the antigen.
m Wash the plate, so that the unbound antibody-
enzyme conjugates are removed
m A substrate is added to know the binding of
conjugated antiserum to antigen-antibody complex
m In case of binding (positive result), an enzyme acts
on substrate to produce colour, intensity of which
can be read by Elisa reader
m Colour detection can also be seen by naked eye.
This type of Elisa test is also known as sandwich
ELISA
m Positive and negative controls should always be
inclued in the test
m At every steps of ELISA test, incubation and
washing is done to wash off unbound reagents.
m ëor antibody detection, the wells of microtitre plate are coated with
antigen
m Sera to be tested are added in these coated wells. If antibody is
present in specimen, it bind to coated antigen.
m To detect this antigen-antibody reaction, a goat antihuman
immunoglobulin antibody conjugated with an enzyme is added.
m Enzyme conjugated antihuman immunoglobulin binds to antibody
m To detect this binding , a substrate is added and
enzyme acts on substrate to produced colour in
a positive reaction
m This procedure is also named as indired ELISA
m Positive and Negative controls are always put
up alongwith test sera
m Incubation and washing is done at every step to
wash off unbound reagents.
m O


m A third use of ELISA is through competitive binding.
The steps for this ELISA are somewhat different than
the first two examples:
m Unlabeled antibody is incubated in the presence of its
antigen. (Sample)
m These bound antibody/antigen complexes are then
added to an antigen coated well.
m The plate is washed, so that unbound antibody is
removed. (The more antigen in the sample, the less
antibody will be able to bind to the antigen in the well,
hence ˜competition.˜)
m The secondary antibody, specific to the primary
antibody is added. This second antibody is
coupled to the enzyme.
m A substrate is added, and remaining enzymes
elicit a chromogenic or fluorescent signal.
m ëor competitive ELISA, the higher the sample
antigen concentration, the weaker the eventual
signal. The major advantage of a competitive
ELISA is the ability to use crude or impure
samples and still selectively bind any antigen that
may be present
m 96 well plate
m Made of plastic on which protein can be adsorbed
(bind) easily
m Special buffer used that will not denature Ab and
maximize binding
m Blocking step ensures no empty spaces are left
m Blocking reagent is often 10% ëBS
m Measure antibody levels (allergies, vaccines)
m Detect viruses (hepatitis, HIV, venereal diseases)
m Detect hormonal changes (pregnancy)
m Detect circulatory inflammatory markers
(cytokines)
m Detection of rotavirus in faeces
m Detection of mycobacterial antibodies in
tuberculosis
THANK YOU