The Enzyme-Linked Immunosorbent Assay (ELISA) / (EIA) involves coating (binding) of an antigen (Protein) to a solid support such as a membrane (as used in immunoblotting/western blotting) or a 96-well micro plate (ELISA Plate). The coating is done using bicarbonate / carbonate coating buffer. Proteins which have not been separated by electrophoresis can be bound to membranes and analyzed with primary and secondary antibodies as in the immunoblotting procedure. Such analysis are often called dot blots. The more common format is to absorb the antigen to the wells of a 96-well microplate and to use substrates that produce a colored product. The ELISA is suitable for the analysis of large numbers of samples and most of the procedure can be automated.

1. ELISA Protocol- Types
of ELISA- Advantages
& Applications of ELISA
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2. ELISA
 The Enzyme-Linked Immunosorbent Assay
(ELISA) / (EIA) involves coating (binding) an
antigen (Protein) to a solid support such as a
membrane (as used in immunoblotting) or a 96-
well micro plate (ELISA Plate). The coating is
done using bicarbonate / carbonate coating
buffer.

3.  Proteins which have not been separated by
electrophoresis can be bound to membranes and
analyzed with primary and secondary antibodies as
in the immunoblotting procedure. Such analysis are
often called dot blots. The more common format is
to absorb the antigen to the wells of a 96-well
microplate and to use substrates that produce a
colored soluble product. The ELISA is suitable for
the analysis of large numbers of samples and most
of the procedure can be automated.

4. Antigen Coating
 The first step is to bind the antigen to the micro
well plate. The antigen used can be a purified
protein , peptide or a crude protein extract.
 The choice of antigen will depend on the application
and the nature of the antibodies being tested.
Binding to 96-well micro plates is achieved by
incubating the wells with a solution containing the
antigen.
 Protein binding to the plate is due to hydrophobic
interactions between the protein and the plastic.
coating incubation may vary from 2hrs at room
temperature or overnight at 4oC.

5. Blocking
 Once antigen is bound to the plates, it is important to block
the remaining surface of the plate / membrane to prevent
nonspecific binding and the detection antibodies during
subsequent steps.
 Many Blocking buffers are available to block the free sites
on the plate, BSA, non fat dry milk powder etc in
PBS(phosphate buffered saline) or TBS (Tris buffered
saline) with minute percentage of tween 20 or Triton X-100
can be used as blocking buffer.
 The protein in the blocking solution will attach to the
membrane in all places where the target proteins have not
attached. thus when antibody is added which will bind only
to the target protein so the background interference will be
reduced.

6. Incubation with Primary &
Secondary Antibodies
 After blocking the, excess blocking agent is removed by washing the plate / membrane
with PBS and tween 20 (wash buffer) for sometime Then the antigen-containing wells
are incubated with the primary antibody. Appropriate antibody dilutions need to be
made which will better results, Serial dilutions are often carried out and the minimum
titer which is giving a good response can be used. Optimization of the antibody
concentration need to be done for getting better results. Antibody dilution are made in
PBS or TBS. ELISAs are typically carried out in duplicate or triplicate. A positive and a
negative control is also included along with the test samples. Primary antibody
incubation may vary from 2 hours or even more incubation periods are required to get
good results. After incubation with the primary antibody the wells are washed
extensively to remove any unbound antibody. Similarly membrane / plate is incubated
with 2o antibody with suitable dilutions. incubation period varies from 2 hrs or
sometimes even more, secondary antibody will bind to the primary antibody, secondary
antibodies are conjugated with enzyme which on reaction with substrate in the
developing solution will yield colour. excess antibodies are washed off with wash buffer.
there are variety of tags or enzyme labels can be conjugated to the secondary antibody.
Most widely used enzymes are Horse Radish Peroxidase(HRP) and Alkaline Phosphatase
(AP).

7. Developing
 A developing solution having chromogenic substrate is added to the
wells, the enzyme reacts with the substrate and gives colour. The
intensity of the color will be proportional to the amount of
secondary antibody which proportional to the amount of specific
primary antibody in the sample. Alternatively a constant amount of
a defined antibody can be used to quantify the amount of a specific
protein in the sample. ELISA plate readers is basically a
spectrophotometer designed to read the individual wells in a 96-well
microplate. ELISA plate readers are interfaced with a computer to
assist in data management.

 most widely used substrates are pNPP (p-nitrophenyl phosphate);
BCIP (5-bromo-4-chloro-3-
 indolyl phosphate); NBT (nitro blue tetrazolium); TMB (3,3',5,5'-
tetramethyl-benzidine); DAB (3,3-diaminobenzidine)

8. Types of ELISA
 Indirect ELISA
 Competitive ELISA
 Sandwich ELISA

9. Advantages and Applications of
ELISA
 ELISA can be used to quantify Antibody in the
sample
 ELISA can be used to quantify Antigen in the
sample
 Large no of samples can be processed at a time.
 Highly sensitivity.

10.  ELISA has many applications in invitro disease
diagnostics, some of the are
 Detection of HIV antibodies in serum
 Diagnosing Lyme Disease
 Diagnosing Lyme Disease, etc
 Detect plant diseases

11. References
 Methods in Cell Biology, Mark F. Wiser
http://technologyinscience.blogspot.com/2011/12/el
isa-protocol-types-of-elisa.html

12. Click here to Visit Bio-Resource
for more detailed information on
ELISA, TYPES, APPLICATION &
ADVANTAGES
http://technologyinscience.blogspot.com