ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antigens or antibodies in a biological sample. It uses antibodies and color changing enzymes to identify a substance in the sample. There are different types of ELISA including sandwich, indirect, and competitive ELISA. ELISA is widely used in areas like immunology, diagnostics, and food testing due to its accuracy, sensitivity, and ability to analyze multiple samples at once.

1. Sachu Elsa Mathew
PG-2 Botany

3.  ELISA:ENZYME LINKED IMMUNOSORBENT
ASSAY
 Term Was Coined By ENGVALL and PEARLMANN
in 1971.
 It is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample..
 The biological samples which are used during
this test are usually blood, urine or some cell
extracts.

4.  Antigens from the sample are attached to a
surface.
 Then, a further specific antibody is applied over
the surface so it can bind to the antigen.
 one antibody is specific for a particular antigen.
 The sample with an unknown amount of antigen
is immobilized on a solid support (usually a
polystyrene microtiter plate) either non-
specifically (via adsorption to the surface) or
specifically (via capture by another antibody
specific to the same antigen, in a "sandwich"
ELISA)

5.  After the antigen is immobilized, the detection
antibody is added, forming a complex with the
antigen.
 The detection antibody can be covalently linked to
an enzyme, or can itself be detected by a
secondary antibody that is linked to an enzyme
through bioconjugation.
 Between each step, the plate is typically washed
with a mild detergent solution to remove any
proteins or antibodies that are aspecifically bound.
 After the final wash step, the plate is developed by
adding an enzymatic substrate to produce a visible
signal, which indicates the quantity of antigen in
the sample.

6.  The qualitative "reading" usually based on detection of
intensity of transmitted light by spectrophotometry, which
involves quantitation of transmission of some specific
wavelength of light through the liquid
 The sensitivity of detection depends on amplification of the
signal during the analytic reactions.
 Since enzyme reactions are very well known amplification
processes, the signal is generated by enzymes which are
linked to the detection reagents in fixed proportions to
allow accurate quantification - thus the name "enzyme
linked".
 The technique is divided into
1- Competitive ELISA
2- Sandwich ELISA (also called direct
ELISA)
3- Indirect ELISA

7.  Prepare a surface to which a known quantity of capture
antibody is bound.
 Block any non specific binding sites on the surface
 Apply the antigen-containing sample to the plate.
 Wash the plate,with PBS(phosphate-buffered saline) so
that unbound antigen is removed.
 Apply enzyme linked primary antibodies as detection
antibodies which also bind specifically to the antigen.
 Wash the plate, so that the unbound antibody-enzyme
conjugates are removed.
 Apply a chemical which is converted by the enzyme into
a coloured product.
 Measure the absorbency of the plate wells to determine
the presence and quantity of antigen

9.  The protein antigen to be tested for is added to each
well of ELISA plate, where it is given time to adhere to
the plastic through charge interactions
 A solution of non-reacting protein is added to block
any plastic surface in the well that remains uncoated
by the protein antigen
 Then the serum is added, which contains a mixture of
the serum antibodies, of unknown concentration, some
of which may bind specifically to the test antigen that
is coating the well.
 Afterwards, a secondary antibody is added, which will
bind to the antibody bound to the test antigen in the
well. This secondary antibody often has an enzyme
attached to it

10.  A substrate for this enzyme is then added. Often,
this substrate changes colour upon reaction with
the enzyme. The colour change shows that
secondary antibody has bound to primary antibody,
which strongly implies that the donor has had an
immune reaction to the test antigen.
 The higher the concentration of the primary
antibody that was present in the serum, the
stronger the colour change. Often a spectrometer is
used to give quantitative values for colour strength

12.  A third use of ELISA is through competitive binding. The
steps for this ELISA are somewhat different from the first
two examples:
 Unlabeled antibody is incubated in the presence of its
antigen (sample).
 These bound antibody/antigen complexes are then added to
an antigen-coated well.
 The plate is washed, so 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".)
 The secondary antibody, specific to the primary antibody, is
added. This second antibody is coupled to the enzyme.
 A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.
 The reaction is stopped to prevent eventual saturation of
the signal.

13.  The basic procedure of this technique is to
use an antibody, which finds an antigen
corresponding to this antibody and binds to
it. This bond of antibody-antigen is
recognized by another antibody. This
antibody is joined to an enzyme which is
responsible for catalyzing the mixture of the
reaction.

14.  It can be applied to determination of serum
antibody concentrations in a virus test (such
as HIV).
 It also used in the food industry when
detecting potential food allergens such as
milk, peanuts, walnuts, almonds and eggs.
 It also used in toxicology as a rapid
presumptive screen for certain classes of
drugs
 They are widely used in various of areas such
as Immunology, Biological Pharmacy,
Diagnostic industry and so on

16.  The fluorescent antibody techniques are extremely
valuable qualitative tools, but they do not provide
quantitative data.
 This drawback has been overcome by the development of
flow cytometry.
 In biotechnology, flow cytometry is a laser-based,
biophysical technology employed in cell counting, cell
sorting, biomarker detection and protein engineering,
by suspending cells in a stream of fluid and passing them
by an electronic detection apparatus.

17.  To carry out this technique, dividing cells are carefully
broken open so that a mixture of intact chromosomes
is obtained
 The chromosomes are then stained with a fluorescent
dye.
 The amount of dye that a chromosome binds depends
on its size, so larger chromosomes bind more dye and
fluoresce more brightly than small ones
 The chromosome preparation is diluted and passed
through a fine aperture, producing a stream of
droplets, each one containing a single chromosome

18.  The droplets pass through a detector which
measures the amount of fluroscence, and
hence identifies which droplets contain the
particular chromosome being sought.
 An electric charge is applied to these drops,
and no others enabling the droplets
containing the desired chromosome to be
deflected and separated from the rest

20.  high speed analysis (>100.000 s-1)
 Measures single cells
 Measures large number of cells
 simultaneous analysis of multiple parameters
(up to 20)
 Identifies small subpopulations
 quantification of fluorescence intensities
 sorting of predefined cell populations (up to
70.000 s-1

22.  FISH (fluorescent in situ hybridization).
 It is a cytogenetic technique developed by biomedical
researchers in the early 1980s that is used to detect
and localize the presence or absence of specific DNA
sequences on chromosomes.
 FISH uses fluorescent probes that bind to only those
parts of the chromosome with which they show a high
degree of sequence complementarity.
 Insitu hybridisation is the annealing of probe DNA
fragments with the complementary sections of
chromosomes

23.  Fluorescence microscopy can be used to find out
where the fluorescent probe is bound to the
chromosomes.
 FISH is often used for finding specific features in
DNA for use in genetic counseling, medicine, and
species identification.
 FISH can also be used to detect and localize specific
RNA targets (mRNA, lncRNA and miRNA) in cells,
circulating tumor cells, and tissue samples. In this
context, it can help define the spatial-temporal
patterns of gene expression within cells and tissues.

24. Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the
dark

25.  To perform FISH, a probe of optimum length is
constructed first. The probe must be large enough
to hybridize specifically with its target but not too
large to impede the hybridization process.
 The probe is tagged directly with fluorophores, with
targets for antibodies or with biotin. Tagging can be
done in various ways, such as nick translation,
or PCR using tagged nucleotides.
 Then an interphase or mataphase chromosome
preparation is produced
.

26.  The chromosomes are firmly attached to a glass
substrate
 Repetitive DNA sequences are blocked by adding short
fragments of DNA to the sample
 The probe is then applied to the chromosome on the
glass substrate and incubated for approximately 12hrs
to allow hybrisization.
 All unhybridized or partially hybridized probes are
removed by several wash steps
 The results are then visualized and quantified using a
fluorescent microscope that is capable of exciting the
dye and recording images

27.  If the fluorescent signal is weak, amplification of
the signal may be necessary in order to exceed the
detection threshold of the microscope
 Fluorescent signal strength depends on many
factors such as probe labeling efficiency, the type
of probe, and the type of dye
 These secondary components, i.e., the fluroscently
tagged antibodies are selected so that a strong
signal is achieved

29.  From a medical perspective, FISH can be applied to
detect genetic abnormalities such as characteristic
gene fusions, aneuploidy, loss of a chromosomal
region or a whole chromosome or to monitor the
progression of an aberration serving as a
technique that can help in both the diagnosis of a
genetic disease or suggesting prognostic outcomes.
 FISH can also be applied to such research
applications as gene mapping or the identification
of novel oncogenes or genetic aberrations that
contribute towards various cancers.

30.  SMITA RASTOGI, NEELAM PATHAK; Genetic
Engineering, Oxford university press
 GAMBORG O.L, PHILIPS G.C, Plant cell, tissue and
Organ culture, Narosa Publishing House
 SINGH B.D, Biotechnology, Kalyani publishers
 BROWN.T.A ,Genomes 3,3rd Edition,garland Science
Publishing.Usa
 www. Wikipedia.com