1. PREPARED BY: ASMA MA
FIRST M.PHARM

2. ELISA
(ENZYME-LINKED
IMMUNOSORBENT ASSAY)

3.  ELISA (Enzyme-linked Immunosorbent Assay) is a
quantitative immunological procedure in which the Ag-Ab
reaction is monitored by enzyme measurements.
 It is a plate-based assay technique designed for detecting
and quantifying substances such as peptides, proteins,
antibodies, and hormones.

4.  The term ELISA was first used by Engvall and Perlma
in 1971.
 The basic steps in ELISA includes:
1. Immobilization of Antigen to a solid surface,
2. Complexation of Antigen with specific antibody
(primary),
3. Attachment of primary antibody to enzyme linked
secondary antibody,
4. Reaction of substrate with conjugated enzyme to
produce a coloured product,
5. Measurement of coloured product.

5.  ELISA is a popular format of "wet-lab" type assay that
uses a solid-phase enzyme immunoassay (EIA) to
detect the presence of a substance, usually an antigen, in
a liquid sample or wet sample.
 It involves detection of an "analyte" (i.e. the specific
substance whose presence is being quantitatively or
qualitatively analyzed).
 The analyte is also called the ligand and it will
specifically bind to a detection reagent.

7.  The ligand-specific binding reagent is "immobilized”
onto a stationary solid phase /solid substrate known as
the "ELISA plate".
 It is then complexed with an antibody that is linked to
an enzyme.
 Detection is accomplished by assessing the conjugated
enzyme activity via incubation with a substrate which
form a coloured product.
 The most crucial element of the detection strategy is a
highly specific antibody-antigen interaction.

10.  ELISAs begin with a coating step, where the first layer, either
an antigen or an antibody, is adsorbed to a well in an ELISA
plate.
 Coating is followed by blocking and detection steps as shown
in the simple schematic diagram.
 Since the assay uses surface binding for separation, several
washes are repeated between each ELISA step to remove
unbound materials.
 During this process it is essential that excess liquid is
removed in order to prevent the dilution of the solutions
added in the next stage.
 The most complex and varying step in the overall process is
detection, where multiple layers of antibodies can be used to
amplify signal.

12. ▪ ELISA plates
▪ Buffers
▪ Antibodies
▪ Enzymes
▪ Sample handling and preparation
▪ Substrate

13.  Plate format
 Flat-bottomed, 96-well plates,
made from polystyrene or
polyvinyl chloride, are coated with
either inactivated Ag/Ab.
 The coating act as the binding site
for the Ag/Ab present in the
sample.
 Further variants are 384-well and
1536-well plates.

14.  Plate characteristics
 It maintain consistency,
minimizing edge effects and
providing optimal optical
conditions for data collection.
 Low to medium binding type
(100–200 ng of IgG/cm2).
 High-binding plates (400–500
ng of IgG/cm2).
 Antigen or antibody pre-coated
plates are also commercially
available.

15.  Standard buffers
 Several different buffers are used during an ELISA:
one for coating, another for blocking, another for
washing, and perhaps another for sample and
antibody dilution.
 Buffers can be produced in house or sourced from a
variety of commercial antibody and reagent suppliers.

16.  Coating buffers
 Coating is the process where a suitably diluted antigen
or antibody is incubated until adsorbed to the surface
of the well.
 Adsorption occurs passively as the result of
hydrophobic interactions between the amino acids side
chains on the antibody or antigen used for coating, and
the plastic surface.
 It is dependent upon time, temperature, and the pH of
the coating buffer, as well as the concentration of the
coating agent.

17.  Typical coating conditions involve adding 50-100 μl of
coating buffer, containing antigen or antibody at a
concentration of 1-10 μg/ml, and incubating overnight
at 4°C or for 1-3 hours at 37°C.
 Coating buffers stabilize the antigen or antibody which
is used to coat the ELISA multiwell plate, maximizing
adsorption to the plate and optimizing interactions
with the detection antibody.
 The two most common coating buffers are bicarbonate
buffer at pH 9.6 or PBS.

18.  Blocking buffers
 To prevent the non-specific binding of detection
antibodies.
 There are two main types of blocking agents, proteins
and detergents.
 Proteins-permanent blocking agents and hence added
after the capture antibody has adsorbed to the well
surface.
 Detergents-block temporarily, blocking function
disappears during washing steps.
 The most basic blocking buffer contains 1% BSA or milk
proteins dissolved in PBS.
 Usually 150 μl of blocking buffer is added to the well to
incubate for 1 hour at 37°C in order to fully block the
plate.

19.  Washing buffers
 To remove unbound materials.
 Usually PBS, with a small concentration of a non-
ionic detergent such as Tween-20 is used.
 Washing is typically repeated 3-5 times between
each step in the ELISA to thoroughly remove
unbound material.
 Excess wash solution must be removed in the final
wash step to prevent the dilution of the reagents
added in the subsequent stage.

20.  The antibodies used in ELISA assays can be monoclonal,
polyclonal, or a combination of both.
 The interaction between antibodies and their antigens is
described in three ways: specificity, affinity, and avidity.
 Specificity is an indication of whether an antibody binds
solely to a unique antigen.
 Affinity describes the strength of binding of an antibody to
a single antigen.
 Avidity accounts for the total stability of the antibody-
antigen interaction.

21.  Monoclonal antibodies
 Monoclonal antibodies can be used for all antibody-
containing steps in all types of ELISAs.
 Monoclonal antibodies are homogeneous by definition,
with specificity for a single epitope or small region of a
protein.
 Polyclonal antibodies
 Polyclonal antibodies are complex antibody pools which
represent a collection of specificities to various
epitopes found in a single antigen.
 They can also be used as capture and detection
antibodies.

22.  The most commonly used enzyme labels are
▪ Horseradish peroxidase (HRP)
▪ Alkaline phosphatase (AP)
▪ β-galactosidase,
▪ Acetylcholinesterase,
▪ Catalase

23.  The specimen samples for ELISA includes:
▪ Serum
▪ CSF
▪ Sputum
▪ Urine
▪ Semen
▪ Supernatant of culture
▪ Stool
 In the simplest case, ELISA samples are diluted in PBS,
wash buffer, or other specialty buffers and applied in a final
volume of 100 μl.

24.  A large selection of substrates is available for performing
the ELISA with an HRP or AP conjugate.
 HRP- ABTS (2,2-azinobis {3-ethylbenzothiazoline-6-sulfonic
acid}-diammonium salt), OPD (o-phenylenediamine
dihydrochloride), TMB (3,3,5,5-tetramethylbenzidine).
 AP- PNPP (p- nitrophenyl phosphate), Disodium salt.
 The choice of substrate depends upon the required assay
sensitivity and the instrumentation available for signal
detection (spectrophotometer, fluorometer, etc).

25. 1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competition/Inhibition ELISA

26.  An antigen is immobilized in the well of an ELISA plate.
 The antigen is then detected by an antibody directly
conjugated to an enzyme such as HRP.

27.  Advantages:
 Direct ELISA detection is much faster than other
ELISA techniques.
 The assay is also less prone to error since fewer
reagents and steps are needed.
 Best for analyzing the immune response to an
antigen.
 Disadvantages:
 Antigen immobilization is not specific.
 Less flexible.
 No signal amplification- reduces assay sensitivity.

28.  Antigen is adsorbed to a well in an ELISA plate.
 Detection is a two-step process. First, an unlabeled primary
antibody binds to the specific antigen.
 Second, an enzyme conjugated secondary antibody that is
directed against the host species of the primary antibody is
applied.

29.  Advantages:
 Economical
 High sensitivity
 Greater flexibility
 Best for determining total antibody concentration
in samples.
 Disadvantages:
 Possibility of background noise
 Longer procedure than direct ELISA technique.
 Additional incubation step for secondary antibody
needed.

30.  Sandwich ELISAs require the use of matched antibody pairs
(capture and detection antibodies).
 Each antibody is therefore specific for a different and non-
overlapping region or epitope of the antigen.
 The capture antibody, binds the antigen that can then be
detected in a direct ELISA or in an indirect ELISA
configuration.

31.  Advantages:
 High sensitivity - 2-5 times more sensitive than direct
or indirect ELISA.
 High specificity - two antibodies are involved in
capture and detection.
 Flexibility - both direct and indirect detection can be
used.
 Best for analysis of complex samples, since the
antigen does not need to be purified prior to
measurement.
 Disadvantages:
 Antibody optimization can be difficult-cross-
reactivity may occur between the capture and
detection antibodies.

32.  The competition/inhibition ELISA, also known as a
blocking ELISA, is the most complex of all the ELISA
techniques.
 Used to measure the concentration of an antigen or
antibody in a sample by detecting interference in an
expected signal output.
 Sample antigen or antibody competes with a reference for
binding to a limited amount of labeled antibody or antigen,
respectively.
 The higher the sample antigen concentration, the weaker
the output signal, indicating that the signal output
inversely correlates with the amount of antigen in the
sample.

34.  Advantages:
 No sample processing is required and crude or impure
samples can be used.
 More robust - less sensitive to sample dilution and sample
matrix effects than the sandwich ELISA.
 More consistent - less variability between duplicate
samples and assays.
 Maximum flexibility - it can be based on direct, indirect or
sandwich ELISA.
 Commonly used when only one antibody is available for the
antigen of interest. It is also suitable for detecting small
antigens that cannot be bound by two different antibodies
such as in the sandwich ELISA technique.

36.  Used for determining serum antibody concentrations.
 Monoclonal Antibody Screening.
 For Virus test (HIV, West Nile Virus, NDV).
 For Home Pregnancy Test.
 In Food industry for detecting potential food allergens
E.g. Milk, Peanuts, Almonds, Eggs and Walnuts.
 Disease diagnosis E.g. HIV, bird flu, common, colds,
cholera, STD etc.
 Detection of enterotoxin of E. coli in feces.
 To study drug toxicity.

38.  Fluorescence polarization immunoassay (FPIA) is a
class of in-vitro biochemical test used for rapid
detection of antibody or antigen in sample.
 It is a competitive homogenous assay, that consists of a
simple prepare and read method, without the
requirement of separation or washing steps.
 The basis of the assay is fluorescence anisotropy, also
known as fluorescence polarization.

39.  If a fluorescent molecule is stationary and exposed
to plane-polarized light, it will become excited and
consequently emit radiation back to the polarized-plane.
 If the excited fluorescent molecule is in motion (rotational
or translational) during the fluorescent lifetime, it will emit
light in a different direction than the excitation plane.

40.  The rate at which a molecule rotates is indicative of its
size.
 When a fluorescent-labelled molecule (tracer) binds to
another molecule the rotational motion will change,
resulting in an altered intensity of plane-polarized light,
which results in altered fluorescence polarization.
 Fluorescence polarization immunoassays employ
a fluorophore bound antigen that when bound to
the antibody of interest, will increase fluorescence
polarization.
 The change in polarization is proportional to the amount
of antigen in sample, and is measured by a fluorescence
polarization analyzer.

42.  FPIA quantifies the change in fluorescence polarization of
reaction mixtures of fluorescent-labelled tracer,
sample antigen, and defined antibody.
 Operating under fixed temperature and viscosity allows
for the fluorescence polarization to be directly
proportional to the size of the fluorophore.

43.  Free tracer in solution has a lower fluorescence
polarization than antibody-bound tracer.
 The tracer and the specific antigen will compete to bind to
the antibody and if the antigen is low in concentration,
more tracer will be bound to the antibody resulting in a
higher fluorescence polarization and vice versa.

44. 1. A specific quantity of sample is added to reaction buffer
and the solution is allowed to equilibrate at room
temperature for approximately 2 minutes.
2. The solution is evaluated in a fluorescence polarization
analyzer to gather a baseline measurement.
3. A specific quantity of antigen conjugated with fluorophore
is added to the solution and equilibrates for approximately
2 minutes.
4. The fluorescence polarization value for the tracer
containing solution is compared to the baseline and
magnitude of difference is proportional to quantity of
target analyte in sample.

45.  FPIA has emerged as a viable technique for
quantification of small molecules in mixtures, including:
 Pesticides,
 Mycotoxins in food,
 Pharmaceutical compounds in wastewater,
 Metabolites in urine,
 Serum indicative of drug use (cannabinoids,
amphetamines,barbiturates, cocaine, benzodiazepines, met
hadone, opiates) and other small molecule toxins.
 The analysis of hormone-receptor interactions.