Transcript ELISA - ASAB-NUST

(Enzyme Linked Immunosorbent Assay)
What is ELISA?
 diagnostic method
 determining protein concentrations
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Purpose
to determine if a particular protein is present in a sample
and if so, how much.
samples in solution (i.e., biological fluids, culture media or cell
lysates)
 blood plasma, serum or cell/tissue extracts in a multi-well plate format
 Advantages
 ELISAs are quick and simple to carry out, and since they are designed to rapidly
handle a large numbers of samples in parallel, they are a very popular choice for the
evaluation of various research and diagnostic targets
 Antibodies specific for the protein of interest are used
to probe the plate.
 Background is minimised by optimising blocking and
washing methods (as for IHC),
 specificity is ensured via the presence of positive and
negative controls.
 Detection methods are usually colorimetric or
chemiluminescence based.
Types
 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
 Direct Elisa
 Indirect Elisa
 Sandwich Elisa
 Direct ELISAs
 involve attachment of the antigen to the solid phase, followed by an
enzyme-labeled antibody. This type of assay generally makes
measurement of crude samples difficult, since contaminating
proteins compete for plastic binding sites.
 Indirect ELISA
 involve attachment of the antigen to a solid phase, but in this case,
the primary antibody is not labeled. An enzyme-conjugated
secondary antibody, directed at the first antibody, is then added. This
format is used most often to detect specific antibodies in sera.
 Sandwich ELISA
 The last type of assay is the sandwich ELISA. Sandwich ELISAs
involve attachment of a capture antibody to a solid phase support.
Samples containing known or unknown antigen are then added in a
matrix or buffer that will minimize attachment to the solid phase.
An enzyme-labeled antibody is then added for detection.
Microtitre plate
 flat plate
 typically has 6, 24, 96, 384 or even 1536 sample wells
 Made of : most common is polystyrene, used for most
optical detection microplates.
 Plate coating is achieved through passive adsorption of
the protein to the plastic of the assay microplate.
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This process occurs though hydrophobic interactions between
the plastic and non-polar protein residues.
 Positive control – sample containing protein
 Negative control- sample not containing protein
 Standard
A sample containing a known concentration of the
target protein from which the standard curve can be
obtained.
 ELISAs begin with a coating step,
where the first layer - either an
antigen or an antibody - is adsorbed to
a polystyrene 96 well plate.
(Adsorption is the passive attachment
of a liquid to a solid surface creating a
thin film.)
 Coating is followed by blocking and
detection steps
 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
sample
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Negative
standard
Positive
control
ELISA Machine
 the entire plate is
placed into a plate
reader
 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
ELISA Results
 The ELISA assay yields three different types of data output:
 1) Quantitative:
ELISA data can be interpreted in comparison to a standard curve
(a serial dilution of a known, purified antigen) in order to
precisely calculate the concentrations of antigen in various
samples.
 2) Qualitative:
ELISAs can also be used to achieve a yes or no answer indicating
whether a particular antigen is present in a sample, as compared
to a blank well containing no antigen or an unrelated control
antigen.
 3) Semi-quantitative:
ELISAs can be used to compare the relative levels of antigen in
assay samples, since the intensity of signal will vary directly with
antigen concentration.