ELISA
There are four major types of ELISA:
Direct ELISA (antigen-coated plate; screening antibody)
Indirect ELISA (antigen-coated plate; screening antigen/antibody)
Sandwich ELISA (antibody-coated plate; screening antigen)
Competitive ELISA (screening antibody)
Direct ELISA
In a direct ELISA, the antigen is immobilized to the surface of the multi-well
plate and detected with an antibody specific for the antigen The antibody is
directly conjugated to HRP or other detection molecules.
Indirect ELISA
Is a technique that uses a two-step process for detection, whereby a
primary antibody specific for the antigen binds to the target, and a labeled
secondary antibody against the host species of the primary antibody binds
to the primary antibody for detection. As for direct ELISA assays, the
antigen is immobilized to the surface of the multi-well plate.
The method can also be used to detect specific antibodies in a serum
sample by substituting the serum for the primary antibody.
Sandwich ELISA
is the most commonly used format. This format requires two antibodies
specific for different epitopes of the antigen. These two antibodies are
normally referred to as matched antibody pairs. One of the antibodies is
coated on the surface of the multi-well plate and used as a capture
antibody to facilitate the immobilization of the antigen. The other antibody
is conjugated and facilitates the detection of the antigen.
Competitive ELISA
Also known as inhibition ELISA or competitive immunoassay, competitive
ELISA assays measure the concentration of an antigen by detection of
signal interference. Each of the previous formats can be adapted to the
competitive format.
The sample antigen competes with a reference antigen for binding to a
specific amount of labeled antibody. The reference antigen is pre-coated
on a multi-well plate and sample is pre-incubated with labeled antibody and
added to the wells. Depending on the amount of antigen in the sample,
more or less free antibodies will be available to bind the reference antigen.
This means the more antigen there is in the sample, the less reference
antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled
antibody. The labeled antigen and the sample antigen (unlabeled) compete
for binding to the primary antibody. The lower the amount of antigen in the
sample, the stronger the signal due to more labeled antigen in the well.
Which type of ELISA should I use?
Advantages Disadvantages
Direct ELISA Short protocol: saves Potential high
time and reagents. background: all proteins
No cross-reactivity from in the sample bind to
secondary antibody. the surface.
No signal amplification.
Low flexibility: the
primary antibody must
be conjugated.
Indirect ELISA Signal amplification: Long protocol if
several secondary compared to direct
antibodies will bind to ELISA.
the primary antibody. Potential cross-
High flexibility: the same reactivity from
secondary antibody may secondary antibody.
be used for several
primary antibodies.
Sandwich ELISA High specificity: Demanding design:
involves two antibodies finding two antibodies
detecting different against the same target
epitopes on the same that recognize different
antigen. epitopes and work well
Suitable for complex together can be
samples. challenging at times.
High flexibility and
sensitivity: both direct
and indirect methods
can be used.
Competitive ELISA Depends on base ELISA Depends on base ELISA
selected. selected
Suitable for small
antigens.
There are four main general steps to completing an ELISA
immunoassay. These steps are:
1-Coating (with either antigen or antibody)
2-Blocking (typically with the addition of bovine serum albumin [BSA])
3-Antibody Incubation
4-Detection
Why is blocking important in ELISA?
Blocking buffers remove the possibility of non-specific binding and
therefore help to increase stability, reduce background noise and make the
assay more sensitive. ELISA blocking buffers should not interfere with the
assay itself, and should only contain compounds that do not themselves
react with the epitope.
Why are the washing steps necessary in the ELISA test?
Washing steps are necessary to remove non-bound reagents and decrease
background, thereby increasing the signal to noise ratio. Insufficient
washing will cause high background, while excessive washing might result
in decreased sensitivity caused by elution of the antibody and/or antigen
from the well.
What is conjugate in ELISA?
A conjugated antibody binds to detect the target protein when added. Then
an ELISA substrate is added, producing a signal proportional to the given
analyte in a sample
Which enzyme is most commonly used in ELISA?
Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most
commonly used enzyme labels, mainly due to the large selection of
substrates available for ELISA
Does ELISA measure enzyme activity?
In ELISA, various antigen-antibody combinations are used, always including
an enzyme-labeled antigen or antibody, and enzyme activity is measured
colorimetrically. The enzyme activity is measured using a substrate that
changes color when modified by the enzyme.
What is the substrate in an ELISA test?
TMB (tetramethylbenzidine) is a chromogenic substrate that is common in
numerous staining procedures used at the Horseradish Peroxidase (HRP)
detection step. One of its most common applications is in the ELISA
procedure.
ELISA tests which used alkaline phosphatase conjugated antibodies for the
detection system as it used the substrate p-nitrophenyl phosphate (pNPP)
which produces a water-soluble yellow reaction product
