Versatile: Many systems using different combinations of reagents can be used. Allows passive immobilization of reagents to a solid phase and easy separation of bound/unbound reactants using washing steps.
Simple: Multiple samples can be tested in a variety of formats using microtiter plates and compatible equipment that provides high capacity, rapid and inexpensive assays feasible to kit development.
Sensitive: Readouts come from enzyme catalysts or fluorescent tags that amplify signal efficiently and provide range of detection optimal for critical assays including diagnostics.
Quantitative: Signal is detected by multichannel spectrophotometers and therefore the collected data can be stored and analyzed statistically.
Although all the different systems operate within the common parameters of immobilization to a solid phase, separation of bound and free reagents by washing steps and readout from colorimetric, fluorescent or luminescent signals, each configuration differ in the nature and number of stages needed as well as their possibilities for specialized applications like detection and quantification of antibodies, proteins, peptides and even small molecules.
The following are the core systems that constitute the basis to the many possible configurations:
1. Direct ELISA: It is the simplest configuration in which the antigen is bound by passive adsorption to the solid phase, washed to remove any unbound molecules and then directly incubated with a conjugated antibody. Following the incubation period and additional washing, substrate is added to produce signal that is allowed to develop. After certain time, the substrate reaction is stopped and the resulting signal quantified. It is commonly used for tittering conjugated secondary antibodies and very useful to estimate antigen cross-reactivity.
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ELISA: In this system,
initial antigen binding and washing steps are the same as the direct method. The
main difference in this case is the use of unconjugated antibody to bind the immobilized antigen upon
incubation at optimal temperature (usually 37⁰C). Following a washing step to
remove unbound antibodies, the remaining antigen-bound antibodies are targeted
by a conjugated secondary antibody that will generate the readout signal as
described for direct ELISA. This system has been widely applied in diagnostics
because it allows large number of samples to be screened with a single conjugated
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ELISA: This assay requires a compatible antibody pair that
recognize different antigenic targets (epitopes) on the same antigen. The first
antibody, called capturing antibody,
is coated on the plate and used to immobilize the antigen upon binding during incubation
with the sample. Free antigen is removed by a washing step and then a detecting antibody is added to bind the
captured antigen and enable subsequent detection. Sandwich ELISA is divided
into two main systems:
Each of the core systems described
above can be further modified to measure molecules based on their ability to
interfere with a well-known pre-titrated assay. Such assays can be used to
study either antigens or antibodies and they can be competitive or inhibitory
based on the specific conditions of each assay. In both types of assays, a
pre-titrated system is challenged with the presence of the testing sample whose
binding activity is then determined from the degree of the resulting
interference in the established system.
Below is the description of the main
steps in the most common type of ELISA assays and the recommended reagents to
use throughout the whole assay. Details on recommended protocols and technical tips are also provided.
1. The choice of blocking buffer
is critical in obtaining genuine intensity signals and reduced backgrounds
For this purpose and
to account for unspecific binding between conjugated secondary antibody and the
antigen, it is also necessary to include controls of antigen coated wells
incubated with the conjugate only. Efficient blocking buffers include
BSA and gelatin.
2. Optimize the amount of conjugated
secondary antibody to be used
will provide different ranges of signal depending on how much antibody they
will bind. Because the goal is to bind the entire antigen-antibody complex
present in each well, you want to make sure you will use the right amount of
conjugate in your assay by performing a serial dilution assay in advance (see
tip #2). Always use high quality antibody conjugates. For alkaline phosphatase
conjugates replace use TBS
3. Adjust Incubation times to improve the performance
of your assay
In general, antigen-antibody complexes will form within two hours
incubation at room temperature but in some cases significant stronger specific
signal might be obtained with longer incubation times. Following the addition
of the substrate, it is important to read the plate
within the recommended time for the specific reporter system being used.
Chromogenic and chemiluminescent substrates, particularly those used by
peroxidase-conjugated antibodies can saturate the signal intensity and reduce
the dynamic range of the assay.
4. Once your assay is optimized, always
keep the conditions unmodified throughout all steps
Because Enzyme-Linked Immunosorbent Assays are very sensitive, even minor changes in buffer composition, volumes,
washing times, antigen/antibody concentrations, temperature or incubation times
can have a significant impact in the performance of the assay. It is highly
advisable to run all assays in duplicate and include controls that help to
account for plate to plate variability.
Rockland Immunochemicals Inc.Limerick, PA 19468E-mail: email@example.comPhone: 800.656.7625