2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 18
Chapter 2: Assay Kits
ELISA 101
Enzyme Linked Immuno-Sorbent Assay (ELISA) is a powerful technique for detection and quantification
of biological substances, such as proteins, peptides, antibodies, and hormones. By combining the specificity of
antibodies with the sensitivity of a simple enzyme assay, the ELISA can provide a quick and useful
measurement of the concentration of an unknown antigen or antibody.
Currently, there are three major types of ELISA assays commonly used Optimizing an ELISA assay requires the careful selection of antibodies
by researchers. They are: indirect ELISA, typically used for screening and enzyme-substrate reporting system. Once optimized, the
antibodies; sandwich ELISA (or antigen capture), for analysis of sandwich ELISA is fast and accurate. If a purified antigen standard is
amount of antigen present; and competitive ELISA, for antigen available, this method can be used to detect the presence and to
specificity. PBL's ELISA kits use the sandwich strategy. quantitate the antigen in an unknown sample. The sensitivity of the
The "sandwich" technique is so called because the antigen being
sandwich ELISA is dependent on 3 factors:
assayed is held between two different antibodies.
a) The number of molecules of the first antibody that are bound to the
In this method:
solid phase, namely, the microtiter plate;
1. Plate is coated with a capture antibody.
b) The avidity of the antibodies (both capture and detection) for the
2. Sample is then added, and antigen present binds to the capture
antigen; and,
antibody.
c) The specific activity of the detection antibody that is in part
3. The detecting antibody is then added and binds to a different
dependent on the number and type of labeled moieties
region (epitope) of the antigen.
it contains.
4. Enzyme-linked secondary antibody is added and binds to the It is important to note that while an ELISA is a useful tool to detect the
detecting antibody. presence and the quantity of an antigen in the sample, it does not provide
5. The substrate is then added and the reaction between the information concerning the biological activity of the sample. ELISAs are
substrate and the enzyme produces a color change. not generally used to discriminate between active or non-active forms of
The optical density (OD) values can be measured a protein. It may also detect degraded proteins that have intact epitopes.
spectrophotometrically. If you are interested in measuring interferon activity, please see page 29
6. The signal generated is directly proportional to the amount of for information on our iLite
TM
Alpha kit or page 38 to learn more about our
antibody-bound antigen. bioassay services.
HRP-Linked Antibody
HRP
TMB
Substrate
Detection Antibody
Target Protein
Capture Antibody
Sandwich ELISA
PBLInterferonSource Toll Free: 1 877- PBL-8881
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