The quality of an antibody can make or break an assay.
Catalog reagents are fine when they’re available and when they work. Yet the majority of commercially available
antibodies were created with little thought to end use, and simply aren’t up to
the more complex tasks — such as flow cytometry, sandwich ELISA,
immunofluorescence or functional assays — that they’re often asked to perform.
Since the quality of an antibody can make or break an assay, it’s important for
it to reliably discriminate antigen from artifact — especially for proprietary
or critical targets in research, development, diagnostics, and therapeutics.
How the antibody is generated will not only impact its
specificity and sensitivity, but even the likelihood of being able to generate
any useful reagent at all. The type and design of the immunogen, the species from
which the immunogen is derived, the protocol under which the host is exposed to
the immunogen, and how the resultant immunoglobulin are screened and purified,
are vitally important to obtaining antibody that will recognize the right
target in the right context.
SDIX Genomic Antibody Technology™
Using a carefully designed protein domain as an antigen is an exciting alternative that navigates
between immunizing with unstructured peptide antigen on the one hand, and
immunizing with complex protein on the other. A revolutionary method, SDIX Genomic Antibody Technology results
The creation of a SDIX Genomic Antibody begins with a
deep examination of the target epitope in the context of its end use
applications. By utilizing a combination of bioinformatics, structural analysis
(including x-ray crystal studies, if available), and literature reviews, a
self-contained target domain is selected. A host of criteria — from topology,
the existence of paralogs and orthologs, and repeats, to the presence of
alternative splice sites, point mutations, functional and other interaction
sites, as well as cleavage and other post-translational modifications including
disulfide bonds, glycosylation and phosphorylation — are all taken into account
in the design of an approminately 100 amino acid protein domain that will function as the immunogen.
While immunizing with protein antigens is generally
considered the “gold standard” for generating antibodies to conformational
epitopes, the vast majority of antibodies are generated against peptide
antigens. Each of these techniques has its place, but may not be ideal for
every application.
Peptides’ linear strings of 10-20 amino acids can’t adopt
the three-dimensional conformation that a native protein would normally assume,
and certainly can’t mimic epitopes that are made up of sections of different
parts of the folded protein. Instead, the immune system is presented with
something that looks nothing like the target epitope (unless that epitope
happens to be located on the N- or C-terminus and is loosely structured). Even for
western blotting, which displays denatured epitopes, protein- derived
antibodies or SDIX Genomic Antibodies generally
yield far more sensitivity than do assays using peptide-derived antibodies.
This is perhaps because the larger immunogens can engender truly polyclonal
antibodies able to recognize multiple epitopes on the target protein. Or
perhaps because rotation around the chemical bonds of a peptide are
unconstrained, antibodies are formed against many different conformations of
the same sequence — most of which are not found in the target protein — resulting
in a lower specific activity.
Sometimes protein immunization, too, can be less than
optimal — even beyond the time, expertise, and cost required to produce
high-quality protein antigen (if it can be made at all). Many human proteins in
other mammals are non-immunogenic and
will simply be tolerated by a host rather than cause an antibody response, or
they may contain domains that are toxic or immunosuppressive. Other proteins elicit
antibodies against a wide variety of epitopes, some of which are identical with
other proteins and can result in cross-reactivity in immunoassays. And the
challenges of using whole protein to generate antibodies against membrane-bound
antigens — the protein’s hydrophobic regions are highly unlikely to adopt their
native conformation in the absence of the lipid bilayer in which they are found
— are notorious.
What the Antibody Sees
An antibody’s footprint — the size of the antigen
recognition site — is about 30 Angstroms in diameter, allowing it to make
contact with about 10-20 amino acids on the target protein. These residues are
adjacent to each other in three dimensions space — scattered across alpha
helices, beta pleated sheets, and other structural elements — yet discontinuous
in terms of the protein’s primary (linear) makeup. The more complementary the
fit between antibody and antigen, the more chemical interactions are made, the
tighter the bond is, and thus the lower the limit of detection that can be
achieved.
Design for Purpose
One size does not fit all. An antibody reagent that works
well for western blotting may be useless in sandwich ELISAs or flow cytometry.
A protein is denatured when it’s prepared for western blotting. The epitopes an
antibody might recognize are essentially linear sequences of amino acids, with
no particular structure to them.
Conversely in flow cytometry it’s generally the native
three-dimensional protein displayed on a cell surface that the antibody sees.
The 3D protein is on display in ELISAs, too — either bound to a plate or ready
to be captured from a samplelike serum or plasma by a plate-bound antibody — as
it is in functional assays and as a therapeutic target. Those epitopes recognized by the antibodies
are generally amino acids found on the exposed surface of the folded protein.
Residues buried inside remain incognito, although their presence may likely be
reflected in the overall conformational structure of the protein.
The SDIX Genomic Antibody Technology presents the host
with a single domain containing a few, carefully-chosen epitopes in their
native conformation, against which the immune system can mount a highly
effective and focused B cell response. The polyclonal
pool of antibodies produced collectively has specificity for various facets of
the target antigen including the conformational epitopes and disordered loops, allowing
for recognition of both denatured and native structures.