Better Antigens. Better Antibodies. Better Assays.
At SDIX we understand the importance of knowing what your end use application will be for your antibody when developing the antigen. Not all antibodies work the same in all applications. To learn more about what sets SDIX apart and our uncommon competency, watch a few of our videos.
We also offer complete custom immunoassay services and can develop
and produce a wide variety of assays to your specifications.
Not all antibodies work optimally in all applications.
For instance an antibody that works well in a western blot may not
perform as optimally in flow cytometry or sandwich assays.
In some instances a polyclonal antibody may work in a broader range
of applications than a monoclonal that was not selected by screening in
each of those applications.
In all cases antigen design and quality used for generating the
antibody along with immunization protocols will strongly influence the
suitability of the resultant antibody for application.
Commonly Used Assay Procedures and Antibody Selection
The following are a list of commonly used assay procedures, along
with considerations in antibody selection for optimal performance:
Western Blot
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In western blot the target proteins are highly and irreversibly
denatured by the treatment with SDS.
Secondary, tertiary, and quaternary structures are destroyed.
Antibodies made against peptides, protein fragments, recombinant
antigens, or GAT
technology may be useful here.
Major issues will be sensitivity and specificity.
Careful titration of the antibody will likely be required to achieve
the desired levels of specificity, generally the appearance of a single
band reactive with the antibody. Both polyclonal and monoclonal
antibodies work well here.
It should be noted that antibodies, particularly monoclonals reactive
with conformationally determined epitopes (epitopes influenced by not
only the primary sequence but also by the proper folding) may not work
well (or at all) in Western blot.
Fortunately with polyclonals made to conformationally correct
proteins a mixture of reactivities to liner and conformational epitopes
may exist and these may work in Western blot as well as more demanding
assays.
Direct
Bind
In direct bind assay target proteins (or peptides) are
immobilized on a surface and antibody titered over that surface.
This is fairly non demanding application commonly used for
following the course of immunizations or as an early screening tool for
hybridoma evaluation, although it does require a pure antigen as
impurities compete with antigen for binding to the solid phase.
Antigen is typically present in large excess in this assay
compared to other methods of evaluation and consequently produces very
high apparent titers.
Titer in direct bind ELISA will typically be 10 to 1000 fold
higher than in other applications, and accurately comparing two
different labs (or manufactures) direct bind titers is difficult.
Since the antigen is bound to the surface, it is possible that
certain determinates are masked by the immobilization.
Specificity may be an issue and appropriate controls are
required.
For evaluating immunizations it is highly desirable to use an
antigen source different than the antigen used for immunization. For
instance when immunizing with recombinant protein it would be highly
desirable to screen against native protein.
Sandwich
Immunoassay
A wide variety of permutations of sandwich assays on a wide variety
of assay platforms are commonly practiced. Briefly, antibody
immobilized to one surface is used to capture the antigen from solution
which in turn reacts with a second antibody to the antigen.
This second antibody often contains a detectable moiety, such as an
enzyme, a fluorescent molecule, a particle label, a label excitable by a
label on the first antibody, etc.
By using two different antibodies one can guarantee specificity of
the reaction. In some instances one of the antibodies may have slightly
broader specificity and the second antibody’s specificity defines the
specificity of the reaction.
The downside of using two antibodies is that sensitivity is limited
by the lower affinity of the two antibodies. However, with carefully
optimized assays with the right antibodies detection into the pM range
is often achievable.
Sandwich assays in laboratories buffers are simple and
straightforward to configure. However, more complex matrices such as
serum/plasma, urine, or other body fluids may pose special
considerations.
Molecular interactions with other target binders within these
matrices may require special buffers to overcome these interactions and
often high affinity antibodies will be required to fully and accurately
measure the target molecule.
Additionally non specific binding may be quite prevalent, depending
on the sample type and special measures may be required to prevent false
positives.
Both monoclonal and polyclonal antibodies work well in sandwich
immunoassays and can even be used together. Generally these antibodies
will need to be directed against conformational epitopes.
Antibodies made against protein fragments, recombinant antigens,
purified native antigen, or GAT technology may be useful here, with
antibodies made against peptides typically not being of much utility.
As previously mentioned high affinity will often required to achieve
desired levels of sensitivity.
IHC
In Immunohistochemistry (IHC), antibodies are used to stain tissue
sections. These tissues have typically been subjected to fixatives,
such as formaldehyde, resulting in antigen alteration and imbedded in
paraffin.
A variety of antigen retrieval protocols may be employed to render
the tissue suitable for staining with antibody and choice of retrieval
protocol may influence reactivity.
Intracellular, membrane, and extracellular antigens may be detected.
A variety of methods for generation of antibodies, including peptides
and recombinant antigens or fragments or GAT
technology , may work to elicit antibodies that work in IHC. As
with Western Blot specificity and appropriate titration will be
critical.
Specificity is usually determined by the staining patterns in a given
tissue or by using a tissue known to be negative for the marker as a
control. In many instances a trained pathologist may be required to
evaluate the accuracy of the staining patterns. It is also possible to
do IHC with frozen tissue sections.
Flow
Cytometry (FACS)
In flow cytometry only antibodies reactive to conformational
determinants exposed on the cell surface will be reactive. Interactions
of molecules on the cell surface may mask other epitopes. It
is critical that the antibodies to be used for flow cytometry be made
against conformationally correct antigens with appropriate post
translational processing. Antibodies made against antigens
expressed on whole cells or plasma membrane fractions, as well as
against recombinant proteins (or GAT
technology ) representing the extracellular portion of the molecule
may work well here.
It will be critical that the antigen used for immunization mimic the
conformation of the antigen as it exist on the cell. For reasons of
specificity (depending on immunization strategy) monoclonal antibodies
may be preferred.
It should also be noted that it is possible to do intracellular
staining with flow cytometry by first fixing the membrane, then
permeabilizing the cell with detergents. In this fashion antibodies
against cytoplasmic portions of the molecule may be detected.
Autofluorescence (background fluorescence of the cell) greatly increases
and appropriate positive and negative controls are required.
Cell
Based Assays
Based on the ability of an antibody to specifically modulate a cells
activity, these are perhaps the most demanding of all applications. At a
minimum the criteria described for antibodies for flow cytometry will
apply.
Since polyclonal antibodies may contain antibodies with both
agonistic and antagonistic activity, highly confusing the readout in
some cases, monoclonal antibodies may be highly desirable for this
application.
Antibodies useful in cell based assays may be taken forward to even
more advanced applications, including humanization and potential use as
therapeutic antibodies.