Validated Protein Standards for Antibody Tests
The problem in employing antibodies in any test is to be confident that they are binding to the correct target protein. But how can you be sure of the identity of a positive control? Our answer is to make the protein standards independently in a recombinant gene expression system. These can then be used in subsequent tests to validate an antibody.
How Will the Service Work?
If you have an antibody you wish to have validated independently, simply provide us with information
about the protein it binds and we will make a synthetic gene for it to be inserted into a baculovirus
expression vector. The protein will also be tagged at either end, usually with a 6-histidine sequence,
to enable subsequent purification if required. By using the appropriate gene promoter, we can then make
the protein in a variety of mammalian cell lines for subsequent tests with the antibody. This is
possible because although baculoviruses only replicate productively in insect cells, they can be used to
transduce mammalian cells and express a gene under the control of an appropriate promoter. Importantly,
we can also provide a negative control for these tests to ensure that the expression system itself does
not produce a false positive. This will comprise a baculovirus expression vector lacking a recombinant
gene. It is often assumed that making recombinant proteins is a lengthy process. However, using our
patented flashBAC™ system, we can usually produce a protein within a calendar month. Furthermore, most
proteins give good yields in either insect or mammalian cells. This renders subsequent purification much
easier than if they were being isolated from original tissues.
The Tests We Can Undertake
The interaction of antibodies with proteins can be studied in a variety of systems.
This analyzes binding of an antibody to a denatured protein in extracts separated using gel
electrophoresis. It is very useful for identifying off target binding to other proteins. It can also
be combined with two dimensional analyses, which separates proteins by charge in the first dimension
before separation by size in the second. This more advanced method identifies if a non target protein
of the same size as the intended protein is binding the antibody.
We have access to one of the most advanced confocal microscopy facilities in the country at Oxford
Brookes University. Confocal microscopes are ideal for studying the interaction of an antibody with
its target in a non denatured state within a cell. Any desired cell culture can be infected (insect)
or transduced (mammalian and avian) with the recombinant baculovirus or the negative control. The
cells are then treated with the primary antibody before subsequent incubation with a labelled
secondary antibody that produces fluorescence in the appropriate wavelength of light.
This is one of the most common applications for antibodies, particularly in diagnostic kits. It is
crucial that any antibody used can be trusted to bind the right protein target. A validated positive
control in such tests is vital. Although crude extracts from cells expressing the recombinant protein
could be used it is better to have purified material. This is also where the provision of a negative
control is important. We can mock extract proteins from these samples and demonstrate that nothing
else from the source of the recombinant protein can bind to the target antibody.
A selection of host cells
Most recombinant protein expression systems are confined to a narrow range of host cells. A major
advantage of the baculovirus system is that it can be used to make proteins in insect cells after a
productive virus infection or in mammalian and avian cell cultures via transduction. Since post
translational modification of proteins can be a key factor in antibody binding, it pays to test this
using material from cells as closely related to the intended target.
The Cost of the Service
The price of making a synthetic gene and recombinant baculovirus has fallen in recent years but still
remains an obstacle for many people needing recombinant protein. Most of our customers request
expression of a gene via the baculovirus system and then retain intellectual property (IP) of the
reagents, including the virus expression vector. We propose a modified service whereby OET will bear a
significant proportion of the cost of making the recombinant virus, but then retain IP on this product.
We are then free to produce the protein for sale to the original customer or to others. Of course, if
the customer wishes to keep the IP themselves they can pay for all the costs in making the virus.