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.

Western blots

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.

Confocal microscopy

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.

ELISA

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.