It can be very difficult to test for PrPSc because it may be present in small amounts and separating it out from PrPC is not trivial. The methods currently being used test dead animal tissue. There are proposed ways to test live animals, such as detecting prions in blood, urine or cerebrospinal fluid, but these methods are not yet in widespread use. More information about the differences between the conformations of the proteins is needed before these other tests can become a reality.
The most conclusive form of testing is the bioassay. This test works by taking a tissue sample of a suspected infected animal and putting it into a mouse or other animal and waiting for the disease to develop in the model organism. Obviously, this is a very slow and labor-intensive method of testing for the disease. A faster form of testing, but still labor intensive, is immunohistochemistry. In this method, antibodies that recognize PrPSc are injected into brain tissue, and then observed on slides under a microscope for the presence of these antibodies. If the antibodies are present, then they have attached to infectious prions and the tissue is infectious. Because each slide must be examined under a microscope, this type of test can take time and energy that makes it hard for mass testing.
Another type of test, the immunoassay, is fast and easier to complete, but only applicable when there are high levels of infectious prions; subsequently, lower levels may go undetected. The immunoassay works by first adding proteases to brain tissue, which will break down all the non-infectious form of prions and leave only infectious prions. Then, antibodies sensitive to prions are added to the solution. The antibodies used are tagged with a visual marker so that the presence of the prions with show up. Testers can also run the solution on a gel and the presence bands will mean that there is infectious prion protein. The reason this only works for high levels of infectious protein is that although many PrPScs are resistant to breakdown by proteases, many are not, especially in the early stages of the disease (it is not known why this is the case). Therefore, after proteases have been applied to the mixture, all of the PrPC is broken down, and some of the PrPSc is broken down as well, so only a little bit remains. Furthermore, it is hard to find antibodies that will only bind to the infectious form when we do not know the exact conformation differences, so proteases are a necessary step before adding antibodies.
Finally, a newer method that is both quick and accurate for low levels of infectious protein is called conformation-dependent immunoassay (CDI). CDI uses tissue taken from a live animal mixed with a chemical that separates infectious from non-infections prions based on their conformations. Next, an antibody tagged with flourescence is added to the separated area, and if the tissue same contains infectious protein, the antibody will fluoresce (Prusiner, 2004).
Brain imaging is another diagnostic tool that can be used for humans, but is not applicable to diagnosing animals that may be fed to humans. Furthermore, brain scans are expensive, and would probably only be used if someone is showing symptoms of a TSE, which often means the disease is very advanced. The development of a wider diagnostic test that could be applied to anyone who might be at risk of a TSE would be incredibly valuable because it might be easier to slow down in an early stage of the disease (Committee on Transmissible Spongiform Encephalopathies, 2004).
One of the obstacles to creating a test that does not require brain tissue, as all of the above tests do, is the low level of prions in other tissues. Formulating a blood or urine test would require a means of amplifying the infectious protein to levels that are detectable. This is difficult because since it is only protein, polymerase chain reaction (a very efficient method for amplifying nucleic acids) is not applicable. However, there is a method being developed that may allow an amplification process that would make a blood test more feasible. This amplification process is called protein misfolding cyclic amplification (PMCA). It has been shown to work experimentally by mixing infected prions with normal prions. The idea behind it is that infectious prions transform normal prions into infectious prions, so the normal prions are there for the infectious to work on changing. However, often the infectious prions form clumps, so they less actively change the normal prion conformations. Therefore, this method uses sonication-pulses of sound waves-to break up infectious prion clumps so that they will spread throughout the tissue mix and transform the normal prions. This seems to work as a method of amplifying infectious protein. Another possible way to get around the low levels of infectious protein is to detect a "surrogate marker" instead of the protein itself. There may be other proteins or molecules that indicate the presence of the infectious prion protein that are easier to detect, and could therefore act as a "surrogate marker" because they, instead of prions, could be tested for in tissue. Another obstacle is how to distinguish among the different strains of TSEs. Knowing this could be integral to determining the source of the disease (inherited, spontaneous, transmitted) and to inventing with more targeted ways of dealing with the different strains disease (Committee on Transmissible Spongiform Encephalopathies, 2004).
The lack of inexpensive testing poses a major issue in the beef processing market because BSE testing cannot catch the disease in its early stages. It is also difficult to test a large proportion of cows going to the slaughterhouse. Scientists have argued back and forth whether all cows should be tested or if only older and downer cows (cows that typically cannot stand on their own due to illness or injury) require the testing. Normile (2004) states that this really comes down to a cost-benefit analysis-is it really worth the enormous cost to test all cows meant for human consumption? The complicating feature, he states, is that the risk is not known. A more recently published article describes initial findings on a new ability to identify BSE markers in the urine of cows (Simon, Lamoureux, Plews, Stobart, LeMaistre, Ziegler, Graham, Czub, Groschup & Knox, 2008). If this ends up working and being a practical solution, it would be a monumental step in the right direction toward having cheaper and subsequently more widespread testing in the cattle industry (Normile, 2004).
Currently, Japan is the only country to incorporate comprehensive BSE testing on all cattle produced for human consumption. Most other countries conduct testing only on downer cows and those slaughtered over 30 months of age (Normile, 2004). While some scientists say that comprehensive testing poses an unnecessary cost, Japan justifies its policy by pointing out that the most recent cattle they found with BSE were 21 and 23 months old: in an age range other testing protocols would have not tested. In comparison, the U.S. conducts the fewest number of tests-about 20,000 are cattle tested per year. Compare this with 10.4 million tested cattle in the E.U. (Normile, 2004.) Furthermore, since there is far more beef produced in the U.S., the proportion of tested cattle in the U.S. is shockingly low. American farmers are resistant to more testing because of the cost. The general mindset in America is bigger, faster, cheaper, which does not help improve safety or good animal handling practices. More research is being done to create faster, cheaper BSE tests, but additional research and more testing regulations need to be put into place to ensure there are not additional vCJD outbreaks in humans.