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Originally Published IVD Technology September 2004

Beyond Clinical Diagnostics

Figure 1. A schematic illustration of the Seprion assay for rogue prion protein (click to enlarge).

The detection of prion diseases in animals and their diagnosis and treatment in human medicine is an area of recent intense scientific investigation. Tests are now available for brain tissue testing, and rapid progress is expected in the development of improved diagnostic technologies for detecting the presence of the infectious agent (the prion, a protein particle lacking nucleic acid) in the blood of infected animals and in donated human blood. A blood test for prion diseases in animals, when it is developed, will be an important element in the control and eradication of these diseases. And, of course, a test for donated blood will ensure the safety of the blood supply in human healthcare.

Currently, commercial diagnostic assays for prion diseases, or transmissible spongiform encephalopathies (TSEs), can detect the infection only in postmortem brain samples. These assays are used widely in food safety applications, principally to look for bovine spongiform encephalopathy (BSE). A number of immunoassays for BSE have been evaluated by the European Union (EU); three have been approved for routine use in testing laboratories.¹ The approved tests include two enzyme-linked immunosorbent assays (ELISAs) using prion protein–specific antibodies and peroxidase labels in the microplate format, as well as reagents for a Western blot analysis that is often employed as a confirmatory test.

Following a brief review of the TSE problem for animal and human populations, this article discusses research efforts to develop assays using synthetic ligands that could simplify the assay procedure. Success with assays for brain tissue is described, and the prospect of a blood assay for living animals and human patients is considered.

Scope of the Disease Family

Nearly 20 years have passed since BSE was first recognized. Since then, as many as a million cattle in the United Kingdom have been infected, of which some 80% managed to enter the human food chain. An unexpectedly large number of cases are still being recorded each year in that country, despite implementation of safety procedures deemed appropriate, such as excluding animals more than 30 months old from slaughter for food and removing specified risk material (brains and other organs) from the food supply.

More and more BSE cases are appearing across Europe as well, with infected animals being reported in France, Germany, Spain, Greece, and Hungary. In fact, Germany, will probably not see its epidemic peak until 2005.

Moreover, while BSE has been incubating in European cattle over the past five years, meal made from bovine by-products has continued to be exported from Europe to feed cattle around the world. Cases of BSE have been recorded in the United States and Canada, while Japan has implemented a major BSE screening program following the discovery of infected animals in the national herd. Japan now tests every animal sent for slaughter.

As for variant Creutzfeldt-Jakob disease (vCJD), the suspected human form of BSE, it has been eight years since the first case was officially recorded. A link between vCJD and the consumption of BSE-infected meat has been strongly suggested. More than 140 people have been diagnosed as having died of this disease in the United Kingdom and other European countries. Reports have suggested that everyone in the United Kingdom over the age of 4 has been exposed to beef or beef-derivative products from BSE-infected cattle and on average has eaten 50 meals containing tissues from infected cattle.² The incubation period of vCJD could be as long as 30 years, however, so estimates of the likely number of infected humans vary widely.

The picture is further clouded by another factor. While eating meat from infected cattle is the most likely cause of vCJD, it is now accepted that a risk of contracting vCJD infection through contaminated surgical and dental equipment and donated blood exists. The recent report that scrapie infection was able to be transmitted from infected sheep to healthy animals through blood transfusion could be an indicator of a potential problem of great significance for the safety of the blood supply.³ The first probable incidence of vCJD infection by this route was reported in the United Kingdom in December 2003. Studies have suggested that the number of infected people who might develop vCJD could range from 1000 to more than 1 million worldwide.⁴

Postmortem Prion Disease Testing

Commercially available diagnostic assay tests for BSE, scrapie, and chronic wasting disease (CWD) in animal brain tissue seek to differentiate the normal prion protein PrPsen (the sen indicating protease sensitivity) from the abnormal, aggregated, and infectious form PrPres (res for protease resistance) by means of a proteinase K digestion step performed prior to conducting an ELISA or a Western blot analysis. The protease digestion step is used because the monoclonal antibodies employed in these tests are not specific for the abnormal form of the protein. All of the normal protein present in the sample—which substantially exceeds the amount of the abnormal form—must be removed prior to the ELISA by this method. The digestion process can be rather cumbersome and unreliable, however. False-positives resulting from underdigestion and false-negatives resulting from overdigestion can be a problem.

Figure 2. A comparison of diagnostic test results for BSE in bovine brain homogenates obtained with the Seprion affinity ligand-based microplate assay and with an EU-approved ELISA that uses proteinase K. The negative brain samples are clustered, with an optical density (OD) generally below 0.11. The lowest OD recorded with a positive sample in the Seprion assay was 0.46 (click to enlarge).

Obviously, this procedure could be simplified considerably if antibodies specific for the abnormal PrPres form of the prion protein were to be used. Antibodies having such properties have been reported.^5,6 However, they have not yet been incorporated in commercial kits.

The approach taken in the research reported in this article has been to investigate the use of synthetic ligands with prion protein–binding properties. The research aim was to establish whether a differential binding capability could be demonstrated. Such capability would allow selection between the two conformational variants of the protein.

It has been known for some time that certain anionic materials are useful in histochemistry applications. For example, the dyes Sirius red and Congo red are observed to accumulate in the regions of a TSE-infected brain where PrPres is present in large quantities. These dye compounds can inhibit prion propagation in both animals and cell culture, according to reports.^{7, 8} Recent unpublished work by the present investigators has shown that dyes of this type do not exhibit useful selectivity for PrPres in an in vitro diagnostic test format.

An alternative to the anionic dyes was sought. The investigators decided to screen polymeric/polyionic compounds in order to identify potential ligands with specificity for the native conformation of the rogue prion protein. It is now well established that the prion protein in its normal PrPsen form is a globular, alpha-helix-rich, membrane-bound protein of unknown function. When misfolded in the PrPres conformation, the protein is predominantly beta-sheet in structure, exhibiting a propensity to aggregate into oligomers and then into large insoluble aggregates. The researchers sought compounds that would bind directly to the abnormal conformation.

Earlier studies provided hints. Recombinant prion protein produced in Escherichia coli has been found to contain binding sites for heparan sulfate and related polyanions.⁹ Other polyanions (for example, pentosan polysulphate) and polycationic dendrimers reduce concentrations of PrPres produced by TSE-infected cell lines.^{10, 11} However, the nature of the interaction between the abnormal, aggregated form of the protein and these polyions is not yet known.

The research reported here led to identification of several polyionic compounds that, under the appropriate reaction conditions, are able to bind the PrPres present in BSE-, scrapie-, CWD-, and vCJD-infected brain tissue with high selectivity in the presence of a much larger quantity of PrPsen.^{12, 13} These prion-selective reagents have been named Seprion ligands (Microsens Biotechnologies; London). Owing to the lack of information on the detailed structure of the PrPres protein aggregates, the mechanism by which PrPres binds to the Seprion ligands is unclear. The optimal ligands were found to be those with a high molecular weight of more than 50,000 daltons. Their weight is presumed to facilitate multiple ionic interactions with the aggregated beta-sheet protein structure, which would probably have multiple repeating regions of positive or negative charge. The Seprion ligands are mixed-mode in character and predominantly polyionic; however, they are also able to bind strongly to solid-phase materials in a simple procedure compatible with large-scale manufacturing.

The original intention was to develop the Seprion affinity ligands as a diagnostic technology to help in the detection of vCJD in human blood samples and thus make possible a blood screening program. But early research and development work identified a group of these polymeric compounds that were able to bind abnormal prion protein with high selectivity in BSE- and scrapie-infected bovine and ovine brain tissue, so a new direction was followed. An assay procedure for brain homogenate that is much simpler than existing proteinase K digestion methods was developed.

Suitable for high-throughput automation on a standard ELISA processor, the assay involves an immunometric format. The Seprion ligand is immobilized directly to the surface of a 96-well microplate. The brain homogenate is then added to the well without any further processing, and the direct capture of PrPres by the affinity ligand is detected using a commercially available prion protein–specific antibody/peroxidase conjugate (see Figure 1).

Samples of BSE-positive bovine brain tissue from 35 infected animals and 12 noninfected BSE-negative brain material controls were supplied by the Veterinary Laboratories Agency (Weybridge, Surrey, UK), with positives confirmed by histopathology, and tested with the Seprion assay and an EU-approved ELISA. A comparison of results is graphed in Figure 2. The EU-approved procedure was carried out according to the manufacturer’s instructions and included a proteinase K digestion step followed by centrifugation and denaturation of the sample by boiling.

Because the tissue samples obtained from heavily infected animals were very heterogeneous, a formal correlation analysis of the data was not carried out. However, with the bovine brain samples, 100% correlation—defined as a positive or a negative sample with reference to the recommended cutoff for both methods—between the two methods was observed. This result was confirmed subsequently with more than 100 other samples (data publication is forthcoming).

Significant signal differences between the two methods were observed with some positive samples (for example, the outliers in Figure 2). They may be attributable to over- or underdigestion problems with the proteinase K step. These signal differences have not been sufficient to cause misclassification of an animal’s infection status, however.

The animal healthcare market has been the first to realize the potential of the Seprion affinity ligand technology, which has been licensed to IDEXX Laboratories Inc. (Westbrook, ME) and Sanko Junyaku Company, Ltd. (Tokyo). These companies will use Seprion initially as a key component in their next generation of diagnostics for the detection of TSEs in animal brain tissue. Those tests should be simpler, more sensitive, more reliable, and faster than existing products. IDEXX test kits have just been approved by the U.S. Department of Agriculture (USDA) for screening and CWD in the United States, and they are being evaluated by the European Food Standards Agency for use in detecting BSE.

At present, the annual market for postmortem BSE test kits, consisting primarily of Europe and Japan, exceeds $100 million. The United States, in spite of a cattle population of more than 90 million, does not conduct BSE tests in large numbers. However, given the recently announced first U.S. and Canadian case of BSE, along with the growing concern in those countries regarding CWD in deer, the level of postmortem testing for TSE diseases in North America can be expected to increase rapidly.

Blood Testing for the Prion Disease Agent

Unfortunately, efforts to prevent or mitigate the incidence of vCJD, BSE, and the other TSE diseases are now severely hampered by the fact that it is still not possible to detect these diseases in vivo. A necessary and telltale component of all the TSEs is the rogue prion protein; that is known. However, the only reliable ELISA tests for detecting these abnormal proteins have such limited sensitivity that they can be used only for confirmation of the disease in postmortem brain tissue samples, where there can be as many as 109 IU per gram of tissue. Rogue prion proteins in the blood of cattle infected with BSE and of humans with vCJD could be present in concentrations as low as 1 to 10 IU per milliliter, by contrast.

Culling suspect cattle may effect a partial solution to the problem of BSE, but that approach is hardly suitable for tackling vCJD. The long incubation period of these TSEs means that there are likely already a significant number of people in the United Kingdom who are infected but do not exhibit any symptoms. They would harbor the vCJD prions in body tissues, principally in blood, lymph nodes, and the spleen. Authorities admitted recently that the UK blood supply now includes donations from infected individuals, and that hospital and dental surgical instruments are also likely to be vCJD-contaminated after operations performed on people infected by this disease.¹⁴

Therefore, until highly sensitive tests are developed that can determine the presence—or confirm the absence—of BSE in living cattle and screen out infected blood from human blood banks, worldwide uncertainty regarding BSE and vCJD will continue.

Having improved the Seprion ligand technology so as to provide a more robust and potentially more sensitive test that can detect much lower concentrations of infectious particles in brain and other tissues, Microsens now is pursuing the near-term development of blood tests. Studies involving Seprion ligands coated onto magnetic microparticles are under way which focus on the capture of abnormal prion proteins from larger quantities of urine, milk, whole blood, and blood fractions. A major advantage of the microparticle format over the 96-well microplate is that much larger volumes of sample can be processed, thereby maximizing the likelihood of detecting the relatively few abnormal prion proteins present.

The first report of the detection of PrPres or, in this case, PrPsc in scrapie-infected sheep blood was presented recently.15 In the research, the Seprion ligand technology and magnetic particle capture format were used with a 10-ml blood sample. Attention was focused on the non–red cell cellular fraction where the abnormal prion protein may be present in a membrane-associated form. Further studies to adapt the Seprion assay technology for detection of vCJD in human blood are now being conducted.

Conclusion

Soon, perhaps within the next two years, the first diagnostic and screening blood tests for vCJD using the Seprion technology should be available. While concerns have been expressed regarding the ethics of identifying infected individuals when the means to treat them are lacking, a recent report of successful therapy by means of infusing pentosan polysulfate (a drug approved as a mild anticoagulant) directly into the brain suggests that the disease may now be treatable.16 Thus, large-scale screening for vCJD is now fully justified as a way to protect the blood supply. Such practice would make it possible for the first time to acquire a clear understanding of the scale of the danger to humans.

References

1. BJ Bennion and V Daggetta, “Protein Conformation and Diagnostic Tests: The Prion Protein,” Clinical Chemistry 48 (2002): 2105–2114.
2. S Dealler, “BSE/vCJD: A Calm in the Storm?” Microbiology Today 28 (2001): 228.
3. N Hunter et al., “Transmission of Prion Diseases by Blood Transfusion,” Journal of Genetic Virology 83 (2002): 2897–2905.
4. JN Huillard d’Aignaux, SN Cousens, and PG Smith, “Predictability of the UK Variant Creutzfeldt-Jakob Disease Epidemic,” Science 294 (2001): 1792–1931.
5. C Korth et al., “Prion (PrPsc)-Specific Epitope Defined by a Monoclonal Antibody,” Nature 390 (1997): 74–77.
6. E Paramithiotis et al., “A Prion Protein Epitope Selective for the Pathologically Misfolded Conformation,” Nature Medicine 9 (2003): 893–899.
7. R Demaimay, B Chesebro, and B Caughey, “Inhibition of Formation of Protease-Resistant Prion Protein by Trypan Blue, Sirius Red and Other Congo Red Analogs,” Archives of Virology. Supplement. 16 (2000): 277–283. 
8. L Ingrosso, A Ladogana, M Pocchiari, “Congo Red Prolongs the Incubation Period in Scrapie-Infected Hamsters,” Journal of Virology 69, vol. 1 (1995): 506–508.
9. RG Warner et al., “Identification of the Heparan Sulfate Binding Sites in the Cellular Prion Protein,” Journal of Biological Chemistry 277 (2002): 18421–18430.
10. S Supattapone et al., “Elimination of Prions by Branched Polyamines and Implications for Therapeutics,” Proceedings of the National Academy of Sciences USA 96 (1999): 14529–14534.
11. B Caughey and GJ Raymond, “Sulfated Polyanion Inhibition of Scrapie-Associated PrP Accumulation in Cultured Cells,” Journal of Virology 67 (1993): 643–650.
12. A Lane et al., “Polymeric Ligands with Specificity for Aggregated Prion Proteins,” Clinical Chemistry 49 (2003): 1774–1775.
13. AR Lane, CJ Stanley, and SM Wilson, “Binding of Pathological Forms of Prion Proteins,” PCT/GB03/00858, 2003.
14. CA Llewelyn et al., “Possible Transmission of Variant Creutzfeldt-Jakob Disease by Blood Transfusion,” Lancet 363, vol. 9407 (2004): 417–421.
15. J Oliver et al., “Application of the Seprion Ligand System for Detection of PrPsc in Ovine Blood” (poster presented at the International Prion Disease Conference, Munich, October 8–10, 2003).
16. S Dealler, N Rainov, and C Pomfret, “Variant CJD Case Treated by Intraventricular PPS: Symptomatic Improvements over the Following Six Months” (poster presented at the International Prion Disease Conference, Munich, October 8–10, 2003). 

Stuart Wilson, PhD, is the founder and scientific director of Microsens Biotechnologies (London). Amin Lane is a senior research scientist and Josephine Oliver a research scientist with Microsens. Christopher Stanley, PhD, is CEO of the company. The authors can be contacted at stuart.wilson@microsens.co.uk,  amin.lane@microsens.co.uk, josephine.oliver@microsens.co.uk, and chris.stanley@microsens.co.uk,  
respectively.

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