Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone,
Prion is by definition a “proteinaceous infectious particle,” responsible for transmissibility of a group of fatal neurodegenerative diseases that affect humans and many other mammals. The so-called protein-only hypothesis, which postulated that the aberrantly folded protein is able to infect and replicate, made prion diseases (at that time quite heretically) distinct from infections caused by microorganisms [
Prion (PrPSc) has an endogenous cellular counterpart, named prion protein (PrPC), which is expressed on the surface of various cell types, most abundantly in the central nervous system. PrPSc and PrPC share the same amino acid sequence, but differ substantially in the secondary, tertiary, and quaternary structures. PrPSc is believed to be acting like a mold for converting endogenous PrPC molecules into new prions. However, not only one, but several prion strains have been characterized so far, differing in their structure and biochemical characteristics [
In humans, the so-called transmissible spongiform encephalopathies (TSEs) or prion diseases have been known to either occur sporadically (sporadic Creutzfeldt-Jakob disease (sCJD)) or can be inherited (familial Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Sträussler-Scheinker syndrome). The transmissibility of these diseases was first demonstrated by Gajdusek et al., who successfully transmitted kuru to chimpanzees [
Due to the remarkable biochemical diversity among prions on one hand and the disturbing presence of PrPC on the other, as well as due to the absence of specific nucleic acids, TSEs testing has remained one of the biggest challenges of diagnostics until now.
One way to assess the problem is to search for surrogate markers. For antemortem diagnosis of CJD, different liquor proteins, such as 14-3-3, Tau, phospho-Tau, amyloid-
Another way to approach TSE diagnostics is to exploit the physicochemical differences between PrPC and PrPSc. Namely, PrPSc, being richer in beta sheet content, was found to be much more resistant to denaturation and proteolytic degradation than PrPC. Ever since, PrPSc has been detected either by immunohistochemistry (IHC) after special pretreatments of tissue slices, which destroyed relevant PrPC epitopes, or by western blotting of brain homogenates after degradation of PrPC by proteinase K (PK). Many other commercially available diagnostic immunoassays that have been developed still relay on PK digestion of PrPC. Contemporary options of discrimination between PrPC and PrPSc exploit the aggregation-prone nature of PrPSc molecules in confrontation with to the monomeric PrPC.
PrPSc-specific monoclonal antibodies (mAbs) have always represented an ideal approach for prion diagnostics development. However, with the knowledge of various infectious prion strains and fragments, the idea of producing one mAb that would detect them all appears less credible.
In the present paper we have reviewed immunoassays designed to detect pathological form of prion protein as a diagnostic or research tool, discussing their evolution, their advantages, and their weaknesses. Because of the abundance of PrPSc, brain tissue is the most common and reliable diagnostic material. Routine testing of brain tissue is a good way to identify and remove diseased animals from the food chain, and many important advances have been achieved in this area in recent years. Nevertheless, detection of prions at presymptomatic levels of the disease in samples other than brain is the ultimate goal for which researchers still strive.
Several types of ELISA or similar immunoassays have been developed for detection of PrPSc in brain tissue (Table
Summary of the methods for detection of PrPSc in brain tissue.
Reference | PrP source | PK | Denaturation | Antibodies | Detection method | Sensitivity* |
---|---|---|---|---|---|---|
[ |
rMoPrP, rOvPrP, rBoPrP, rHuPrP, and mice brain | − | − | C: 11G5 |
Sandwich ELISA | 6 ng of aggregated PrP |
[ |
BSE bovine brain and scrapie ovine brain | + | − | C: 6H4 |
Sandwich ELISA | 6 pg rPrP/well |
[ |
Scrapie sheep brain and tonsils, BSE bovine brain, and scrapie hamster brain | + | − | D: SAF70 |
ELISA | 3 ng rBoPrP |
[ |
sCJD human brain | + | − | C: 1E5 |
IPCR | n.r. |
[ |
Scrapie hamster brain | + | − | C: 8b4 or 7A12 |
IPCR |
|
[ |
BSE bovine brain | − | 0.1 M GdnSCN | C: 6H4 |
Sandwich ELISA | 1 ug PrPSc/mL |
[ |
BSE bovine brain | − | 1 M GdnHCl |
C: FH11 |
DELFIA | 36 pg PrP/well |
[ |
BSE bovine brain, CWD white-tailed deer, mule deer, and elk brains | + | 4 M GdnHCl | C: Fab D18 |
DELFIA | 1 ng rec |
[ |
Scrapie mouse and hamster brain | − | 8 M GdnHCl |
C: 11G5 |
Sandwich ELISA | 0.05–5 ng rHuPrP |
[ |
Scrapie sheep brain | − | 6 M GdnHCl | C: FH11 |
DELFIA | 200 pg rOvPrP/well |
[ |
vCJD human spleen and brain | − | 2 M GdnHCl |
C: FH11 |
DELFIA | 10 pg rHuPrP/mL |
[ |
BSE ovine brain and scrapie ovine brain | + | Heath | C: SAF34 |
Sandwich ELISA | n.r. |
[ |
Scrapie hamster and sheep brain, CWD-infected white-tailed deer brain | − | 1% SDS | C: 11F12 |
SOFIA | 10 ag rHaPrP, rMoPrP, rOvPrP, and rDePr |
[ |
TME hamster brain | + | 3 M GdnSCN | D: 3F4 |
ELISA | n.r. |
[ |
Paraffin-embedded scrapie ship, CWD white-tailed deer and TME cattle brains | − | Denaturation buffer (not specified) | HerdChek BSE-Scrapie Ag Test |
ELISA | n.r. |
[ |
CJD human brain | − | 3 M GdnSCN | C: V5B2 |
DELFIA | n.r. |
rHuPrP: recombinant human prion protein, rMoPrP: recombinant mouse prion protein, rOvPrP: recombinant ovine prion protein, rBoPrP: recombinant bovine prion protein, rDePrP: recombinant deer prion protein, rHaPrP: recombinant hamster prion protein.
ICSM is not an acronym but a name of two anti-prion antibodies (ICSM 35, ICSM 18).
Schematic representation of described PrPSc immunoassays. Dashed lines indicate optional steps of sample pretreatment.
During the transition from PrPC to PrPSc, and more importantly during the aggregation of PrPSc molecules, certain epitopes become inaccessible. Upon denaturation of PrPSc, immunoreactivity is greatly enhanced presumably because the structure of the aggregates loosens and buried epitopes become accessible again [
Denaturation has been used in numerous studies, in most cases with an important simplification of the original CDI method, although the main principle and the name of the method were retained [
In a different set of assays, denaturation step was employed for differential extraction of PrP [
An important issue of immunoassaying brains is the fact that different parts of brain may vary greatly in the abundance of PrPSc, which was shown for animal and also for human brain [
In sandwich ELISA capture, mAb is adsorbed to the bottom of the well and detector mAb is used to detect antigen bound to the capture Ab. This format requires two mAbs directed against two different epitopes on one antigen molecule. But in a case of aggregated proteins such as PrPSc, it is reasonable to assume that certain epitopes are represented more than once. This assumption is the basis of the so-called aggregation-specific ELISA (AS-ELISA) that detects only PrP aggregates in brain samples [
Ligands other than Abs can be used for the purpose of capturing PrP. Glycosaminoglycans (GAGs) that have been found to bind PrP in the cell [
The above-mentioned methods all rely on frozen tissues that are sometimes not available. As IHC is still the golden standard for definite diagnosis of TSE, much of the tissue taken for analysis is paraffin embedded. Because IHC is not a high-throughput method, protocols for detection of PrPSc from paraffin-embedded tissue by WB have been developed [
Sensitivity of an immunoassay depends not only on the sample preparation and treatment, but largely also on the detection system. The simplest and most easily accessible is the ELISA format where detection of PrP is achieved via anti-PrP mAb coupled directly or indirectly to an enzyme which produces visible signal after the addition of the substrate. In more sensitive DELFIA, anti-PrP antibody is labeled with lanthanide chelates, most commonly Europium, that emit stable fluorescent signal. DELFIA was used in a number of studies described in this review [
Yet another method that was proved to be more sensitive than WB and IHC is immuno-polymerase chain reaction (IPCR). Original protocol exploits the benefits of both specific antigen recognition in ELISA and exponential amplification of DNA in polymerase chain reaction (PCR) [
The reports of the development of PrPSc-specific mAb based immunoassay are very limited. Despite of the use of PrPSc—or aggregate-specific mAb—, these immunoassays are still based on denaturation or PK digestion of samples. The V5B2 mAb, first described by our group in 2004 [
All the above-mentioned methods were developed for analysis of human and animal brain tissues, and can thus be applied only for postmortem diagnostics. For an
Summary of the methods for detection of PrPSc in blood.
Reference | PrP source | PK | Denaturation | Antibodies | Detection method | Sensitivity* |
---|---|---|---|---|---|---|
[ |
Scrapie mice blood and CWD deer and elk blood | − | − | n.r. | FACCT | n.r. |
| ||||||
[ |
CJD human blood | + | − | C: 6H4 |
DELFIA | 50 ul recPrP/mL PK-digested plasma |
| ||||||
[ |
Scrapie sheep blood and CWD white-tailed deer blood | − | 1% SDS | C: 11F12 |
SOFIA | n.r. |
| ||||||
[ |
Healthy human blood spiked with vCJD brain | Thermolysin | 4 M GdnHCl | C: ICSM 10 |
Sandwich ELISA | 2.8 pg PrPSc/well |
| ||||||
[ |
vCJD human blood and healthy human blood spiked with vCJD brain | − | Heat | D: ICSM 18-biotin | ELISA | 1010-fold dilution of vCJD brain homogenate in whole blood |
rHuPrP: recombinant human prion protein, rMoPrP: recombinant mouse prion protein, rOvPrP: recombinant ovine prion protein, rBoPrP: recombinant bovine prion protein, rDePrP: recombinant deer prion protein, rHaPrP: recombinant hamster prion protein.
ICSM is not an acronym but a name of two anti-prion antibodies (ICSM 35, ICSM 18).
As stated before, the main problem of detecting prions in blood or plasma is the extremely small quantity of prions and a high background of other proteins and PrPC; therefore, extreme sensitivity and specificity is a necessity for a blood test. Apart from that, samples of prion infected blood are rare, limited, and only accessible to few laboratories. To overcome that problem, many test developers make use of spiking brain homogenates or PrPSc isolated from brain into the blood of healthy persons. This might not be the optimal solution of the problem since pathological PrP, if present in blood, not necessarily possesses the same characteristics as that of brain derived. Nevertheless, such studies are important as they represent an insight into the detection limits we are currently able to reach [
Instead of the immunoprecipitation, a precipitation on solid-state capture matrix can be performed [
Besides PrP precipitation, another way to approach to the sensitivity issue is
Two recent reports have shown the use of
The knowledge about prions that has accumulated in the last three decades and the use of routine testing of bovine brain for BSE had great impact on reducing the risk of prion transmission. However, for complete prevention on prion transmission through food, drugs, and blood-derived products, the sensitivity of the methods for prion detection must be greatly improved and designed for analyzing low-content prion material.
The latest advances in PrPSc immunoassaying set the course of development of testing in different directions, all headed for the same goal—the maximal sensitivity and specificity of the method.
Accumulating reports on PK-sensitive strains of prions have reflected unfavorably on the use of PK-based diagnostics and therefore in novel prion immunoassays, PK is being avoided.
For routine antemortem testing of potential TSE transmitters, a blood test would be most appropriate. In an effort to develop such a test, different obstacles need to be overcome. Firstly, testing systems, developed for brain tissue, cannot be transferred directly to blood because quantities of PrPSc in blood are much lower than in brain. Secondly, little is known about biophysical properties of PrPSc in blood which may differ from PrPSc in brain. Moreover, samples of infected human blood are limited in number and availability, which is an important drawback. However, a recent study by Edgeworth et al. shows that it is possible to detect prions in the blood of symptomatic vCJD patients [
A simple, inexpensive, high-throughput, and at the same time highly sensitive blood test for prions does not seem to be available in the near future. A more likely solution seems to be large-scale screening for TSE surrogate markers in combination with an extremely sensitive prion test applied only to the identified risk samples.
A. Lukan and T. Vranac contributed equally to this work.