title / Antibodies specific for the abnormal isoform of the prion protein; their application to TSE diagnostic tests.
/ DEFRA
project code / SE1776
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Antibodies specific for the abnormal isoform of the prion protein; their application to TSE diagnostic tests.
DEFRA project code / SE1776
Contractor organisation and location / The Veterinary Laboratories Agency,
Woodham Lane,
New Haw, Addlestone
Surrey
Total DEFRA project costs / £ 323,303
Project start date / 01/08/02 / Project end date / 31/07/04
Executive summary (maximum 2 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (Rev. 6/02) 3
Projecttitle / Antibodies specific for the abnormal isoform of the prion protein; their application to TSE diagnostic tests.
/ DEFRA
project code / SE1776
The most effective marker of TSE disease diagnosis is the abnormal conformer of the host prion protein, PrPsc. Commonly, its relative resistance to digestion by proteinase K is used to distinguish this mis-folded, disease-associated protein from the normal cellular form of the protein in diagnostic tests.
Until recently, no diagnostic antibody or ligand, had been produced that could differentiate between the disease-associated abnormal prion protein (PrPsc) and its normal, cellular form (PrPc). The lack of appropriate reagents restricted the sensitivity and specificity of tests resulting in their only being applied post mortem to central nervous system tissue and requiring protease treatment of the sample to remove interfering PrPc. The development, production and evaluation of antibodies, along with other ligands, at IDEXX Laboratories Inc. have major TSE diagnostic implications, which this project aimed to exploit.
The development of rapid and reliable methods for screening of post-mortem samples to identify TSE-infected carcasses, thus preventing spread and entry into the food chain, remains of great importance. This project describes the development of a rapid post-mortem diagnostic that is simple in its sample preparation, high in sensitivity, and easily adaptable to automated platforms.
Originally, the primary targets for a PrPsc-specific binding reagent were the antibodies developed by N.Cashman at Caprion Pharmaceuticals Inc. The common factor between all the antibodies produced is that they are raised to a tyrosine-tyrosine-arginine (YYR) epitope, although this epitope is expressed in varying forms for immunisation. Intensive studies were undertaken in order to determine their ability to recognise abnormal PrP from various species. Antibody recognition was assessed via three techniques, immunocapture of brain material using antibody coated magnetic beads, capture of mouse PrPsc in a 96-well bead format and mouse PrPsc immobilisation in an EIA format. The resultant data showed that even with the same immunogen differences exist in the resultant antibody properties with the comprehensive immunisation / testing undertaken has led to numerous examples of species differentiation.
Having undertaken exhaustive testing of the antibodies and test formats, it was concluded that, despite obvious species differentiation, a highly sensitive assay could be developed for the detection of the abnormal prion in murine samples only. No such test could be produced, within the permitted timescale, for BSE, scrapie or chronic wasting disease (CWD) using the YYR antibodies raised to date. Whilst it remains a genuine possibility that these antibodies could, with more time, be used in the development of a post-mortem assay and may yet be suitable for the ante-mortem test, the decision was taken to focus, for the immediate future, on a novel PrPsc-extraction reagent (SEPRION) developed by Dr Stuart Wilson of Microsens Biotechnologies as an alternative capture ligand. Indications of PrPsc–specificity using SEPRION immobilised on paramagnetic beads had previously been confirmed at the VLA under project SE1762. Considerable testing and development, performed by IDEXX, has realized the potential of the synthetic ligand, SEPRION, for the selective capture of PrPsc even in the presence of excess PrPc.
Using this synthetic ligand, a simple test system that eliminates the requirement for proteinase K and uses a PrPsc–selective capture system in a microtitre plate-format has been developed by IDEXX Laboratories as part of this project. Sample preparation, for the assay developed, is limited to homogenisation of tissue and the addition of diluent, with no other processing required. The PrPsc is captured onto SEPRION-coated wells of a microtitre plate and then detected in traditional manner by a peroxidase-labelled anti-PrP antibody conjugate.
The test system has been evaluated on characterized bovine and ovine brains, and cervine lymph nodes. To date, specificity is 100% in all species and tissues tested. Sensitivity is >99% for non-obex bovine tissue (versus immunohistochemical (IHC) localization of PrPsc in the corresponding obex) and 100% for the limited number of IHC-positive cervine lymph nodes. A 100% correlation was also obtained between the IDEXX test and a commercial, EU-evaluated BSE ELISA kit used on a population of bovine brainstem samples. Further field trials and evaluations are underway.
The SEPRION-based test developed by IDEXX is a post-mortem test and studies are currently being directed towards the development of an ante-mortem assay. Although not proven, it is assumed that the abnormal prion protein will be found in a less-aggregated state in body fluids suitable for such a test. With this in mind, much work has been undertaken at the VLA in preparing a “less-aggregated” spiking reagent that could be used in the study of body fluids. It remains uncertain, however, whether a format similar to that used in the post mortem assay could be used for the analysis of body fluids as it remains a genuine possibility that the specificity demonstrated by SEPRION is a result of its recognition of prion aggregates, rather than an absolute specificity for the PrPsc molecule itself.
Development of a “less-aggregated” spiking reagent, believed to resemble more closely that present in body fluids, was undertaken using a 55,000g centrifugation step. Purity and stability of the resultant reagent was assessed, with western blot analysis demonstrating the presence of a less-aggregated abnormal prion protein, that could be stored at –80oC for in excess of 3 months. The preparation could be immunoprecipitated using both the YYR antibodies and SEPRION. Equivalent preparations from TSE-Negative brain contained no detectable prion protein molecules. The development of this spiking reagent will be of enormous benefit in controlling development of rapid body fluid assays, for which naturally incurred samples are not always readily available.
CSG 15 (Rev. 6/02) 3
Projecttitle / Antibodies specific for the abnormal isoform of the prion protein; their application to TSE diagnostic tests.
/ DEFRA
project code / SE1776
Scientific report (maximum 20 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (Rev. 6/02) 3
Projecttitle / Antibodies specific for the abnormal isoform of the prion protein; their application to TSE diagnostic tests.
/ DEFRA
project code / SE1776
Objective 1 was to complete screening, identification and production of candidate antibodies for use in a post-mortem and subsequently an ante-mortem test. The common factor between all the antibodies produced is that they are raised to a tyrosine-tyrosine-arginine (YYR) epitope, although this epitope is expressed in varying forms for immunisation. Table 1 summarises the exhaustive evaluations carried out on a series of monoclonal antibodies arising from immunisations based on the YYR target recognition structure.
The basic method used for the evaluation was immunocapture (or immunoprecipitation). Candidate antibodies were immobilised on paramagnetic beads (eg Dynabeads) and the coated beads mixed with homogenates of brain material derived from scrapie-infected mouse, hamster or sheep, BSE-infected cattle or CJD-infected human. Appropriate TSE-negative species controls provided disease-free comparisons. Immunocaptured abnormal PrP was eluted and analysed by traditional PAGE / Western immunoblotting for proteinase K resistant prion protein. Subsequently, two different 96 well capture formats were developed to speed up the screening process, one in which the use of the magnetic beads was retained but the analysis for PrPsc was accomplished by second antibody detection in an microtitration plate format. The second screening process was more akin to a traditional ELISA with the antibody / ligand under evaluation directly coated to 96-well plates. The 96-well procedures were used to screen ligands against murine TSE-infected brain only.
Performance of the antibodies was scored as negative (-), weak positive (w+), positive (+) or strongly positive (++). Selection on this basis allowed a number of antibodies to be targeted at assay format development. In particular, those denoted in green and yellow in the tables below were the focus of further study.
Table 1 shows that even with the same immunogen differences exist in the resultant antibody properties. 9A4 and 12B1 for example show comparable recognition of mouse, sheep, hamster and cattle PrPsc whereas 12A12 fails to recognise hamster PrPsc and only weakly recognises the ovine form. The comprehensive immunisation / testing undertaken has led to numerous examples of such species differentiation.
Table 1: Performance of YYR monoclonal antibodies
CONFIDENTIAL
Monoclonal AntibodiesFusion/subclone / Immunocapture (brain) / Mouse-PrPsc capture
Mouse / hamster / Sheep / Bovine / Human / Bead 96well / EIA Plate
KLH (YYRRYYRYY)
3A1 / W+
3A3 / -
3A4 / W+
3A6 / W+
7A1 / W+
7A2 / -
8A.16 / - / - / -
8A.5 / ++ / ++ / +/- / -/W+ / -
16A.18 / ++ / ++ / + / + / + / ++ / -
20A.13 / + / + / W+ / -/W+ / -
9M1 (YYRRYYRYY)
1A7 / ++ / + / - / W+ / W+
3A2 / W+ / ++ / - / W+
3B2 / W+ / - / -
3C2 / W+ / W+ / -
4A1 / -
4A9 / -
7A9 / W+ / -
8A4 / W+ / +/-
9A4 / ++ / ++ / ++ / ++ / ++ / +
10A10 / -
10A2 / -
10A8 / -
12A5 / ++ / ++ / + / + / +
12B1 / ++ / ++ / ++ / ++ / ++ / +
9M2 (YYRRYYRYY)
3B1 / W+ / - / -
4A3
5C.14.8
6C4.12 / W+ / - / -
12A12 / ++ / W+ / + / -
14A1 / W+ / W+ / -
15A1 / W+ / - / -
16A14 / + / - / -
9B1 / - / - / -
1B1 bo(158-183)
3A15 / - / - / -
3A19 / W+ / - / W+
13A6 / W+ / W+ / + / +
13A4 / W+ / W+ / + / +
1B2 Cbo(158-183)
2A1 / W+
2A2 / W+
2A4.2 / -
2A4.4 / -
2A4.7 / -
2A5 / W+
14A1 / W+
14A3 / W+
14A4 / W+
14A5 / W+
15A1 / -
15A6 / -
17A1 / -
17A2 / -
17A5 / -
17B1 / ++ / ++ / + / W+ / ++ / ++
17B4 / W+
1B3 Cbo(158-183)
23A1 / -
23A18 / ++ / - / -
23C3 / -
25A2 / ++ / W+ / W+
25B1 / + / - / -
25B2 / -
27A3 / W+
27C1 / ++ / ++ / W+ / W+ / ++ / ++
28A1 / -
28A2 / -
28B1 / ++
28B2 / ++
29B5.1 / -
29B5.2 / -
29B9 / W+ / - / - / W+
31A1 / +
31A4 / ++ / - / +
31A8 / ++
32B1 / -
32B2 / W+
34A3 / ++ / ++ / + / + / + / +
34A8.1 / +
34A8.2 / ++
34A8.3 / -
39A1 / W+ / - / -
39A3.1 / W+
39A3.12 / W+
39A3.14 / -
40A8 / + / +/- / -
4M1 (4map-YYR)
1A20 / - / - / -
2A6 / - / - / -
9A20 / + / - / -
12A1 / + / - / +
12B5 / + / W+ / + / +
DM1 (YYR dimer)
26A4 / - / - / -
28A2 / - / - / -
34A13 / W+ / - / -
35A13 / W+ / - / -
1S2 C(VYYRP)K
3C1
4C4
4C2
4S2 C(YYRPMD)K
22A2 / + / -
25C1 / + / -
37A5 / + / -
37A1 / + / +
W+= weak positive
Objective 2 aimed to confirm PrPsc specificity and select antibodies for test development. All the antibodies produced underwent substantial testing, with specificity for the abnormal prion being universally confirmed. Figure 1 shows, as an example, how 3 of these antibodies behaved when assayed on normal and scrapie mouse brain. The specificity of these antibodies for the abnormal isoform can be shown clearly with the isotype matched control (4E4) showing no PrP recognition.
Figure 1: Bead assay: PrPsc capture with YYR antibodies 16A18, 17B1 or 27C1 (4E4 isotype
negative control) and detection with 1B2 and 1B3 monoclonal antibodies