1 Virology, Aventis Behring GmbH, PO Box 1230, 35002 Marburg, Germany
2 Industry and Health Policy, Aventis Behring SA, 46 Quai de la Rapée, 75601 Paris Cedex 12, France
Correspondence
Albrecht Gröner
albrecht.groener{at}aventis.com
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ABSTRACT |
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MAIN TEXT |
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Monoclonal antibody (mAb) 1120-64-9 and mAbs 1120-63-33 and 1120-2214 were produced as described (Peters et al., 1985): one 6-week-old BALB/c mouse was immunized with minute amounts of human PrPC immunopurified from brains of transgenic (tg) mice expressing human PrP by intraperitoneal and, before recovery of the spleen, by intravenous injections with a total of five boost injections several weeks apart. The resulting hybridoma cells were deposited at DSMZ (Germany) as ASM DCC 2523 (and ASM DCC 2522 and 2524, respectively).
A 10 % (w/v) vCJD brain homogenate, prepared in PBS, was diluted in human plasma in 0·5 log10 steps and Sarcosyl (20 %, w/v, stock solution in PBS) was added to a final concentration of 2 % and processed according to Safar et al. (1998). After incubation of the diluted samples at 37 °C for 15 min, phosphotungstic acid (PTA) was added to a final concentration of 0·32 % (w/v). Samples were then incubated at 37 °C and precipitates collected by centrifugation at 14 000 g for 30 min at room temperature. Pellets were further resuspended in H2O/0·2 % Sarcosyl and split into two aliquots: one aliquot was denatured by adding guanidinium hydrochloride (4 M final concentration) and heating at 83 °C for 6 min, while the other aliquot was left untreated. Both aliquots were diluted and adjusted to a final guanidinium concentration of 0·205 M. For the originally reported direct CDI, microtitre plates were pre-activated with glutaraldehyde. For the modified sandwich CDI, microtitre plates were coated with 10 µg mAb 1120-64-9 ml-1, which binds specifically to human PrP. Denatured and native aliquots were split and loaded in triplicate on to the plates. PrP bound either by glutaraldehyde or by mAb was detected using Europium-conjugated mAb 3F4 as described (Safar et al., 1998
) and TRF counts of the denatured aliquots were divided by the counts of corresponding native aliquots to give the d/n ratio. The cut-off value was calculated from the mean d/n ratio of at least 12 non-spiked plasma samples plus three times the standard deviation.
In the direct CDI procedure, binding of PrPSc to the plate was not very efficient and, as a result, higher than cut-off d/n ratios were recorded up to 10-2 dilutions. In the sandwich CDI, positive d/n ratios were recorded at dilutions higher than 10-4 (Fig. 1a) because the first antibody specifically enriched prion proteins on the plate. Pretreating the vCJD-spiked plasma samples with 250 µg proteinase K (PK) ml-1 for 60 min, followed by addition of 1 mM PMSF to stop digestion before addition of PTA, further increased the sensitivity of the sandwich CDI by greater than threefold (0·5 log10), because background signals resulting from co-precipitated PrPC were further eliminated by enzymatic digestion (Fig. 1a
). Thus, the sandwich CDI detected vCJD with 100-fold higher sensitivity in plasma without protease treatment than the direct CDI.
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In order to allow comparison of the sandwich CDI sensitivity with other established immunoassays, WHO CJD/vCJD reference samples were sequentially diluted in human plasma, treated with PK and analysed using the sandwich CDI. The WHO reference samples included one sporadic or sCJD type 1, one sCJD type 2 and one vCJD brain homogenate (Minor et al., 2000). The experiments were performed by three independent operators on two different days. Immunoreactivity endpoints were used to calculate the relative CDI titres ml-1 (CDI-Unit50 ml-1) of brain homogenate (based on an input of 167 µl brain homogenate per test), resulting in similar PK-resistant PrPSc concentrations for sCJD type I and the vCJD brains of 106·0 CDI-Unit50 ml-1 and 106·2 CDI-Unit50 ml-1, respectively (Table 1
). The sCJD type 2 sample showed 10-fold lower PrPSc concentrations (CDI titre 105·0 CDI-Unit50 ml-1). These results demonstrated that sandwich CDI was able to detect sCJD as well as vCJD. This study further showed that 1 nl of the 10 % vCJD and sCJD type 1 brain homogenates, respectively, was the minimum volume detectable in a single microtitre well by sandwich CDI.
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Despite reported evidence for the appearance of very low levels of prion infectivity in blood or plasma during the clinical phase of prion disease in experimental rodent models (Brown et al., 1998, 2001
; Cervenakova et al., 2001
) and preliminary data on transmission of prion disease by transfusion in sheep (Houston et al., 2000
; Hunter et al., 2002
), transmission of CJD/vCJD by transfusion has never been documented in humans. Epidemiological studies (Baron & Prusiner, 2000
) as well as failed attempts to transmit sporadic CJD prion disease to animals by blood transfusion (Brown et al., 1994
) suggest that prions either are not present in blood of sCJD patients or are there at levels that are insufficient to elicit prion infection following transfusion. However, with the occurrence of prions in lymphoid tissues of vCJD patients and the limited information on epidemiology of vCJD, the theoretical risk of vCJD transmission by blood or blood products, though never substantiated, is more difficult to rule out at this time. Although prions could not be detected in buffy coats or plasma from vCJD patients using PTA precipitation and immunoblotting (Wadsworth et al., 2001
) or infectivity bioassays (Bruce et al., 2001
), questions as to the sensitivity of the methods and the biophysical properties remain. The degree of PK resistance of the theoretical prion contaminant in blood or plasma could not be assessed when procedures such as PK pretreatment of samples were used in prion detection assays (Wadsworth et al., 2001
). The potential presence of a PK-sensitive, PrP-like protein in the urine of prion-infected animals, which becomes protease-resistant after dialysis, has been reported (Shaked et al., 2001
). This uPrP could be blood-derived and might only be identified using procedures without prior proteolysis of samples. Due to the sandwich CDI's ability to detect low levels of PrPSc in the presence of 1000 times higher concentrations of PrPC, even without the use of PK, such questions can now be addressed by evaluating tissues and body fluids of clinically affected vCJD patients for PrPSc distribution. Only if PrPSc can be reproducibly detected in blood, buffy coat or plasma by the sandwich CDI in persons who later develop (v)CJD could plasma screening be considered. Implementation of a plasma screening system requires a true positive control for validation of the assay. The closest substitute for a patient's blood might be endogenous infectivity in CJD-inoculated transgenic mice as described for human prions adapted to wild-type mice (Brown et al., 1998
). The CDI is currently being used to evaluate the prion clearance capacity of purification and concentration steps used in the manufacturing processes of plasma-derived products (Vey et al., 2002
).
In order to determine the human PrP epitope to which mAb 1120-64-9 binds, overlapping 15-mer peptides covering the PrP 90231 sequence were tested using this mAb in an ELISA-formatted assay with negative results (data not shown), indicating that the epitope is not a linear peptide sequence, although mAb 1120-64-9 can detect human prions in Western blots (data not shown). Furthermore, other covalent modifications present on native, mammalian-derived PrP, such as N-linked glycosylation or C-terminal GPI-anchor linkage, do not seem to be part of the epitope because human recombinant PrP 90231 was detected by mAb 1120-64-9 (data not shown). The only covalent modification that could be involved in antibody binding was the disulphide bridge connecting cysteine-179 with cysteine-214. Antibody binding to human PrP was tested in the absence or presence of the disulphide bridge by treatment of purified human PrPSc with 4 M guanidinium hydrochloride in the absence or presence of 3·3 mM DTT for 6 min, prior to binding it to a microtitre plate and detection with 1120-64-9 and a europium-labelled rabbit anti-mouse IgG antiserum. After reduction, parallel samples were treated with iodacetamide (10 mM final concentration) in order to avoid reoxidation of the disulphide bridge. DTT-treated PrP resulted in a low 1120-64-9 binding signal, in contrast to untreated PrP (Fig. 2). Furthermore, efficient binding of 1120-64-9 to PrP could be restored after reoxidation of reduced samples (not treated with iodacetamide) using oxidized glutathione or incubation at room temperature. From these experiments, we concluded that the epitope to which mAb 1120-64-9 binds depends on a correctly formed disulphide bridge. Previous studies have characterized linear and conformational epitopes on prion protein (Li et al., 2000
) but this is the first time that an epitope has been linked to the presence of the disulphide bridge, suggesting that the disulphide group itself could be an integral part of the peptide sequence or could form a conformational epitope by holding together otherwise distantly located amino acid groups.
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ACKNOWLEDGEMENTS |
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Received 21 November 2002;
accepted 6 March 2003.