Fast and ultrasensitive method for quantitating prion infectivity titre

Abstract

Bioassay by end-point dilution has been used for decades for routine determination of prion infectivity titre. Here we show that the new protein misfolding cyclic amplification with beads (PMCAb) technique can be used to estimate titres of the infection-specific forms of the prion protein with a higher level of precision and in 3-6 days as opposed to 2 years, when compared with the bioassay. For two hamster strains, 263 K and SSLOW, the median reactive doses determined by PCMAb (PMCAb₅₀ ) were found to be 10^12.8 and 10^12.2 per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding median infectious dose (ID₅₀ ) values measured by bioassay. The 10² - to 10³ -fold differences between ID₅₀ and PMCAb₅₀ values could be due to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID₅₀ and PMCAb₅₀ could be due to higher rate of clearance of infection-specific prion protein seeds in animals versus PMCAb reactions. A well-calibrated PMCAb reaction can be an efficient and cost-effective method for the estimation of infection-specific prion protein titre.

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Received 5 Aug 2011 | Accepted 6 Feb 2012 | Published 13 Mar 2012 DOI: 10.1038/ncomms1730

Natallia Makarava1,2, Regina Savtchenko1,2, Irina Alexeeva3, Robert G. Rohwer3,4 & Ilia V. Baskakov1,2

Bioassay by end-point dilution has been used for decades for routine determination of prion infectivity titre. Here we show that the new protein misfolding cyclic amplication with beads (PMCAb) technique can be used to estimate titres of the infection-specic forms of the prion protein with a higher level of precision and in 36 days as opposed to 2 years, when compared with the bioassay. For two hamster strains, 263 K and SSLOW, the median reactive doses determined by PCMAb (PMCAb50) were found to be 1012.8 and 1012.2 per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding median infectious dose (ID50)

values measured by bioassay. The 102- to 103-fold differences between ID50 and PMCAb50 values could be due to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID50 and PMCAb50 could be due to higher rate of clearance of infection-specic prion protein seeds in animals versus PMCAb reactions.

A well-calibrated PMCAb reaction can be an efcient and cost-effective method for the estimation of infection-specic prion protein titre.

Fast and ultrasensitive method for quantitating prion infectivity titre

1 Center for Biomedical Engineering and Technology, University of Maryland, 725 W. Lombard Street, Baltimore 21201, USA. 2 Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore 21201, USA. 3 Medical Research Service, Veterans Affairs Maryland Health Care System, Baltimore 21201, USA. 4 Department of Neurology, University of Maryland School of Medicine, Baltimore 21201, USA. Correspondence and requests for materials should be addressed to I.V.B. (email: [email protected]).

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A

pathognomonic hallmark of the prion diseases is the accumulation of the misfolded isoform of the prion protein (PrPSc). The traditional method for obtaining a quantitative estimate of prion infectivity is end-point dilution titration in animals. A suspension of the tissue or uid of interest is diluted in tenfold serial steps, and then each dilution is inoculated into a group of animals. A dilution at which only a fraction of the inoculated animals develops clinical signs of disease or shows positive evidence of PrPSc on immunoassay is called a limiting dilution. At limiting dilution there are only one or a few infectious doses per inoculation volume. End-point dilution titres are typically expressed as the median infective dose (ID50): the reciprocal of the dilution required to infect only 50% of the animals inoculated as determined by interpolation or other statistical methods. While the end-point bioassay has been the principal method for determining prion infectivity, the assay is extremely long, expensive and laborious. Moreover, the bio-assay works optimally only for prion strains with incubation times well within the lifespan of the host.

Alternatives to end-point titration are biochemical or immuno-chemical assays that assess either the presence, mass or concentration of PrPSc (refs 14). However, establishing accurate quantitative relationships between PrPSc concentration and prion titre has proven to be difficult because of the size heterogeneity of prion particles and uncertainty over whether all prion particles are equally infectious57. Moreover, the size distribution and physical properties of prion particles appear to vary with agent strain and host species6. In 2003, Weissmann and co-workers8 introduced a scrapie cell assay that quantitatively estimates prion infectivity titres within a much shorter time frame than animal bioassay. The scrapie cell assay was shown to be capable of detecting prions in a dilution as low as 1010-fold of scrapie-infected brain material9. Moreover, in recent studies, the scrapie cell assay was adapted for detecting prions from various species10.

Protein misfolding cyclic amplication (PMCA) propagates PrPSc and infectivity in vitro1114. The sensitivity of the PMCA reaction to detect prion particles exceeds that of the bioassay15,16. While PMCA has been proven for detecting and amplifying prions from a broad range of species including human, cow, sheep, cervids, mouse, hamster and others1626, highly robust PMCA amplication has been limited to mostly rodent and rodent-adapted strains. Nevertheless, the efficiency of prion replication in PMCA was shown to mimic cross-species transmission barriers2729 or genetic susceptibility of sheep to scrapie that occurs due to prion protein polymorphisms19. The improvements in the PMCA assay found in PMCA with beads (PMCAb) have resulted in a much faster, more robust, sensitive and cost-efficient way of measuring PrPSc compared with either PMCA or bioassay29,30.

To illustrate the advantages of PMCAb-based end-point titration, we assessed the relative concentrations of PrPSc in brain material of two rodent strains, 263K and SSLOW, which display very short or very long incubation times to symptomatic disease, respectively31. We show that using the PMCAb format PrPSc titration can be performed in only a few days. We also observed strain-specic dierences in the specic infectivity relative to PMCAb-based PrPSc titre ranging from hundreds to thousands of PMCAb-active particles per intracranial infectious dose 50 (ic ID50).

ResultsEnd-point dilution titration using PMCAb. The PMCAb amplication was initiated by seeding a substrate of uninfected brain homogenate (BH) with PrPSc. To determine the concentration of PMCAb seeds, 10% BHs prepared from 263K- or SSLOW-infected animals were diluted in tenfold serial steps, then aliquots from each dilution were used to seed serial PMCAb reactions. Up to ten independent serial PMCAb reactions were conducted for each dilution for each strain. Each PMCAb round consisted of 48 cycles, 30 min each. Three or six serial PMCAb rounds were sufficient

for amplication of even the highest dilutions of 263K or SSLOW, respectively (Fig. 1a,b), to the level detectible by western blot. An increase in the number of PMCAb rounds did not increase the percentage of positive reactions for the most highly diluted samples illustrating that the limiting dilution was reached (Fig. 1c).

In our experience, PMCAb displays a high level of selectivity for infection-specic forms of PrP. To date, we have detected no false-positive amplications in the routine control reactions that are included with every PMCAb titration experiment. These controls consist of six serial rounds of 48 cycles of PMCAb of unseeded substrate only. Other controls include six rounds of serial PMCAb of 10% normal BHs from 700-day-old hamsters (Fig. 2), and PMCAb amplications of hamster substrate seeded with full-length recombinant hamster PrP amyloid brils prepared under a variety of previously described experimental conditions and protocols3235. No positive signals were detected in any of the controls (Fig. 2).

End-point dilution titration in animals. In parallel to PMCAb titration, the infectivity titres of brains from 263K- and SSLOW-infected animals were measured using end-point dilution bioassay. Animals were considered infected if they develop symptomatic disease or if their brains contained PrPSc, as judged by western blot even without symptomatic disease. The fractions of animals infected at each dilution are presented in Table 1.

SSLOW

263K

kDa

10

11

Log10[dilution fold]

Log10[dilution fold]

11

11

11

12

12

12

12

13

13

13

14

9

35

25

PK

kDa

+ + + + + + + + + + + + + +

+ + + + + + + + + + + + + +

+

11

12

13

14

15

11

12

13

14

15

11

12

13

14

35

25

PK

263K

PMCAb: Round #2 Round #3 Round #6

Log10[dilut. fold]

12

13

14

15

12

13

14

15

12

13

14

15

35

25

PK

+

+

+

+ + + + + + + +

Figure 1 | End-point sPMCAb titration. 10% scrapie brain material from SSLOW- (a) or 263K-infected animals (b,c) were diluted in tenfold serial steps, then aliquots from each dilution were used to seed serial PMCAb reactions. Example of a western blot of serial PMCAb (sPMCAb) products amplied for six rounds is shown in plots a and b. Panel c shows products of PMCAb reactions seeded with four dilutions and amplied for 2, 3 or 6 serial rounds. The dilutions shown for PMCAb reactions were normalized per gram of brain tissue, to correct for differences in assay volumes used for PMCAb (100 l) and bioassay (50 l).

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kDa

35

25

PK + + + + + + + + + + + + + +

% Of positive PMCAb reactions or positive animals

No seeds NBH rPrP fibrils

SSLOW 1011

1 2 3 4 5 6 7 9

8 10 11 12 13 14

6 7 10 11 12 13 14 15

9

8

Figure 2 | Testing the specicity of PrPSc detection in sPMCAb. sPMCAb reactions were seeded with 10% NBH from 700-day-old Syrian hamsters (lanes 5 and 6), recombinant PrP (rPrP) amyloid brils type I not subjected to annealing (lane 7); brils type I subjected to annealing in Triton (lane 8), BSA (lane 9) or NBH (lanes 10, 11 and 12); with rPrP amyloid brils type II (lane 13); or 1011-fold diluted SSLOW brain material (lane14). Four non-seeded reactions are shown in lanes 1, 2, 3 and 4. Six rounds of sPMCAb were conducted for each seeded condition and non-seeded reaction. The nal concentration of rPrP amyloid brils in serial PMCAb reactions was10 g ml 1. rPrP brils were prepared using Syrian Hamster full-length rPrP; brils type I were prepared in 2M guanidine hydrochloride (GdnHCl) as described by Bocharova et al.32; brils type II were prepared in 0.5 M GdnHCl as described by Sun et al.34 rPrP brils type I were subjected to annealing in 1% Tritorn X-100, 5 mg ml 1 BSA or 5% NBH as describedby Bocharova et al.33 and Makarava et al. 31. After six sPMCAb rounds, all samples were treated with PK as described in Methods; western blots were stained with 3F4. To rigorously test the specicity of sPMCAb for detecting PrPSc, multiple experiments were performed using four Misonix S-4000 sonicators over a 2-year period. Several independently prepared stocks of rPrP amyloid brils of each type were produced using rPrP from several independent purications. All results on sPMCAb seeding by rPrP brils were negative, conrming that sPMCAb is highly specic for PrPSc.

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

Log10 [dilution fold]

% Of positive PMCAb reactions or positive animals

% Of positive PMCAb reactionsor positive animals

SSLOW

263K

6 7 10 11 12 13 14 15

9

8

Log10 [dilution fold]

Log10 [dilution fold]

Table 1 | Summary of end-point titration of 263K and SSLOW.

Dilution of brain material Number of animals infecteda/total number

263K SSLOW

101 8/8 102 8/8 103 8/8 104 8/8 105 12/12 5/5 106 12/12 8/8 107 11/11 5/8 108 55/59 2/8 109 107/153 0/8 1010 2/12 0/6 1011 0/12 0/8

aAnimals were considered infected if they were symptomatic or positive for PrPSc by western

blotting.

6 7 10 11 12 13 14 15

9

8

Figure 3 | Correlation of prion infectivity titre by end-point titration bioassay with PMCAb activity. Brain homogenate materials from Syrian Hamsters infected with SSLOW (large green symbols) or 263K (smallred symbols) were subjected to ten-fold serial dilution, then each dilution was analysed by animal bioassay or serial PMCAb. Percentage of animals infected (squares) or giving positive PMCAb reactions (triangles) is plotted as a function of dilution. (a) Analysis of the data using the Poisson function, where the solid curves represent the results of nonlinear least-squarebest t. Analysis of the data for SSLOW (b) or 263K (c) using a sigmoidal function. The solid curves represent the results of a nonlinear least-squares best t and the blue curves represent 95% condence intervals. ID50 and

PMCAb50 values were calculated from the results of tting.

Analysis of prion titre. For each dilution, the fraction of animals infected or the fraction of PMCAb reactions with a positive signal on western blot was plotted against the logarithm of dilution for both 263K and SSLOW (Fig. 3). At limiting dilution, PrPSc particles must necessarily assort randomly into the reaction aliquots, and their distribution is described by the Poisson equation. While there was a good t to the Poisson equation by the animal infectivity data for both 263K and SSLOW, the PMCAb titration curves for both strains showed a more gradual slope than predicted by the Poisson equation (Fig. 3a). This is consistent with increasing reaction efficiency at higher dilutions. One possibility is that dilution results in a concentration-dependent dissociation of aggregates, thereby releasing and increasing the concentration of PMCAb reactive centres.

Alternatively, dilution might diminish the eects of an inhibitor. As the eect is at very high dilutions, the inhibitor alternative seems less likely.

An arbitrary sigmoidal function and nonlinear regression analysis were used to calculate ID50 or PMCAb50 values from bioassayor PMCAb-based end-point curves, respectively (Fig. 3b,c, Table 2).

(Note that the Poisson equation is a special instance of the more generalized sigmoidal function used.) The infectivity titres determined in this way were almost identical to those determined by the Poisson equation or by more traditional application of the Reed and Meunch and Spearman and Karber methods (Table 2). Analogous to the bioassay, a PMCAb50 is the reciprocal of the concentration at which only 50% of the PMCAb reactions were positive. As judged

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Table 2 | ID50 values measured by end-point bioassay or PMCAb.

Assay50 units per gram of tissue Ratio Bioassaya ID50 Bioassayb ID50 PMCAbb PMCAb50 bPMCAb50/bID50 263K 1010.5 1010.6 1012.8 160

SSLOW 108.7 108.6 1012.2 4000

aThe ID50 values were estimated using the Poisson equation.

bThe ID50 values were estimated using the sigmoidal equation.

from ID50 and PMCAb50 values, PMCAb was more sensitive than bioassay by ~4,000-fold for SSLOW and ~160-fold in the case of 263K. Importantly, PMCAb titration was completed in a few days, whereas the bioassay required nearly 2 years.

Discussion

We show here that PMCAb, when well calibrated against bioassay, can be used to obtain an estimate of PrPSc titre in only 36 days, ~100 times faster than the bioassay. At the same time, PMCAb oers ethical advantages of reducing the need for animal use. The precision of the measurements in PMCAb is limited only by the number of replicates performed. The new PMCAb-based assay can be used as a fast, efficient and ultrasensitive method for determining PrPSc titre and is uniquely benecial for samples that have extremely low levels of infectivity and for determining infectivity concentrations for prion strains with long incubation times. On the other hand, while PMCA has been used for amplication of PrPSc from a wide range of species16,1825, the most robust amplication was achieved, so far, for a limited number of rodent or rodent-adapted strains. Further technical improvements are needed for PMCA to be used as a platform for quantitative estimates of PrPSc from a broad range of species.

In recent studies, Soto and co-workers16 estimated the amount of PrPSc based on the number of PMCA rounds necessary to amplify prions to a detectible level. While PMCAb and PMCA methods have similar sensitivities for detection of 263K, PMCA requires 18 days to reach sensitivity comparable with that achieved by PMCAb in 3 days. Furthermore, PMCA did not work well for amplication of SSLOW PrPSc (ref. 29). PMCAb is also far less sensitive to inhibitors and small variations in sonication conditions that have plagued conventional PMCA.

It is of interest to consider the molecular relationship of PMCAb titre to prion infectivity. A PMCAb50 titre represents the number of

PMCAb-active, PrPSc-particles capable of initiating PMCAb amplication per gram of material sampled. Both PMCAb amplication products and PrPSc are heterogeneous in at least size, and perhaps structure, and exactly which structures represent infectivity is not yet clear. Furthermore, contradictory data exist regarding the specic infectivity of PMCA-generated versus that of brain-derived PrPSc (refs 36,37).

Close comparison of the two sets of data, obtained from the end-point dilution titration on animals and PMCAb, can be used to establish a quantitative relationship. The ratio of PMCAb50/ID50

is the number of PMCAb-active particles that corresponds to one prion infectious dose or unit. The PMCAb50/ID50 ratio was 160 and 4,000 particles per one infectious dose for 263K and SSLOW, respectively (Table 2). There are several ways to explain the 102-to 103-fold excess in the number of PMCAb-active particles over the number of the infectious doses. The large PMCAb50/ID50

ratio could be interpreted as only a few or one out of each 160 or 4,000 PrPSc particles being truly infectious, whereas the majority of particles while detectible and ampliable by PMCAb have little or no infectivity. Consistent with this interpretation, recent studies reported a dissociation between PMCA seeding ability and biological infectivity for PrPSc seeds produced from PrP constructs with deletions of polybasic domains38. Furthermore, the studies by

Klingeborn et al.36 showed that PrPSc generated in PMCA had considerably lower infectivity titre than that of brain-derived PrPSc (ref. 36). This work suggested that two competitive PMCA pathways that amplify infectious PrPSc and non-infectious particles exist. In contrast, the work by Shikiya and Bartz37 reported that PMCA- generated PrPSc had a titre similar to that of brain-derived PrPSc. Further studies are needed to clarify these contradictory results.

Alternatively, the dierences between ID50 and PMCAb50 values might reect substantial dierences in obstacles to successfully initiate prion infection in an animal versus PMCAb reaction. It has been reported that prions inoculated into animals are subject to intensive proteolytic degradation and clearance39. In contrast, the PMCAb amplication is conducted in the presence of protease inhibitors to prevent degradation of the normal prion protein substrate for conversion. It is conceivable that this environment also preserves a greater fraction of infectious PMCAb-active particles, thereby accounting for the ~102- to 103-fold greater sensitivity of the PMCAb assay.

Strain 263K (short incubation, short clinical duration) and SSLOW (very long incubation and clinical duration31) represent the two extremes of prion hamster disease. Nevertheless, the concentration of PMCAb-reactive particles was similar in brains of animals infected with 263K or SSLOW (Table 2). No PMCAb-reactive particles were found in brains from aged animals, conrming that these particles are disease-specic. In contrast to having similar PMCAb50

values, the infectivity titre was ~100-fold lower for SSLOW than for 263K (Table 2). The PMCAb50/ID50 ratio was 160 and 4,000 particles for 263K and SSLOW, respectively (Table 2). If the hypothesis that

PMCAb amplies infectious and non-infectious prion protein seeds is correct, then PMCAb50/ID50 value reects a strain-specic ratio of non-infectious, disease-associated PMCAb-reactive particles per infectious PrPSc seeds. Alternatively, the dierence in the PMCAb50/

ID50 ratio could also reect strain-specic dierences in the amount of PMCAb-active particles required to infect an animal. The difference in efficiency of infection between the two strains could be attributed to several factors. First, the dierences could be due to the dierences in strain-specic rates of PrPSc clearance on inoculation. Second, the PrPSc species that is the most reactive in PMCAb might be not the one that is the most toxic. In fact, previous studies pointed to an uncoupling of prion infectious titre and neurotoxicity40. While PMCAb presumably counts PrPSc particles, the read-out parameter for bioassay is the presence of clinical or subclinical disease, which results from an accumulation of neurotoxic PrP species. Therefore, if infectious and neurotoxic PrP species are two dierent entities, the dierences in PMCAb50/ID50 value could reect a complex strain-specic relationship between a PMCAb-active PrPSc species and a neurotoxic species.

The strain-specic dierence in the PMCAb50/ID50 ratio was mainly due to the substantially lower ID50 titre of SSLOW in comparison with that of 263K. We do not know whether we would have eventually seen an ID50 for SSLOW equivalent to that of 263K, if hamsters had longer lifespans in which to develop infections from low titre inocula. In this respect, PMCAb oers advantages over the bioassay for titering slow prion strains, as the read-out parameter in PMCAb is not limited by the lifespan of an animal.

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Further comparison of end-point titration data from bioassay and PMCAb will test whether PMCAb50/ID50 ratio represents an inherent strain-specic property. Fortunately, uncertainties as to the relationship of PMCAb-active particles to infectivity do not diminish the power or usefulness of PMCAb as a quantitative assay. PMCAb can be used as is, as a relative indicator of infectivity titre between two similar samples. With careful calibration and sample control, it can be used to estimate PrPSc infectivity titre itself.

Methods

End-point titration bioassay. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore (assurance number: A32000-01; permit number: 0309001).

Ten percent scrapie BH was prepared in PBS, pH 7.4, by sonication and serially diluted up to 1011-fold in PBS, as previously described41. SSLOW-inoculated animals from the second passage of SSLOW31 were used for both bioassay and PMCAb. Before inoculation, samples were dispersed by 90 s of maximum power ultrasonication in PBS. Each hamster received 50-l inoculum intracerebrally under general anaesthesia (2 LPM O2/4 MAC isourane). The inoculated animals were observed closely for up to 660 days post-inoculation or until they developed clinical signs of prion disease. For SSLOW-inoculated animals, the clinical signs were observed as early as 318 10 days post-inoculation for the 10-fold dilution of brain material or as late as 560 10 days for the 106-fold dilution of brain material. Only one out of eight animals inoculated with the 107-fold dilution showed clinical signs, whereas no clinical signs were observed for dilutions 108-fold and higher. Aected animals were euthanized and their disease status was conrmed by western blot analysis of their brains. At the end of the incubation (660 days post-inoculation) all remaining animals were euthanized, and all brains were assessed for the presence of PrPSc by western blot. Animal brains that contained PrPSc but had not yet developed symptomatic disease were considered infected.

End-point titration using PMCAb. Healthy hamsters were euthanized and immediately perfused with PBS, pH 7.4, supplemented with 5 mM EDTA. Brains were dissected, and 10% BH (wt/vol) was prepared using ice-cold conversion buer and glass/Teon tissue grinders cooled on ice and attached to a constant torque homogenizer (Heidolph, RZR2020). The brains were ground at low speed until homogeneous, and then ve additional strokes completed the homogenization. The composition of conversion buer was as previously described11: Ca2 + -free and Mg2 + -free PBS, pH 7.4, supplemented with 0.15 M NaCl, 1.0% Triton and 1 tablet of Complete protease inhibitors cocktail (Roche, cat. no. 1836145) per 50 ml of conversion buer. The resulting 10% normal BH (NBH) in conversion buer was used as the substrate in PMCA reactions. To prepare seeds, scrapie-infected brains were homogenized as for inoculation (above), and 100 l aliquots were sonicated in MISONIX S-400 microplate (Misonix) horn for 30 s at 50% power before serial dilution from 10- to 1014-fold in conversion buer. Then, 10 l of each dilution were used to seed 90 l of NBH for PMCAb. Teon beads (2.38 mm diameter, McMaster-Carr, Los Angeles, CA, USA) were placed into the 0.2 ml tubes rst, and then NBH and seeds were added. Samples in 0.2 ml thin-wall PCR tubes (Fisher, cat. no. 14230205) were placed in a oating rack inside a Misonix S-4000 microplate cup horn lled with 350 ml water. Two coils of rubber tubing attached to a circulating water bath were installed for maintaining 37 C inside the sonicator chamber. The standard sonication program consisted of 30 s sonication pulses delivered at 50% efficiency applied every 30 min during a 24-h period. For each subsequent round of serial PMCAb, 10 l aliquotes from a previous round were used to seed the reactions in the next round. At limiting dilutions, up to ten independent serial PMCAb reactions were used for each dilution to accumulate statistics.

Data analysis. To correct for dierences in assay volumes used for PMCAb(100 l) and bioassay (50 l), the fraction of positive PMCAb reactions or positive animals, presented in Fig. 3, were normalized per gram of brain tissue.

Analysis using Poisson equation. For the probability that a given aliquot received no active particle, P(0), the Poisson equation reduces to P(0) = e m, where m is the concentration of the active particle at that dilution in particles per inoculation volume. The probability that there will be at least one active particle to initiate infection, P(1), is P(1) = 1 e m. The infectivity data were t to the expression:

F x

Analysis using sigmoidal equation. ID50 and PMCAb50 values were determined by regression analysis in Sigma Plot using nonlinear least squares tting of both sets of data to the sigmoidal equation:

F A B x A B x

= + + +

( * ( ( * /( ( ( * )

100 1

exp - ))) exp - ))

where F is percent of positive PMCAb reactions or infected animals, x is logarithm of the dilution fold, A and B are two tting parameters that dene the position of a limiting dilution transition on the x axis and the slope of the transition, respectively. ID50 and PMCAb50 were calculated according to the equation:

PMCAb or ID

50 50 = A B

/

Proteinase K assay. To analyse the PMCAb end-point titration reactions, 10 l of each sample was supplemented with 5 l SDS and 5 l proteinase K (PK), to a nal concentration of SDS and PK of 0.25% and 50 g ml-1, respectively, followed by incubation at 37C for 1 h. The digestion was terminated by addition of SDS sample buer and boiling for 10 min. Samples were loaded onto NuPAGE 12% BisTris gels, transferred to polyvinylidene diuoride membrane, and stained with 3F4 antibody.

To analyse scrapie BHs, an aliquot of 10% BH was mixed with an equal volume of 4% sarcosyl in PBS, supplemented with 50 mM Tris, pH 7.5, and digested with 20 g ml 1 PK for 30 min at 37C with 1,000 r.p.m. shaking (Eppendorf Thermo-mixer). The reaction was stopped by SDS sample buer. Samples were boiled for10 min and loaded onto NuPAGE 12% BisTris gels. Aer transfer to polyvinylidene diuoride membrane, PrP was detected with 3F4 antibody.

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(3)(3)

(4)(4)

(1)(1)

(2)(2)

=

1-exp a )

( *

where F is fraction of infected animals, x is dilution and a is the tted parameter equal to the undiluted concentration of infectivity. ID50 was calculated by solving the tted equations for F = 0.5 (infectivity), which reduce to the expression:

ID ln(0.5)/a

50 =

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Acknowledgements

We thank Pamela Wright for editing the manuscript. This work was supported by NIH grant NS045585 to I.V.B., and VA Merit Award to R.G.R.

Author contributions

N.M., R.S. and I.A. carried out the work. N.M., R.G.R. and I.V.B. designed the study and analysed the data. I.V.B and R.G.R. wrote the manuscript. All authors commented on the nal manuscript.

Additional information

Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Makarava, N. et al. Fast and ultrasensitive method for quantitating prion infectivity titre. Nat. Commun. 3:741 doi: 10.1038/ncomms1730 (2012).

NATURE COMMUNICATIONS | 3:741 | DOI: 10.1038/ncomms1730 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.