detection and quantification of tau aggregation using a membrane filter assay

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Analytical Biochemistry 373 (2008) 330–336

ANALYTICAL

BIOCHEMISTRY

Detection and quantification of tau aggregation usinga membrane filter assay

Edward Chang a, Jeff Kuret b,*

a Integrated Biomedical Sciences Graduate Program, Department of Molecular and Cellular Biochemistry, The Ohio State University

College of Medicine, Columbus, OH 43210, USAb Center for Molecular Neurobiology, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, Columbus,

OH 43210, USA

Received 8 August 2007Available online 19 September 2007

Abstract

Aggregation of the microtubule-associated protein tau contributes to the formation of neurofibrillary lesions in Alzheimer’s diseaseand is a useful marker of disease progression. Although filter trap assays have been employed to assess the extent of tau aggregation incells and tissues as well as in vitro, their performance relative to other assay modalities has not been reported. To clarify this issue, theability of the filter trap approach to quantify aggregation of purified recombinant full-length tau protein in vitro was examined as a func-tion of membrane chemistry in a 96-well format. Results showed that nitrocellulose yielded the greatest assay sensitivity relative to poly-vinylidene fluoride or cellulose acetate at equal membrane porosity. However, all combinations of filter chemistries, porosities, andmonoclonal detection antibodies yielded nonlinear correlations between signal intensity and analyte concentration. When correctedfor nonlinearity, the filter trap assay determined a value for the critical monomer concentration for tau aggregation that was statisticallyidentical to determinations made by electron microscopy assay. The data suggest conditions under which filter trap assays can be used toestimate tau aggregation kinetics.� 2007 Elsevier Inc. All rights reserved.

Keywords: Tau; Aggregation; Alzheimer’s disease; Filter assay

Abnormal aggregates of the microtubule-associated pro-tein tau are found in several progressive neurodegenerativediseases, including Alzheimer’s disease (AD)1 and fronto-temporal dementia [1]. In AD, clinical progression ofsymptoms correlates well with the temporal and spatialspread of tau aggregates in the brains of affected individu-als [2–4]. Thus, tau aggregation is a useful marker of dis-ease, and characterization of its mechanism of formationcan yield information on underlying pathological pro-cesses. In addition, in vitro modeling of tau fibrillization

0003-2697/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2007.09.015

* Corresponding author. Fax: +1 614 292 5379.E-mail address: [email protected] (J. Kuret).

1 Abbreviations used: AD, Alzheimer’s disease; PVDF, polyvinylidenefluoride; HRP, horseradish peroxidase; IgG, immunoglobulin G; IgM,immunoglobulin M; SDS, sodium dodecyl sulfate; ECL, enhancedchemiluminescence; SEE, standard error of the estimate.

can be used to clarify the kinetics of the process and toidentify compounds potentially capable of modulatinglesion formation [5].

Tau aggregation has been quantified using variousmethods, including dye-based fluorescence spectroscopy[6,7], laser light scattering [8,9], high-speed centrifugation[10], and electron microscopy [11]. Each approach has itsown advantages and disadvantages. For example, trans-mission electron microscopy provides direct visualizationof the aggregates, which establishes morphology, and pro-vides the length distribution of filaments, which reflects theaggregation mechanism. However, this method is lowthroughput and may be subject to measurement biasdepending on conditions of experimentation [11].

Recently, filtration methods have been used to quantifythe products of protein aggregation reactions [12,13]. In

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Detection and quantification of tau aggregation / E. Chang, J. Kuret / Anal. Biochem. 373 (2008) 330–336 331

this approach, reaction products are filtered through amembrane that traps and retains large protein aggregateswhile small species, including protein monomers, passthrough. When combined with solid-phase immunodetec-tion, the approach can yield a highly sensitive estimationof protein aggregation. Early versions of the assay used cel-lulose acetate as the capture membrane [13], which provedto be capable of trapping tau aggregates in extracts ofhuman and transgenic mouse brain tissue [14]. Subsequentassay of tau aggregation in cultured cell extracts, as well asin vitro, using purified protein preparations employed fil-ters with greater protein binding affinity, including nitrocel-lulose [15] and polyvinylidene fluoride (PVDF) [16].However, a full characterization of any filter-based assayfor tau, including the effect of membrane compositionand porosity, has not yet been reported. Moreover, the rel-ative sensitivity and linearity of these assays has not beendisclosed.

Here we characterize a vacuum-based 96-well format fil-ter assay for assessment of tau fibril formation in vitro.Results indicate that although assay sensitivity is a functionof both filter composition and porosity, analyte concentra-tion dependence is nonlinear under all conditions tested.However, control for nonlinearity through the use of cali-bration standards yields a quantitative assay capable ofestimating aggregation parameters with a precision equalto microscopy-based methods.

Materials and methods

Materials

Recombinant htau40 was prepared as described previ-ously [17]. Primary mouse monoclonal antibodies Tau1[18] and Tau5 [19] were gifts from L. I. Binder (Northwest-ern University), whereas Alz50 [20] was a gift from P.Davies (Albert Einstein College of Medicine). Horseradishperoxidase (HRP)-linked goat anti-mouse immunoglobulinG (IgG) and goat anti-mouse immunoglobulin M (IgM)were obtained from Kirkegaard & Perry Laboratories(Gaithersburg, MD, USA). Filter membranes usedincluded 0.45 lm cellulose acetate from Sterlitech (Kent,WA, USA), 0.2 and 0.45 lm nitrocellulose from Bio-RadLaboratories (Hercules, CA, USA), and 0.45 lm PVDFfrom Millipore (Billerica, MA, USA). Formvar/Carbon-coated copper grids (300 mesh), glutaraldehyde, and uranylacetate were obtained from Electron Microscopy Sciences(Fort Washington, PA, USA). Thiazine red was obtainedfrom ICN Biomedicals (Aurora, OH, USA). Octadecyl sul-fate detergent (Lancaster Synthesis, Pelham, NH, USA)was dissolved in 1:1 isopropanol/H2O before use.

Tau fibrillization

Tau protein was incubated (50 ll final volume) at 37 �Cfor up to 24 h in assembly buffer (10 mM Hepes [pH 7.4],100 mM NaCl, and 5 mM dithiothreitol) in the presence

and absence of either thiazine red (100 lM) or octadecylsulfate (50 lM) fibrillization inducer. Reactions wereimmediately subjected to either filter or electron micros-copy assays described below.

Filter assay

Tau fibrillization reaction products were diluted up to10-fold in 2% sodium dodecyl sulfate (SDS) to prepare aseries of descending tau filament concentrations. Allsamples underwent a further 1:3 dilution in 2% SDS beforevacuum filtration through a 96-well dot blot apparatus(Bio-Rad Laboratories) containing nitrocellulose, PVDF,or cellulose acetate membranes. The resultant membraneswere washed twice with 2% SDS, blocked in 4% nonfatdry milk dissolved in blocking buffer (100 mM Tris–HCl[pH 7.4] and 150 mM NaCl) for 2 h, and then incubatedwith primary antibody at 1:1000 dilution for 1.5 h. Mem-branes were washed twice in blocking buffer and then incu-bated with HRP-linked secondary antibody for 1.5 h. Themembranes were washed again twice in blocking bufferand then developed with the ECL (enhanced chemilumi-nescence) Western Blotting Analysis System (GE Health-care, Buckinghamshire, UK). Chemiluminescence wasrecorded on an Omega 12iC Molecular Imaging Systemand was quantified using UltraQuant software (UltraLum,Claremont, CA, USA).

Transmission electron microscopy

Tau fibrillization reactions were terminated with theaddition of 2% glutaraldehyde and then were adsorbedonto 300-mesh Formvar/carbon-coated copper grids for1 min. The grids were rinsed with water, negatively stainedfor 1 min with 2% uranyl acetate, and washed again withwater. Images were captured on a Tecnai G2 Spirit Bio-TWIN transmission electron microscope (FEI, Hillsboro,OR, USA) operated at 80 kV and 23,000·magnificationand then analyzed with ImageJ software (National Insti-tutes of Health). Average total filament length was deter-mined as described previously [11].

Analytical methods

Regression analysis was performed with SigmaPlot soft-ware (Systat Software, San Jose, CA, USA). Analyte con-centration dependence of the filter assay was fit to thepower function

y � y0 ¼ axb; ð1Þwhere y is the signal intensity produced in the presence ofaggregation inducer at tau concentration x, y0 is the back-ground signal produced in the absence of tau aggregationinducer at tau concentration x, and a and b are constants.

The critical concentration for fibrillization was esti-mated from the abscissa intercept after least squares linearregression and is reported ± standard error of the estimate

332 Detection and quantification of tau aggregation / E. Chang, J. Kuret / Anal. Biochem. 373 (2008) 330–336

(SEE) [9]. The Z 0 factor for evaluating assay performancewas calculated as described previously [21].

Results

To generate a population of tau filaments for testing infilter assays, 1 lM full-length, four-repeat tau protein(htau40) [22] was incubated for 16 h at 37 �C in the pres-ence and absence of thiazine red inducer. Stable plateaulevels of fibrillization are induced under these conditions[23]. In contrast, tau protein incubated in assembly bufferwithout inducer does not produce detectable aggregates[24]. In a preliminary test of the filter trap assay, tau sam-ples prepared as described above were diluted in 2% SDS toform a descending concentration series, and then equalvolumes of each dilution were vacuum filtered through a0.2-lm nitrocellulose membrane in a 96-well format. SDSwas used as diluent because authentic tau filaments arerelatively stable in detergents [25], including SDS [14].Trapped tau protein was then labeled with Tau1 monoclo-nal antibody in conjunction with an HRP-linked secondaryantibody and chemiluminescent substrate. Chemilumines-cence was captured using an Omega 12iC Molecular Imag-ing System. Tau1 was used as the labeling antibody becauseit binds to a well-characterized linear epitope in nonphos-phorylated tau protein with high affinity [17,26]. Resultsshowed that the nitrocellulose filter detected much strongerchemiluminescent signals from the tau sample treated withthiazine red compared with the nontreated control reaction(Fig. 1), indicating that retention of unaggregated mono-mer on the membrane was minor relative to trapping of fil-aments under these conditions. The mean signal-to-background ratio (i.e., signal from sample with thiazinered inducer compared with sample without inducer) was29.7 ± 6.9 (n = 3 replicates) for the neat sample anddecreased in parallel with the amount of tau aggregate sub-jected to filtration (Fig. 1). The Z 0 factor for assays span-ning the fivefold relative concentration range of 0.2 to 1(Fig. 1) was 0.66 ± 0.15 (n = 9 concentrations). These dataindicate that the tau filter trap assay is adequate for high-

Fig. 1. Results from the filter trap assay. Purified recombinant htau40(1 lM) was incubated for 16 h at 37 �C in the presence and absence of100 lM thiazine red inducer and then was diluted in 2% SDS to create adescending concentration series of reaction products. These dilutions werevacuum filtered through a 0.2 lm porosity nitrocellulose membrane andthen were stained with Tau1 primary and HRP-conjugated secondaryantibodies. Tau immunostaining (chemiluminescence) was then visualizedon an Omega 12iC Molecular Imaging System. Under these conditions,substantially more tau is trapped in the presence of aggregation inducerthan in its absence.

throughput screening applications under these experimen-tal conditions [21].

To assess assay linearity, net chemiluminescence (i.e.,the difference between thiazine red-induced tau samplesand noninduced tau samples) was determined by densitom-etry and then plotted as a function of relative tau concen-tration. The resultant curves were nonlinear (Fig. 2A) butcould be fit to a simple power function (Eq. (1)). Nonlin-earity did not result from choice of inducer given that sim-ilar data were obtained for tau filaments prepared in thepresence of anionic surfactant octadecyl sulfate (data notshown). These data suggest that tau aggregates can beselectively trapped and detected by nitrocellulose filtersbut that chemiluminescent signal is not linearly related totau filament concentration under these experimentalconditions.

Membrane chemistry

Published filter trap assays have employed cellulose ace-tate [13,14], nitrocellulose [15], and PVDF [16] membranes.To compare the performance of these membranes at con-stant porosity, each was subjected in parallel to the assaydescribed above using Tau1 monoclonal antibody in con-junction with an HRP-linked secondary antibody andchemiluminescent substrate. Net chemiluminescence wasthen plotted as a function of relative tau concentration(Fig. 2A). Because dependence of chemiluminescence onanalyte concentration was nonlinear under all conditions,data were fit to Eq. (1), with parameter a taken as a mea-sure of relative sensitivity (Fig. 2B). Results showed that0.45 lm cellulose acetate had by far the weakest ability totrap and retain tau aggregates. In contrast, 0.45 lm PVDFimproved retention by more than an order of magnitude(Fig. 2B). Filters of the same porosity but composed ofnitrocellulose improved sensitivity by a further 1.5 ±0.1-fold. Finally, narrowing nitrocellulose porosity to0.2 lm increased sensitivity 1.7 ± 0.1-fold more. Thesedata suggest that filaments prepared in vitro from purifiedfull-length tau preparations are best captured and retainedby membranes with high protein binding activity such asnitrocellulose and PVDF. In addition, although smallerpore sizes are more efficient at trapping tau aggregates inthe context of nitrocellulose, they do not improve assaylinearity.

Primary antibodies

Many monoclonal antibodies are available for detectionof tau protein, including those that bind epitopes depen-dent on conformation. To assess relative performance ofthe filter trap assay, two additional antibodies, Tau5 andAlz50, were tested as detection reagents. Tau5 is an IgGthat binds a linear tau epitope independent of phosphory-lation state [17,27], whereas Alz50 is an IgM with anti-taubinding affinity selective for filamentous tau [17]. A com-parison of the performance of Tau1, Tau5, and Alz50 is

Fig. 2. Influence of membrane composition and porosity on detection oftau aggregates. The dilution series described in Fig. 1 were vacuum filteredthrough 0.2 lm nitrocellulose, 0.45 lm nitrocellulose (NC), 0.45 lmPVDF, or 0.45 lm cellulose acetate (CA) membranes. Membrane-boundtau was then detected with Tau1 monoclonal antibody, followed bydensitometric quantification of chemiluminescence. (A) Net chemilumi-nescence, corresponding to the difference between signals generated in thepresence of thiazine red inducer and those generated in its absence, wasplotted versus relative tau filament concentration (where undiluted analytehas a concentration of 1). Each data point represents the mean ± SD oftriplicate densitometric measurements, whereas the solid curves representbest fits of the data points to a power function (Eq. (1)). All membranesyielded nonlinear concentration dependence curves. (B) Parameter a

estimated from fits to Eq. (1) were replotted ± SEE as a measure of assaysensitivity. Assay sensitivity depended on membrane composition andporosity.

Fig. 3. Comparison of anti-tau monoclonal antibodies for detection of tauaggregates. The dilution series described in Fig. 1 was vacuum filteredthrough a 0.2-lm nitrocellulose membrane. Net chemiluminescence wasquantified using Tau1 (A), Tau5 (B), or Alz50 (C) primary antibodies andappropriate HRP-conjugated secondary antibodies (goat anti-mouse IgGfor Tau1 and Tau5, goat anti-mouse IgM for Alz50) and was plottedversus relative tau filament concentration (undiluted analyte = 1). Eachdata point represents the mean ± SD of triplicate measurements, whereasthe solid curves represent best fits of the data points to a power function(Eq. (1)). All primary antibodies yielded nonlinear concentration curves.


shown in Fig. 3. All three antibodies displayed parabolicconcentration dependence curves, with only minor differ-ences in sensitivity. These data suggest that selection of pri-

mary antibody has only minor effects on the performanceof the filter trap assay.

Comparison with electron microscopy

When corrected for nonlinearity, the filter trap assaycould potentially provide sensitive and high-throughput


measurement of tau aggregation parameters. To test thisapproach, tau critical concentration was estimated by bothelectron microscopy and corrected filter trap assays. Criti-cal concentration is the highest concentration of tau pro-tein that does not support fibrillization; therefore, itcorresponds to the abscissa intercept when aggregate massor total length is plotted as a function of bulk protein con-centration [28]. To determine tau critical concentration,varying concentrations of tau protein (0.3–0.7 lM) wereincubated in the presence and absence of thiazine red indu-cer, with one aliquot being used to determine total filamentlength by electron microscopy and an identical aliquotbeing subjected to the filter trap assay (with 0.2 lm nitro-cellulose as the filter and Tau1 as the primary detectionantibody). For the latter measurements, net chemilumines-cence was corrected for nonlinearity using Eq. (1) fit to adescending concentration series similar to that shown inFigs. 1 and 2. Under these conditions, total filament lengthas determined by electron microscopy was linearly relatedto bulk tau concentration (linear regression r2 = 0.995),with an abscissa intercept of 0.23 ± 0.02 lM (Fig. 4). Whencorrected using Eq. (1), net chemiluminescence also waslinearly related to bulk tau concentration (linear regressionr2 = 0.994), with an abscissa intercept of 0.20 ± 0.02 lM(Fig. 4). The critical concentration values determined byelectron microscopy and corrected filter trap assay were

Fig. 4. Use of calibrated filter assay to estimate critical concentration.Varying concentrations of purified recombinant htau40 (0.3–0.7 lM) wereincubated for 16 h at 37 �C in the presence and absence of 100 lM thiazinered inducer. One aliquot of each reaction product was subjected to thefilter trap assay (s), whereas an equal aliquot was subjected to thetransmission electron microscopy assay (d). Net chemiluminescencemeasured in the filter assay was corrected for nonlinearity using a dilutionseries, whereas total filament length was measured by electron microscopy.Both were then plotted as a function of bulk tau concentration. Each datapoint represents the mean ± SD of triplicate measurements, whereas thesolid lines represent best fits to a linear regression. Critical concentrations,which were determined from the intercepts of these regressions with theabscissa (dotted line), were not statistically different under these conditions(P = 0.37).

in good agreement with previous determinations [23] andwere not statistically different from each other (P = 0.37).This result suggests that the precision of the filter assay iscomparable to that of the electron microscopy assay fortau aggregation and that it may be used to quantify aggre-gation kinetics provided that a standard curve is used tocorrect for nonlinearity.

Discussion

Filter trap assays can play an important role in the char-acterization of tau aggregation reactions owing to theirsensitivity and high-throughput capability. The results pre-sented here identified areas of concern in employing thismethod. First, the amounts of analyte trapped fromin vitro aggregation reactions did not correlate linearlywith the amounts applied to the filter. Nonlinearity wasindependent of the nature of tau aggregation inducer,membrane composition, membrane porosity, and detectionantibody isotype or conformational binding selectivity,suggesting that it was intrinsic to the assay methodology.Nonlinear immunoassays can result from concentration-dependent aggregation of analyte as found, for example,with prion protein PrPC [29]. However, the tau aggregatesemployed in the current study were harvested at reactionplateau and, therefore, were no longer rapidly changingaggregation state [23]. In addition, the dissociation rateof tau filaments is slow relative to sample preparationand assay time [30], suggesting that changes in tau aggrega-tion state do not contribute to nonlinearity. Alternatively,nonlinearity may stem from the use of immunologicaldetection methods. Both the IgGs and the IgM used fordetection of bound tau aggregates are multivalent and,therefore, are subject to avidity effects. Indeed, immobiliza-tion of antigen on a solid support is known to enhanceavidity effects [31]. Finally, it is conceivable that trappedtau aggregates help to recruit and retain additional aggre-gates on the filter, resulting in apparent bindingcooperativity.

Regardless of its source, nonlinear concentration depen-dence of signal intensity was well modeled as a power func-tion. Therefore, it was possible to correct raw signalintensity by including a dilution range of tau fibrils andinterpolating the resultant standard curve. When so cor-rected, the relationship between analyte concentrationand filter trapping became linear, and the filter trap assaywas capable of determining a value for critical concentra-tion in the presence of thiazine red inducer that was statis-tically indistinguishable from the value estimated bytransmission electron microscopy. In terms of precision,the ability of the corrected filter assay to quantify theextent of tau fibrillization at reaction plateau was compara-ble to that of microscopy methods.

Although different membrane chemistries can be usedfor trapping tau aggregates, the most sensitive detectionwas found with nitrocellulose, followed closely by PVDF,when porosity effects were controlled. Cellulose acetate


performed poorly with tau filaments prepared from puri-fied recombinant tau, although it efficiently captures tauaggregates from human and animal brain extracts [14].The difference may be attributable to porosity (0.2 lm vs.the 0.45 lm used here) as well as the size and complexityof the aggregates. For example, aggregates trapped frombrain samples contain b-amyloid as well as tau protein,suggesting that trapped aggregates represent a heteroge-neous mixture of protein species. In contrast, tau filamentsformed in vitro are free of other proteins and, as a result,may be retained less efficiently by cellulose acetate.

Of the antibodies tested in this assay, Tau1 was the mostsensitive at lower concentrations of aggregates; however,all three of the antibodies tested showed similar signalcurves with minimal backgrounds. Thus, labeling antibodymay be chosen to fit the needs of a particular application(e.g., phosphorylation- or conformation-sensitiveantibodies).

When corrected for nonlinearity, and using Tau1 asdetection antibody, the performance of the filter trapassay was comparable to that of electron microscopy[11] and laser light scattering [9] methods. However, fil-tration has special utility for high-throughput quantita-tion of tau aggregation. Potential applications includedetermination of the potency and efficacy of aggregationinhibitors [16], the structure–activity relationship ofaggregation inducers [32], and critical concentration (asshown here). The commonality in these measurementsis constant incubation time. In principle, the filter assaycould also be used for time-dependent applications(e.g., estimation of lag time) provided that reactionsare stopped at the appropriate time points before theyare filtered through the membrane.

Regardless of the application, trapping of aggregatesproduced in vitro is limited by the porosity of the mem-brane. As a result, the assay will underestimate the extentof aggregation depending on the length distribution ofreaction products. For example, retention of small solubleoligomers is expected to be poor. These species are mostabundant at very early time points during filament forma-tion [33] but can appear at any time owing to off-pathwayreactions [34]. If the latter were large enough to be retainedon filters, it might be possible to selectively detect themusing conformation-selective antibodies [35]. Alternatively,the fibrillized state could be selectively detected in the pres-ence of off-pathway aggregates with fibril-selective antibod-ies such as Alz50 [17]. The difficulty of detecting smallaggregates is common to electron microscopy, light scatter-ing, and centrifugation methods as well [28].

In summary, the filter trap assay described here canyield rapid and quantitative assessment of tau aggregationstate. The assay is suitable for a number of membranes andantibodies and so can be modified to fit the particular needsof the experimenter. Quantification can be improved bycalibrating against dilution standards, which facilitates esti-mation of aggregation parameters such as criticalconcentration.

Acknowledgments

We thank Lauren Crissman for assistance with tauexpression and purification. This work was supported bygrants from the National Institutes of Health (AG14452)and from the Alzheimer’s Association.

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detection and quantification of tau aggregation using a membrane filter assay

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