Antigen-Antibody Dissociation in Alzheimer Disease: A NovelApproach to Diagnosis

Katarzyna A. Gustaw1,2, Matthew R. Garrett3, Hyoung-gon Lee3, Rudy J. Castellani4,Michael G. Zagorski5, Annamalai Prakasam6, Sandra L. Siedlak3, Xiongwei Zhu3, GeorgePerry3,7, Robert B. Petersen3, Robert P. Friedland1, and Mark A. Smith3

1Department of Neurology, Case Western Reserve University, Cleveland, Ohio USA 2Departmentof Neurodegenerative Diseases, IMW, Lublin, Poland 3Department of Pathology, Case WesternReserve University, Cleveland, Ohio USA 4Department of Pathology, University of MarylandSchool of Medicine, Baltimore, Maryland USA 5Department of Chemistry, Case Western ReserveUniversity, Cleveland, Ohio USA 6Department of Neurosciences, Medical University of SouthCarolina, Charleston, South Carolina USA 7College of Sciences, University of Texas at SanAntonio, San Antonio, Texas USA

AbstractWith the ever-increasing population of aged individuals at risk of developing Alzheimer diseasethere is an urgent need for a sensitive, specific, non-invasive, diagnostic standard. The majority ofefforts have focused on auto-antibodies against amyloid-β protein, both a potential treatment, anda reliable biomarker of Alzheimer disease pathology. Naturally occurring antibodies againstamyloid-β are found in the cerebrospinal fluid and plasma of patients with Alzheimer disease aswell as healthy control subjects. To date, differences between diseased and control subjects hasbeen highly variable. However, some of the antibody will be in pre-formed antigen-antibodycomplexes and the extent and nature of such complexes may provide a potential explanation forthe variable results reported in human studies. Thus, measuring total amounts of antigen orantibody following unmasking is critical. Here, using a technique for dissociating antibody-antigen complexes, we found significant differences in serum antibodies to amyloid-β betweenAlzheimer disease and aged-matched control subjects. While the current study demonstrates therelevance of measuring total antibody, bound and unbound, against amyloid-β in Alzheimerdisease, this technique may be applicable to diseases such as AIDS and hepatitis B wheredetermination of antigen and antibody levels are important for disease diagnosis and assessingdisease progression.

KeywordsAlzheimer disease; amyloid-β; antigen-antibody complexes; biomarker; diagnosis

IntroductionWith the ever-increasing population of aged individuals who are at risk for Alzheimerdisease (AD), the insensitivity and cost of psychometric cognitive assessment and therelative ineffectiveness of treatment following diagnosis, there is an urgent need for asensitive, specific and preferably, non-invasive, diagnostic standard. Unfortunately, to date,

Correspondence to: Mark A. Smith, Ph.D., Department of Pathology, Case Western Reserve University, 2103 Cornell Road,Cleveland, Ohio 44106 USA, Tel: 216-368-3670, Fax: 216-368-8964, mark.smith@case.edu.

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Published in final edited form as:J Neurochem. 2008 August ; 106(3): 1350–1356. doi:10.1111/j.1471-4159.2008.05477.x.

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despite intensive efforts, there is no definite surrogate marker for the accurate diagnosis ofeither prodromal, or actual, disease. While genetic risk factors and neuroimaging willcertainly have important roles as biomarkers of AD, much attention is focused ondiagnostics based on blood and cerebrospinal fluid (CSF), for example. In the search for abiomarker, the AD-associated amyloid-β (Aβ) protein, and in particular Aβ1–42, has been afavored target. Although some of the initial results were promising, longitudinal studieshave not shown a consistent change in plasma Aβ levels in AD patients. In studies to date,crude plasma Aβ concentrations do not differ enough between AD and controls to be used asa diagnostic parameter (Andreasen et al. 1999, Andreasen et al. 2001).

Aβ is the major protein component of the abnormal brain pathology, the senile plaque, thataccumulates in specific brain regions of patients and is used for a definitive postmortemdiagnosis (Murayama & Saito 2004, McKeel et al. 2004). While Aβ1–42 failed to be areliable biomarker in plasma, attention was drawn to the potential of measuring auto-antibodies directed against Aβ. Thus, the majority of recent efforts have focused on auto-antibodies against Aβ, not only as a potential treatment for AD, but as a reliable biomarkerof AD (Blennow 2004). Naturally occurring antibodies against Aβ are found in the CSF andplasma of patients with AD, as well as in healthy control subjects. Immunization of micewith Aβ1–42 and subsequent administration of these antibodies against Aβ into amyloid-βprotein precursor transgenic mice (an animal model of AD) dramatically reduced amyloidplaque deposition, neuritic dystrophy, and astrogliosis, most likely by enhancing Aβ1–42clearance from brain (Schenk et al. 1999, Wilcock et al. 2001).

A number of reports show that patients with AD have lower levels of serum anti-Aβantibodies than healthy age-matched individuals (Weksler et al. 2002, Du et al. 2001). Otherstudies, however, indicate that the level of anti-Aβ antibody may be much higher in AD ascompared to control. Mruthinti and colleagues (2004) for example reported that affinitypurified IgGs binding the peptide Aβ1–42, exhibited nearly four-fold higher titers in ADpatients versus unaffected individuals. In addition, Aβ antibody titers were negativelycorrelated with cognitive status such that more cognitively impaired individuals tended toexhibit higher anti-Aβ IgG titers (Mruthinti et al. 2004). The major difference between thisand previous studies was that Mruthinti used affinity purified IgG. Nonetheless, the vastmajority of studies show little difference in Aβ-antibodies in sera from patients versusunaffected individuals (Hyman et al. 2001).

In biological fluids, antibodies and antigens are in a state of dynamic equilibrium betweenbound and unbound forms that is concentration dependent. Consequently, the antigen mayeffectively mask a proportion of the corresponding antibody, and limit both antibody andantigen detection. Under certain disassociating conditions, this interaction can beinterrupted, thus freeing antibody and antigen and providing a more accurate analysis ofboth specific antibody titers and antigen concentration.

Although antibody titers against a particular antigen in a given disease state may be stronglyelevated, only a fraction of the total amount is likely detectable via ELISA (enzyme linkedimmunoassay) due to interference by antigen-antibody complexes. In this case, if thecomplexes are dissociated, free antibody would be significantly elevated. For example, if 4units of antigen are circulating together with 5 units of antibody in one patient, and 1 unit ofantigen is circulating together with 2 units of antibody in another, assuming that antibodiesare bound, both patients would be said to have 1 unit of free antibody detectable (Figure1A). In contrast, after dissociation of the antigen-antibody complexes (Figure 1B), thesesame patients would display 5 and 2 units of free antibody, respectively.

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The aim of this study is to demonstrate the importance and reliability of measuring totalamounts of antibody following unmasking and to provide an explanation of the discrepancyin the existing data, using both in vitro and in vivo approaches. Specifically, using atechnique for dissociating antigen-antibody complexes, we found significant differences inserum antibodies to Aβ between AD and aged-matched control subjects that may havediagnostic utility. Supported by similar results from a study in transgenic mice (Li et al.2004), we herein present a technique for the measurement of total antibody against Aβ andantigen in plasma for diagnosis of AD. This technique may be broadly applicable to otherdiseases such as AIDS and hepatitis B where antigen and antibody levels are important toolsfor making disease diagnosis and assessing disease progression (Anh-Tuan & Novak 1980,Fabrizi et al. 2005, Ledergerber et al. 2000).

Materials and MethodsIn vitro dissociation of monoclonal Aβ antibody from Aβ peptide

Mouse monoclonal antibody against Aβ (4G8, Endogen) and Aβ1–42 (40, 80 and 160 µg/mlrespectively) (Hou et al. 2004) were incubated for 20 minutes and dissolved in dissociationbuffer (1.5% BSA, 0.2M glycine-HCl, pH 2.5) or a neutral control buffer (1.5% BSA, 0.2Mglycine-HCl, pH 7) for a total volume of 500 µl in the upper chamber of a Microconcentrifugal device (10K molecular weight filter cut-off, Millipore). Samples were incubatedat room temperature for 20 min. Following incubation, samples were centrifuged 20 min at14,100 rpm at room temperature, with antibody retained in upper chambers. Filters werethen inverted and spun in a second tube for 3 min at 5,000 rpm. The retentate sample wascollected from the second tube and brought back to a neutral pH with 15–20 µl 2.5M TrispH 9. Likewise, samples run in neutral control buffer were collected and equal amounts (15–20 µl) of ELISA dilution buffer was added (1.5% BSA, 0.05% Tween-20, in PBS).Following neutralization, all samples were brought back to the initial 500 µl and 1:100dilutions with ELISA dilution buffer. At this point, samples were ready to be plated forELISA analysis. Initial experiments were performed titrating the concentration of 4G8. Itwas determined that a dilution of 1/1000 gave optimal and very reproducible results.

In vivo dissociation of Aβ antibody in human seraSera was obtained from patients diagnosed with AD (52–91 years, n = 54) and age-matchedcontrols (64–90 years, n = 49) through the University Memory and Aging Center at CaseWestern Reserve University. Diagnosis of AD was consistent with the Diagnostic andStatistical Manual of Mental Disorders [4th edition (DSM-IV)] criteria and NationalInstitute of Neurologic and Communicative Disorders and Stroke-AD and Related DisordersAssociation (NINCDS-ADRDA) criteria for possible or probable AD. Modified HachinskiIschemic Score (MHIS) was determined at baseline for patient eligibility (Rosen et al.1980). Patients with a composite score of five and more points in MHIS were excluded fromthe study as their vascular risk was considered to be inconsistent with probable AD asdefined by NINCDS-ADRDA (McKhann et al. 1984). Patients had to have a Mini-MentalState Examination (MMSE) score of < 24 (Folstein et al. 1975).

CT or MRI scans were obtained at screening. Patients had to be otherwise healthy andambulatory or ambulatory aided (i.e., walker or cane), with vision and hearing sufficient forcompliance with the testing procedures. Laboratory test values had to be within normallimits or considered to be clinically insignificant by the investigator. All patients had to havea reliable caregiver. Patients were excluded if they had evidence of clinically significant andunstable, active gastrointestinal, renal, hepatic, endocrine, or cardiovascular system disease,primary neurologic or psychiatric diseases other than AD (notably DSM-IV-defineddepression or vascular dementia), newly treated hypothyroidism, or a known or suspected

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history (within the past 10 years) of alcoholism or drug abuse. Additional reasons forexclusion included evidence of neoplasm, insulin-dependent diabetes or diabetes notstabilized by diet or oral hypoglycemic agents, obstructive pulmonary disease or asthma,recent (<2 years) hematologic/oncologic disorders, pernicious anemia, or vitamin B12 orfolate deficiency as evidenced by blood concentrations below the lower normal limit. Thestudy was carried out in accordance with the local Institutional Review Board agreement.Written informed consent was obtained from the patient (if possible), the caregiver, and thepatient's representative (if applicable) before beginning detailed screening activities.

Sera were diluted 1:100 with dissociation buffer (PBS buffer with 1.5% BSA and 0.2 Mglycine-acetate pH 2.5) to a 500 µl final volume and incubated for 20 min at roomtemperature (RT). The sera were then pipetted into the sample reservoir of Microconcentrifugal filter device, YM-10 (10,000 MW cut-off; Millipore) and centrifuged at 14,100rpm for 20 min at RT. The sample reservoir was then separated from the flow through,placed inverted into a second tube and centrifuged at 5000 rpm for 3 min at RT. Thecollected solution containing the antibody dissociated from the Aβ peptide was adjusted topH 7.0 with 1 M Tris buffer, pH 9.0. The retentate volume was reconstituted to the initialvolume (500 µl) with ELISA dilution buffer (PBS with 1.5% BSA and 0.1% Tween-20).The collected sera were then added to an ELISA plate at several dilutions to determine theantibody titer. As a control, the same serum was treated in an identical process except thatthe sera were diluted into buffer at pH 7.0 instead of dissociation buffer, pH 2.5.

Measurements of antibody titers by ELISANUNC Maxisorp 96 well ELISA plates were coated with 50 µl/well Aβ1–42 5µg/ml in PBS,pH 7 and incubated overnight at 4°C. Plates were washed 5 times with washing buffer(0.45% BSA + 0.05% Tween-20), and then blocked (300 µl/well) for one hour at 37°C with1.5% BSA + 0.05% Tween 20 in PBS. Following blocking, the plates were washed 4 timeswith washing buffer and samples applied (50 µl/well) in duplicate or triplicate and incubatedat 37°C for one hour. The plates were then washed 10 times with washing buffer. Forexperiments using 4G8 as the detection antibody, a mouse IgG conjugated with horseradishperoxidase (HRP) (Sigma, St. Louis, MO) was diluted 1:5000 and added (50 µl/well). Forexperiments using antibody from human samples as detection antibody, anti-human IgG (H+L) antibody (Southern Biotechnology Associates Inc.) was diluted 1:2500 and added at 50µl/well.

Samples were incubated for 1 hour at 37°C. After incubation, the plates were washed 10times, developed with TMB (Pierce 1-step Ultra, Sigma), the reaction stopped with 2Msulfuric acid (50 µl) and the plates were analyzed spectrophotometrically at 450 nm. Datawas analyzed using the student’s t-test. For the analysis comparing the same plasma samplesanalyzed in 2 separate laboratories, the paired t-test was applied.

ResultsIn vitro experiments

Using an in vitro system, we demonstrated that detection of 4G8, a mouse monoclonalantibody against Aβ, is significantly blocked by the addition of free Aβ1–42 in an ELISA,p<0.0002 (Figure 2A). This demonstrates in a simple system the basic premise of ourhypothesis: Aβ-antigen effectively masks the detection of Aβ auto-antibody, but this can bereversed to facilitate measurement of total Aβ auto-antibody. Significantly, followingdissociation, the levels of detected antibody reached 89–98% level of reference (antibodyonly control) (Figure 2A).

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In parallel to antibody detection, Aβ1–42 antigen recovery was also assessed. Using thecorresponding samples (lower than 10kDa MW sample from the first tube aftercentrifugation), Aβ was measured before and after dissociation as previously described(Sambamurti et al. 2007). Following incubation of Aβ1–42 at concentrations ranging from 40µg/ml to 160 µg/ml with 4G8 (1:1000), and subsequent antigen-antibody complexdissociation, significantly greater levels of Aβ are detected (data not shown). However, evenusing the concentration where peptide retrieval after dissociation was the most striking (160µg/ml), the level of antigen detected only reached 60% of the level of reference (pureantigen Aβ1–42 control) (Figure 2B). This may reflect binding of the peptide to the filter,although more stringent washes failed to release the peptide (not shown) or peptideaggregation at low pH. Antigen detection following dissociation was linear and correlatedwith the initial amount of Aβ1–42 in the solution in a concentration dependant manner, r =0.998.

In vivo experimentsSera collected from AD patients and age-matched controls were examined using the samemethodology as in the in vitro experiments above (Figure 3). The level of antibody againstAβ1–42 was detectable both in control and AD before dissociation of Aβ antibody. Statisticalanalysis shows differences between AD and control patients before dissociation, dependingupon the cases used. After dissociation, however, the level of antibody assessed was greaterthan before dissociation. Significantly, as predicted by our model (Figure 1), the increase inAβ auto-antibody levels expressed as the change in optical density (OD) units before andafter dissociation is much greater in AD cases than in controls, p < 0.001 (Figure 3C).Similar results were obtained by using a higher pH dissociation buffer, pH 3.5 (data notshown).

To validate the methodology, the experiments were performed by two separate individualsin two geographically separate laboratories. Each person was given serum samples from ADand control cases, selected randomly from a large number of previously collected samples.Two striking findings were noted. The results from both laboratories demonstrate asignificant increase in antibody titer, expressed in OD units, following dissociation and,importantly, the AD cases show significantly higher Aβ auto-antibody levels compared tocontrols after dissociation (Figure 4). Strikingly, in the non-dissociated state, one lab (A) butnot the other (B) showed AD/control differences. Such variability reflects the literature.However, following dissociation, both laboratories found striking AD/control differences (p< 0.01 and p < 0.0001 respectively). To standardize between labs, 25 of the same cases,sampled from both the AD and control populations, were analyzed in both laboratories.Indeed, when the results from the same cases were compared between the two laboratories,the variability in the detection seen before dissociation is significant (p = 0.006), while theresults following dissociation are similar (p = 0.46). This latter result provides anexplanation for the variability seen in previous studies using non-dissociated samples as wellas providing strong support for the dissociation method as a reliable, robust technique.

DiscussionGiven the changing age demographics and an increasing population at risk for AD, there is aneed for sensitive, ante-mortem AD diagnostics. In addition to diagnosing disease, thesetests will also facilitate rapid evaluation of new treatment modalities. One potentially fruitfulavenue is the detection of Aβ auto-antibodies. Previous studies in mouse models and inhuman samples have failed to demonstrate the utility of Aβ antibodies as a biomarker,however, we suspected that the actual level of the auto-antibody may be masked by theformation of antigen-antibody complexes in the plasma. This may also help to explain the

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variable results previously reported. To test this idea, we developed a simple, robusttechnique for separating the antigen-antibody complex.

In vitro experiments, in which Aβ1–42 antigen was used to block antibody detection,supported the main concept of this study. Following dissociation, detection of the Aβantibody was restored. Employment of this method results in the Aβ peptide passing througha filter after being dissociated from the antibody, while the antibody remains in the upperchamber of the centrifugation filter tube. This effectively separates the antibody from theantigen and prevents rebinding. Both antibody and corresponding antigen are detectable atgreater levels after dissociation in vitro. Based on these results we evaluated the dissociationtechnique using human plasma samples from AD patients and age-matched controls.

Using double antibody sandwich ELISA assays, levels of Aβ1–40 in plasma have beenreported to be between 100–200 pg/ml and found to correlate with disease parameters insome (van Oijen et al. 2006), yet but not other studies (Mehta et al. 2001). In our studies, theconcentration of Aβ42 used in vitro is far higher than that reported in plasma for two distinctreasons. First, the measured concentrations previously reported (Mehta et al. 2001, vanOijen et al. 2006) represent free Aβ42 and, given the premise of our study (Figure 1), theactual concentration is likely far higher. Second, the present work used higher levels Aβ42peptide, as a proof of concept, to mask the amyloid antibodies in an ELISA system. Underthese conditions nearly all of the antibody could be recovered and readily detected followingdissociation (Figure 2B). Dissociation experiments were carried out on AD and controlplasma samples in two different laboratories. Control and AD titers of antibody weresignificantly different after dissociation, but not before (Figure 3 and Figure 4). Thevariability seen between the two laboratories in the non-dissociated samples may explain thediscrepancy between previous studies. In addition, we found that this technique is robust.Whereas nondissociated samples showed significant variation when evaluated in differentlaboratories, after dissociation there was good concordance. On a separate note, it has beenreported that low pH, i.e., pH 2.5, creates spurious signals that are not manifest whendissociation is accomplished at pH 3.5 (Li et al. 2007). We did not obtain a similar resultusing the human samples (data not shown).

This work extends some concepts and techniques to human samples that have consistentlybeen shown to be reliable (Anh-Tuan & Novak 1980, Fabrizi et al. 2005, Ledergerber et al.2000, Li et al. 2004), specifically in transgenic mice where Aβ antigen-autoantibodycomplexes were dissociated (Li et al. 2004). The concept of antigen-antibody dissociationhas also been theorized and demonstrated in diseases such as HIV and various forms of viralhepatitis (Anh-Tuan & Novak 1980). However, this finding from clinical samples ofprobable AD patients represents a novel and independent approach divergent from previouswork for the characterization and potential diagnosis of AD.

Both mice and human sera evaluation, demonstrated the basic premise of our hypothesis:Aβ-antigen effectively masks the detection of Aβ auto-antibody, but can be dissociated, thusincreasing sensitivity of measurement. The fact that the effect is dependent on Aβ level andmore visible in affected, AD individuals may be of exceptional value for further biomarkersearch.

AcknowledgmentsWe are indebted to Dr. Kumar Sambamurti (Medical University of South Carolina) for amyloid-β measurements.Work in the authors’ laboratories is supported by the National Institutes of Health (AG026151 to MAS). RPFacknowledges support from the Fullerton Family, NIH (R01-AG017173), the Joseph and Florence MandelResearch Fund, the Nickman Family, the GOJO Corporation and the Institute for the Study of Aging.

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Abbreviations

AD Alzheimer’s disease

Aβ amyloid-β

CSF cerebrospinal fluid

DSM-IV Diagnostic and Statistical Manual of Mental Disorders

ELISA enzyme linked immunoassay

HRP horseradish peroxidase

MMSE Mini-Mental State Examination

MHIS Modified Hachinski Ischemic Score

NINCDS-ADRDA National Institute of Neurologic and Communicative Disorders andStroke-AD and Related Disorders Association

OD optical density

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Figure 1.In dissociated samples, unbound antigen-antibody complexes reveal increased disease-stateantigens versus non-diseased counterparts. Therefore, while non-dissociated samples offerno diagnostic value, dissociated antigen and antibody values are discordant betweendiseased and controls, thus discriminating diseased and controls.

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Figure 2.A) Detection of 4G8. Using purified Aβ1–42 and mouse monoclonal 4G8, the detection ofantibody at 1/1000 dilution is strongly blocked by the addition of Aβ before dissociation (□).Following dissociation (■), greater levels of antibody are detected, to levels nearly 100% ofreference (antibody+buffer). B) Detection of Aβ1–42. Using the corresponding samples fromA, Aβ was measured before (□) and after (■) dissociation. Incubation with 4G8 significantlyblocked detection of Aβ and following dissociation, greater levels of Aβ were detected, yetonly reached approximately 60% of reference (Aβ1–42 +buffer). ns = not significant.

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Figure 3.Detection of antibody before and after dissociation in representative AD (B) and control (A)samples, plotted by increasing OD difference before and after dissociation. The level ofamyloid antibody detected after dissociation (●) is greater than in non-dissociated samples(○). AD samples show significantly greater differences in antibody detection (OD units)following dissociation than the control samples, p < 0.001 (C). Data is expressed as mean ±SEM.

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Figure 4.When this newly described methodology was applied in two separate laboratories (A and B),only the dissociated samples (■) consistently showed significant differences between the ADand control samples analyzed. Specifically, the levels of amyloid antibodies detected in thenon-dissociated (□) samples give variable results between AD and control, either significant(p < 0.01) or non-significant. However, following dissociation, the differences between ADand control samples are consistently significant with p values ranging from p < 0.01 to p <0.0001. Importantly, 25 of the same cases, randomly selected from both the AD and controlsamples, were blindly analyzed in both laboratories. Comparison of the antibody levelsobtained for each case in the two laboratories using the student’s paired t-test, again revealedvariability in the non-dissociated samples (p < 0.01), and yet the antibody levels determinedfrom the dissociated samples were not significantly different (p = 0.46), data not shown.

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