Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-β 3-42 (pE 3 -Aβ)

Abstract

N-terminally truncated pyroglutamate amyloid-β (Aβ) peptide starting at position 3 represents a significant fraction of Aβ peptides (pE₃-Aβ) in amyloid plaques of postmortem brains from patients with Alzheimer’s disease (AD) and older persons with Down syndrome (DS). Studies in transgenic mouse models of AD also showed that pE₃-Aβ is a major component of plaques, and mouse monoclonal antibody to pE₃-Aβ appears to be a desirable therapeutic agent for AD. Since small peptides do not typically elicit a good immune response in mice, but do so favorably in rabbits, our aims were to generate and partially characterize a rabbit monoclonal antibody (RabmAb) to pE₃-Aβ. The generated RabmAb was found to be specific for pE₃-Aβ, since it showed no reactivity with Aβ₁₆, Aβ₄₀, Aβ₄₂, Aβ_3-11, and pE_11-17 Aβ peptides in an enzyme linked immunosorbent assay (ELISA). The isotype of the antibody was found to be IgG class. The antibody possesses high affinity to pE₃-Aβ with dissociation constant (K_D) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE₃-FRHD. In dot blotting, the optimal detection of pE₃-Aβ was at an antibody concentration of 0.5 μg/ml. The threshold of pE₃-Aβ detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE₃-Aβ in sandwich ELISA. pE₃-Aβ was detected in AD and DS brain extracts in ELISA and immunoblotting. Immunohistological studies showed immunolabeling of plaques and blood vessels in brains from patients with AD, and DS showing AD pathology. Thus, the antibody can be widely applied in AD and DS research, and therapeutic applications.

Keywords

Alzheimer’s disease Down syndrome ELISA partial characterization

INTRODUCTION

In recent years there is a great interest in the development of amyloid-β (Aβ) immunotherapy trials in Alzheimer’s disease (AD), specifically targeting N-terminally truncated aggregates and modified pyroglutamate Aβ. Characteristic neuropathological changes in AD include cerebral neuritic plaques with cores composed primarily of amyloid peptides, amyloid accumulation in meningeal and cerebral blood vessels, and neurofibrillary tangles [1, 2]. The core protein of neuritic plaques is the 4-kDa Aβ peptide which is proteolytically cleaved from the larger transmembrane glycoprotein, namely, amyloid-β protein precursor (AβPP), by β-, γ-, and other secretases [3]. Common isoforms of Aβ peptide generated from AβPP end at C-terminal amino acid residue 40 (Aβ₄₀) and 42 (Aβ₄₂) [2]. In addition to these two Aβ isoforms, N-terminally truncated pyroglutamate Aβ starting at position 3 (pE₃-Aβ) is a prominent form of Aβ peptides; it represents a major fraction in classic and diffuse amyloid plaques, and cerebral vascular amyloid in postmortem brains of patients with AD, and older persons with Down syndrome (DS) [4 –8]. pE₃-Aβ is formed by truncation of the first two N-terminal amino acids of Aβ peptide, and subsequent cyclization of the third amino acid, glutamate by glutaminyl cyclase [9, 10]. Several studies have shown that pE₃-Aβ possesses a higher amyloidogenicity, and neurotoxicity, compared to full length Aβ₄₀ and Aβ₄₂ peptides [11 –14]. In addition, pE₃-Aβ has a higher propensity and stability than full length Aβ peptides [14 –16]. Since it may be involved in initial nucleation or seeding and Aβ oligomerization, pE₃-Aβ appears to be a promising target for the development of Aβ vaccination in AD [17 –22].

Recently, a number of passive immunization studies using mouse monoclonal antibody (mAb) to pE₃-Aβ in transgenic (Tg) mouse models of AD have been reported [18–20 , 23–26]. Investigators showed the reduction of total Aβ and pE₃-Aβ plaque load in Tg mouse model of AD [23]. Others showed reduction in general plaque load, and improvement in cognitive performances in Tg mice [18]. Another study showed that mouse mAb to pE₃-Aβ was plaque specific but showed no reduction in total Aβ levels [24]. Since different results were obtained with different mouse mAbs to pE₃-Aβ in Tg mouse models of AD, it was suggested that the immunization protocol needs the evaluation of a panel of antibody to develop an effective immunotherapy for AD [19, 20].

Several investigators measured pE₃-Aβ levels in brain homogenates of AD, and Tg mouse models of AD using immunoprecipitation (IP) and mass spectrometry (MS) assay [10 , 27–30]. However, the method is semi-quantitative, subject to interference, and not cost-effective. The absolute amounts of pE₃-Aβ in brain homogenates of AD, and Tg mouse models of AD varied between different studies [20]. The availability of ELISA kit from commercial sources to quantitate pE₃-Aβ is very limited, and in its present form cannot be used for quantitation of pE₃-Aβ in body fluids. Thus, development of a sensitive ELISA to detect low levels of pE₃-Aβ in brain homogenates or body fluids using specific and high affinity RabmAb specific to pE₃-Aβ is essential.

Investigators have produced rabbit polyclonal antibody to pE₃-Aβ [31 –33], and partially characterized them. However, one of the major drawbacks of rabbit polyclonal antibodies is the variability of specificity and affinity in antisera bled at different intervals. The commercial availability of reliable mouse mAb or RabmAb specific to pE₃-Aβ is none.

Unlike traditional mouse mAbs, antibodies generated with RabmAb technology are able to recognize epitopes on human antigens that are not immunogenic in mice. The reason for this is that the rabbit immune system generates antibody diversity, and optimizes affinity by mechanisms that are more efficient than those of mice. In addition, rabbits elicit strong immune responses against small molecules and haptens, which is uncommon in mice [34]. Since the need for specific and high affinity antibody to pE₃-Aβ is increasing, we here report the generation and partial characterization of RabmAb to pE₃-Aβ. This specific and partially characterized RabmAb may be useful for a wide spectrum of research, and diagnostic applications, and the development of recombinant antibodies, and a humanized version for therapeutic applications. The objectives of this study were to 1) generate RabmAb specific to pE₃-Aβ, 2) partially characterize the antibody using immunoassay, immunoblot, and immunohistochemical analyses, and 3) develop a sensitive ELISA to quantitate pE₃-Aβ levels in body fluids, and brain extracts.

MATERIALS AND METHODS

Peptides and antibodies

The peptides Aβ_{3 - 11,} Aβ₁₆, Aβ₄₀ and Aβ_42, pE₃-Aβ, and pE_11-17 -Aβ were obtained commercially (Bachem, King of Prussia, PA). Aβ peptides AP-29, AP-72, AP-73, AP-74, and AP-80 were synthesized by American Peptide Co. (Sunnyvale, CA) (Table 1). The following antibodies were purchased: mouse mAbs 6E10 and 4G8 (specific to an epitope present in Aβ amino acid residues 3–11 and 17–24, respectively) (Covance, Princeton, NJ), goat anti-rabbit IgG conjugated to horseradish peroxidase, and goat anti-rabbit IgG conjugated to alkaline phosphatase (Invitrogen, Rockford, IL).

Table 1

Synthetic peptides used for immunization, and specificity determination in ELISA

Peptide	Sequence
Amyloid-β peptide (human and rabbit) (Aβ₄₂)	₁DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA₄₂
Amyloid-β peptide (rodent)	DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
Pyroglutamate amyloid beta (pE_3-42Aβ) called pE₃-Aβ	pE₃-FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
AP-29	pE₃-FRHDSG-aminohexanoyl-C_nh2
AP-80	pE₃-FRHDSGYE_nh2
AP-72	pE₃-FRHDSG_nh2
AP-73	pE₃-FRHDS_nh2
AP-74	pE₃-FRHD_nh2

Generation and purification of RabmAb to pE₃-Aβ

The detailed methods for the immunization of rabbits, hybridization, fusion, selection of clones, and purification of antibodies have been described previously [35 –37]. Briefly, AP-29 peptide was conjugated to keyhole limpet hemocyanin, and was immunized in rabbits. The specificity of the antisera was examined using ELISA. The spleen from a rabbit showing the highest antibody titer, affinity, and specificity to pE₃-Aβ in ELISA, was removed, and shipped on ice to Epitomics (Abcam, Burlingame, CA), which performed fusion, cloning, and initial ELISA screening. Multiclones were screened for high titer antibodies specific to pE₃-Aβ by ELISA in our Institute. Those clones that secreted high titer antibodies specific to pE₃-Aβ, and showing high affinity were selected, and subcloned by the limited dilution method. The antibodies from culture supernatants were purified by passing through a Protein-A sepharose column. The immunoglobulin isotype of RabmAb to pE₃-Aβ was determined by an ELISA as previously described [38].

Indirect ELISA

Wells of microtiter plates (Nunc Maxisorp, Thermo Scientific, Rockford, IL) were coated with 1 μg/ml of peptide in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. After three washes with PBS containing 0.05% of Tween-20 (PBST), plates were blocked with 1% BSA. After extensive washing, 100 μl of culture supernatants or purified antibodies were added to the wells, and the plates were incubated overnight at 4°C. The plates were washed, and 100 μl of goat anti-rabbit IgG conjugated to alkaline phosphatase, diluted 1:1,000, was added, and plates were incubated for 2 h at room temperature. Color development was performed by adding 100 μl of p-nitrophenyl phosphate in 10% diethylamine (DEA). After 30 min, the absorbance was read at 405 nm using a microplate reader.

Epitope mapping by ELISA inhibition assay

The assay was carried out as described previously [36, 37]. Briefly, wells of microtiter plates were coated with 100 μl of 2 μM of pE₃-Aβ in carbonate-bicarbonate buffer, pH 9.6. 100 pm of RabmAb to pE₃-Aβ was mixed with the test inhibitor peptide at 0–10 μM concentrations, and incubated at 4°C overnight. The wells were washed and the amounts of bound antibody were determined. Concentrations of peptides that inhibited antibody binding by 50% were calculated from plots of percent inhibition versus peptide concentration. In addition to pE₃-Aβ, a number of Aβ peptides (Table 1) were used as inhibitors.

Dissociation constant measurements

The K_D of pE₃-Aβ-RabmAb to pE₃-Aβ was determined by using the ELISA method of Friguet et al. [39]. In brief, 100 μl aliquots of a 0.1 nM solution of the antibody were incubated with increasing concentrations (0.1 nM-100 nM) of antigen for 18 h at 4°C. The reactions were conducted in triplicate. Aliquots of these solutions were transferred to wells coated with 22 pmol per/well of pE₃-Aβ. After a second incubation of 1 h at room temperature, the aliquots were removed and the amounts of bound antibody were determined with an anti-rabbit IgG conjugated to alkaline phosphatase. This method may be compromised by the dissociation of the original antigen—antibody complex during the second incubation. To assess this possibility, the aliquot withdrawn after the second incubation was reapplied to a second well and re-assayed. These results were less than 10% of the total antibody in the aliquot; therefore, the initial measurements were considered to be valid.

The data were plotted according to a rearranged expression for the equilibrium constant: Ao/A = 1+α/K_D, where Ao is the absorbance in the absence of inhibitor peptide, A is the absorbance in the presence of the peptide, and α is the concentration of free peptide. Alpha can be calculated from the absorbance readings as follows: α= [Abo] –0.2×(Ao – A)/Ao. The factor 0.2 is the concentration of antibody combining sites in the original mixture.

Detection of pE₃-Aβ by dot blotting

Synthetic pE₃-Aβ, Aβ₄₀, and Aβ₄₂ were used as standards. The dilutions of the peptides were made from the stock solutions of peptides solubilized in hexafluoro-2- propanol (Sigma, St. Louis, MO) at a concentration of 1 mg/ml. The peptides were dispersed in an ultrasonic disintegrator, and were serially diluted in water to 2 fmol/μl. One μl of each peptide solution was applied onto a 0.1 μm nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membranes were boiled in PBS for 5 min to denature peptides, as described previously [40, 41]. The untreated or denatured membranes were probed with RabmAb to pE₃-Aβ at a concentration of 0.5–0.6 μg/ml to detect pE₃-Aβ peptide. To detect Aβ₄₀ and Aβ₄₂ peptides, mouse mAb 6E10 was used. The reactions were developed using goat anti-rabbit IgG conjugated to alkaline phosphatase as described previously, and the intensities of the dots were measured by photo-densitometry using SigmaGel software (Jandel Scientific Software, San Raphael, CA).

Amyloid proteins extraction of DS and controls brain tissue

Frozen samples of frontal cortex were obtained from the Brain and Tissue Bank for Developmental Disabilities and Aging, Staten Island, NY. The brains were collected at autopsy between 3 and 6 h postmortem from two non-demented controls, aged 59 and 68, one non-demented case with cerebral amyloid angiopathy aged 55 and two persons with DS, aged 47 to 59. The frozen brain samples were homogenized in a Potter-Elvehjem homogenizer in 10 X volume of lysing buffer: 10 mM TRIS buffer pH 7.4 containing 0.65% NP-40, 1 mM EDTA and complete protease inhibitor cocktail (Roche, Mannheim, Germany), sonicated for 2 min and filtered through a nylon mesh (75 μm) to remove blood vessels. The blood vessels were not removed from a lysate from a vascular amyloidosis case that was tested in immunoblotting. Protein contents in lysates were measured by BCA assay (Pierce, ThermoFisher Scientific, and Rockford, IL).

The extraction of Aβ water soluble, SDS soluble, and formic acid fractions was carried out according to the described method [42]. Briefly, brain lysates’ samples containing 400 μg of proteins, adjusted with lysing buffer to the volume of 100 μl, were centrifuged at 14,000 g at 4°C for 30 min to obtain the water soluble fraction (supernatant 1) of Aβ peptides. The pellets were dissolved in 2% SDS, sonicated and centrifuged at 14,000 g at 4°C for 30 min to obtain the SDS soluble fraction (supernatant 2). The pellet was solubilized in 70% formic acid, vortexed, sonicated and lyophilized in Speedvac. The formic acid treated pellets were solubilized in 40 μl of 2 X lithium dodecyl sulfate (LDS) sample buffer and heated at 70°C for 5 min.

Detection of pE₃-Aβ in human brains by immunoblotting

The brain supernatant fractions 1 and 2 obtained from lysates’ samples containing 37 μg proteins, and formic acid extract obtained from 75 μg protein of lysates’, were subjected to SDS-PAGE in 8–15% gradient gels and transferred onto nitrocellulose membranes. Synthetic pE₃-Aβ was used as standards. RabmAb 10-2-3 was used at a concentration of 0.4 μg/ml. The secondary antibody was goat anti-rabbit IgG conjugated to alkaline phosphatase. Part of the membranes were separately probed with mAb 6E10. The blots were developed with nitrotetrazolium blue and BCIP [40].

Immunofluorescence and confocal microscopy

Samples of autopsy brain frontal cortex, formalin-fixed from 4 DS cases (aged 41, 54, 59, and 67 years) and 2 non-demented controls (aged 32 and 33 years) were obtained from the Brain Bank and Tissue Bank for Developmental Disabilities and Aging, New York State Institute for Basic Research in Developmental Disabilities Staten Island, NY.

Free-floating 50-μm sections of formalin-fixed and polyethylene glycol–embedded temporal cortex were treated with 70% formic acid for 20 min, and used for detection of Aβ species by immunofluorescence and confocal microscopy [43]. The antibodies used were RabmAb to pE₃-Aβ at a concentration of 1.3 μg/ml, and mouse mAb 4G8 at a concentration of 0.6 μg/ml. Secondary antibodies were affinity-purified donkey antisera labeled with Alexa 555 and 488 specific for rabbit and mouse, respectively (Invitrogen/Molecular Probes, Grand Island, NY, USA). Nuclei were counterstained with TO-PRO-3-iodide (TOPRO-3i) (Invitrogen/Molecular Probes). Images were captured using a Nikon C1 confocal microscope system and the EZC1 software. Images were collected in three channels at the amplifications at which the background staining was minimal. Specificity of immunostaining was confirmed as described previously [43]. The background autofluorescence in the sections was evaluated in unstained sections, and in sections stained lacking primary antibodies.

Immunohistochemistry

Brain hemispheres of three patients diagnosed with AD (aged 73, 82, 86 years), and three persons diagnosed with DS and showing pathology of AD (aged 56, 65, and 67 years) were fixed in 10% buffered formalin, dehydrated in ascending concentrations of ethyl alcohol, embedded in polyethylene glycol (PEG; Merck #807 485), cut into 50 μm-thick sections, and stored in 70% ethyl alcohol [44]. To enhance immunoreactivity of Aβ, the tissue sections were pretreated with 70% formic acid for 20 min [45]. Endogenous peroxidase was blocked with 0.2% hydrogen peroxide. Non-specific binding of antibodies was blocked with a solution of 10% fetal bovine serum in PBS. To detect the pattern of pE₃-Aβ distribution, the sections were incubated overnight at room temperature with rabbit RabmAb to pE₃-Aβ diluted 1:500 in 10% fetal bovine serum. Immunoreactivity of amyloid was compared to reaction in sections incubated with 4G8 diluted 1:50. The sections were treated with an extravidin peroxidase conjugate diluted 1:200 for 1 h, and the reaction was visualized with diaminobenzidine (0.5 mg/mL with 1% hydrogen peroxide in PBS). To distinguish fibrillar and nonfibrillar amyloid deposits, sections were stained with Thioflavin S, and examined in fluorescence microscope [37].

Tissue samples were identified using coded number provided by the New York State Brain and Tissue Bank for Developmental Disabilities and Aging. Methods applied were approved by the Institutional Board at the New York State Institute for Basic Research in Developmental Disabilities.

Collection of CSF and plasma

Cerebrospinal fluid (CSF) from 11 patients with probable AD, and 10 controls with no history of dementia, and plasma from 8 patients with probable AD, and 10 controls were received from the Alzheimer’s Disease Research Center, NYU School of Medicine, NY. In addition, CSF from 11 patients with probable AD, and 11 controls were purchased from PrecisionMed (Solana Beach, CA). All samples were coded, and laboratory personnel were blinded to group assignments.

Amyloid proteins extraction of soluble Aβ from AD brain tissue

Brain diethylamine (DEA) extracts from 2 AD and 2 controls were received from the NYU School of Medicine, Alzheimer Disease Research Center, New York, NY. The extraction of soluble Aβ from AD brain was carried out according to the method described earlier [46]. Briefly, brain homogenates were mixed with an equal volume of cold 0.4% DEA/100 mM NaCl, and subsequently centrifuged at 100,000×g for 1 h at 4°C. The supernatant was neutralized with 1/10 volume of 0.5 M Tris, pH 6.8, and stored at –80°C until used for ELISA.

Quantitation of pE₃-Aβ in plasma, CSF, and brain extracts using chemiluminescent ELISA substrate

Briefly, wells of Nunc white opaque 96 well microtiter plates were coated with 100 μ1 of 2.5 μg/ml of mouse mAb 6EI0 (a capture antibody), and incubated overnight at 4°C [47]. After the plates were washed with PBST, they were blocked for 1 h with 1% bovine serum albumin (BSA) in PBST to avoid non-specific binding. The plates were washed, and 100 μl of pE₃-Aβ standards (ranged from 10,000 pg/ml to 10 pg/ml diluted in PBST+1% BSA), and plasma or CSF samples diluted 1:2 were added, and incubated for 2 h at room temperature, and 4°C overnight. Brain extracts were diluted in a range of 1:2 to 1:30. After washing, plates were incubated with 100 μl of RabmAb specific to pE₃-Aβ (a detection antibody) for 2 h at room temperature. After washing, 100 μl of goat anti-rabbit IgG conjugated to horseradish peroxidase, diluted 1:100,000 in PBST, was added, and plates were incubated for 2 h at room temperature. Plates were washed again, and developed with Super Signal ELISA Femto Maximum Sensitivity Substrate (100 μl/well) according to the manufacturer’s instructions (ThermoScientific, Rockford, IL). The plates were mixed for 1-2 min, and the luminescence signal for each well was measured at emission at 425 nm according to the kit instructions using a Biotech Synergy H1 hybrid reader. The relationship between the luminescence counts and pE₃-Aβ concentration was determined using a 4-feature logistic logarithm function. Nonlinear curve fitting was performed with a commercially available program (KinetiCalc, BioTek Instruments, Inc., Winooski, VT) to convert luminescence counts of plasma, CSF, or brain extracts to estimated protein concentration. A ten-point calibration curve ranging from 10,000 pg/ml to 10 pg/ml was constructed to analyze pE₃-Aβ concentrations in the samples.

RESULTS

Specificity and affinity of RabmAb to pE₃-Aβ

A panel of RabmAb hybridomas were screened for specificity and affinity against pE₃-Aβ peptide using an ELISA. For further characterization, we selected clone 10-2-3, which showed the highest antibody titer to pE₃-Aβ. The clone was specific for pE₃-Aβ, since it showed no reactivity with Aβ₁₆, Aβ₄₀, Aβ₄₂, Aβ_3-11, and pE_11-17 Aβ in ELISA (Fig. 1). The isotype of RabmAb to 10-2-3 was found to be a IgG class.

Fig.1

RabmAb 10-2-3 to pE₃-Aβ specifically recognizes pE₃-Aβ by indirect ELISA. The titer was defined as the highest dilution of RabmAb 10-2-3 showing absorbance of 1.0 at 405 nm in an ELISA reader.

The K_D was calculated from the slope of the data plotted in Fig. 2, and was found to be 1 nM. This value was confirmed by three datasets, which differed in slope by only 2%, which is much less than the variance due to expected experimental errors. Nevertheless, the result was confirmed by replotting the data using a Scatchard plot (data not shown). The K_D may actually be less than 1 nM, because the antibody preparation was assumed to be 100% pure, when it may actually contain some non-specific IgG.

Fig.2

K_D determination for pE3-Aβ—RabmAb 10-2-3 complexes. The antibody (0.1 nM) was incubated with increasing concentrations of pE₃-Aβ and assayed as described in the Methods section. Three datasets (denoted by open circles, closed circles and squares) were analyzed by plotting the data as a double-reciprocal plot of the fraction of free antibody (A/Ao) versus the concentration of free peptide (alpha). The slope of the line corresponds to K_D.

Epitope mapping

RabmAb 10-2-3 was added to a microplate coated with pE₃-Aβ in the presence of varying amounts of inhibitory peptides (Table 1). The amount of peptide that inhibited antibody binding by 50% was calculated, and the extent of inhibition of binding of the antibody to the plate was taken to be a measure of the affinity of the antibody for the peptide (Table 2). pE₃-Aβ and pE₃-₁₁ -Aβ were the strongest inhibitors. AP-29, a synthetic peptide containing the nine amino terminal residues of pE₃-Aβ, was also a good inhibitor, as were peptides AP-80, AP-72, AP-73, and AP-74, which respectively contained nine, seven, six and five C-terminal residues of pE₃-Aβ respectively. pE_11-17 Aβ, a peptide lacking the first eight terminal residues, showed weak inhibition. Aβ_3-11, Aβ₁₆, Aβ₄₀, and Aβ₄₂ showed weak or no inhibition. From these results, we concluded that the epitope of RabmAb 10-2-3 lies within the sequence pE₃-FRHD_nh2.

Table 2

Inhibition of Rabmab 10-2-3 by selected peptides

Peptide	Concentration (nM)	Extent of Inhibition
pE₃-Aβ	1,000	50%
pE_3-11 -Aβ	1,300	50%
pE_{3 - 9} -Aβ	600	50%
pE_{3 - 8} -Aβ	500	50%
pE_{3 - 7} -Aβ	500	50%
pE_{3 - 6} -Aβ	500	50%
pE_11-17 -Aβ	1,200	8%
Aβ_3-11	700	1%
Aβ₁₆	3,900	6%
Aβ₄₀	1,700	0%
Aβ₄₂	1,700	6%

Detection of synthetic pE₃-Aβ

The optimal antibody reaction with synthetic pE₃-Aβ was between concentrations of 0.5 and 1.2 μg/ml, as tested by dot blotting. Denaturation of the peptide by boiling the membrane had no effect on detection of pE₃-Aβ with RabmAb 10-2-3. The threshold of the peptide detection with this antibody was about 2 fmol of pE₃-Aβ. The intensity of the antibody reactions with pE₃-Aβ peptide was proportional to the amounts of the peptide, and ranged from 2-64 fmols (Fig. 3a, b). RabmAb 10-2-3 at a concentration of 0.5 μg/ml did not react with 128 fmol of Aβ₄₀ and Aβ₄₂ (Fig. 3a). The reaction of RabmAb 10-2-3 was not affected by polymerization or aggregation of the peptide, as indicated by similar reactivity with the pE₃-Aβ peptide freshly diluted from HFIP stock solution, and peptide allowed to polymerize in PBS (Fig. 3c). Aggregation or fibrillization of pE₃-Aβ showed no reaction with mouse mAb 6E10 (Fig. 3c).

Fig.3

Characterization of the RabmAb 10-2-3 by dot blotting (a) using synthetic peptides pE₃-Aβ, Aβ₄₀, and Aβ₄₂. The antibody was used at a concentration of 0.55 μg/ml. The graph (b) shows densitometrical measurements of the reaction with the dots. RabmAb 10-2-3 reacts with aggregated pE₃-Aβ (f) and monomeric pE₃-Aβ peptide (m), respectively. However, aggregation or fibrillization of the peptide eliminated the reaction with 6E10 (c).

Detection of pE₃-Aβ in brain cortex

The pE₃-Aβ species were detected by immunoblotting with RabmAb 10-2-3 in the cortex from vascular amyloidosis and DS cases in the SDS insoluble, and formic acid-extractable fractions (Fig. 4). All fractions obtained from cortices of DS cases, that is, the water soluble, membrane bound (SDS extractable) and insoluble (formic acid-extractable) contained other Aβ peptide species, which were detected with mAb 6E10 (Fig. 4). The pE₃-Aβ peptide was not detected with RabmAb 10-2-3 in any of the fractions tested in two control brain cortices (data not shown).

Fig.4

Detection of pE₃-Aβ in brain extracts from patient with vascular amyloidosis (VA) and person with DS by immunoblotting using RabmAb 10-2-3. The lanes contained the water-soluble fraction of brain lysate (s), membrane-bound fraction (m), and formic acid-extractable fraction (f). pE₃-Aβ peptide standard (std) was loaded (10 fmol per lane), and RabmAb 10-2-3 was used at a concentration of 0.4 μg/ml. The fractions were also probed with mAb 6E10 which reacted with pE₃-Aβ, and unmodified Aβ peptides.

Immunohistochemical reactions for pE₃-Aβ in brain sections

Numerous amyloid plaques and vascular amyloid deposits were detected by immunostaining for Aβ with mouse mAb 4G8 in all DS brains. Double immunostaining with 4G8, and RabmAb 10-2-3 revealed the staining for pE₃-Aβ in central parts of plaques (Fig. 5). The data was similar in other DS brain tissues. The fraction of plaque section area immunoreactive for pE₃-Aβ as well as the intensity of the reaction varied greatly among cases, and individual plaques in each case, from small scattered granules to more extensive staining that included the core and more peripheral parts of the plaque. pE₃-Aβ was not detectable in peripheral regions of classical plaques or diffuse plaques (Fig. 5).

Fig.5

Layer 5 of frontal cortex in an individual with DS (aged 59 years). Amyloid plaques were identified by the presence of reaction with 4G8. pE₃-Aβ was detected with rabbit RabmAb 10-2-3. The nuclei were counterstained with TOPRO-3i (blue color).

Immunocytochemistry

Immunostaining with 4G8 and RabmAb 10-2-3 demonstrated similar patterns of immunolabelling of plaques in the hippocampal formation, cerebral cortex, and striatum of individuals with DS and showing AD pathology (56 and 67 years of age), and those diagnosed with AD (73–86 years). However, in general the number of pE₃-Aβ positive plaques, and the extent of saturation with immunoreactive amyloid was less than that compared with 4G8 (Fig. 6). RabmAb 10-2-3 detected fibrillar amyloid in cortical plaques, and in the wall of cortical and leptomeningeal arterioles (Fig. 7). Strong immunoreactivity for pE₃-Aβ was detected in both, diffuse nonfibrillar amyloid deposits in the parvocellular layer of the presubiculum, and in fibrillar plaques in the presubiculum pyramidal layer. The non-uniform immunoreactivity was detected in diffuse nonfibrillar Aβ deposits in the molecular layer of the cerebellar cortex.

Fig.6

Serial sections of the hippocampal formation and fusiform gyrus of a 56-year-old DS subject diagnosed with AD immunostained with mouse mAb 4G8 for Aβ (a, e) or RabmAb 10-2-3 for pE₃-Aβ (b, f). Both antibodies detected numerous amyloid plaques and their characteristic layer-specific distribution in all sectors of the cornu ammonis (CA), the molecular layer in the dentate gyrus (DG) (a, b), and in the cerebral cortex (fusiform gyrus, e, f). Control sections without primary antibody (4G8; c and g; or 10-2-3, d and h) showed no immunoreactivity for Aβ or pE₃-Aβ.

Fig.7

RabmAb 10-2-3 detected pE₃-Aβ not only in fibrillar cortical plaques (temporal cortex; a) and in the wall of cortical and leptomeningeal vessels (arrowheads; b), but also in diffuse amyloid deposits in the presubiculum parvocellular layer (asterisk; d), and in the molecular layer of the cerebellar cortex (asterisk; c).

Development of ELISA

In order to quantitate pE₃-Aβ levels in body fluids a sandwich ELISA was developed as described in the Methods section (Fig. 8). The lower limit of reliable quantitation was 10 pg/ml; this was defined as the pE₃-Aβ concentration that gave a luminescence signal greater than background plus 2 times the standard deviation of the background. Both intra- and inter-day assay variability were less than 20%. The assay was specific for pE₃-Aβ since equal concentrations of Aβ_3-42 or Aβ₄₂ were not detected above background levels in ELISA.

Fig.8

Standard curve from luminescent ELISA performed for the pE₃-Aβ assay. Results are expressed as the mean±SD of 3 individual standard curves.

The specificity of the antibody to pE₃-Aβ was examined by coating the wells of microtiter plates with 6E10, and incubated these wells with pE₃-Aβ in the usual manner. Half of these wells were treated with RabmAb to pE₃-Aβ, and the other half with equivalent amount of normal rabbit IgG. Wells were developed as described earlier. Our data showed that wells treated with RabmAb to pE₃-Aβ showed luminescence intensity as expected. However, wells treated with normal rabbit IgG showed no measurable luminescence.

pE₃-Aβ levels in CSF and plasma

CSF levels of pE₃-Aβ were detected in 3 of 19 AD and 3 of 20 non-AD controls. Since a majority of CSF showed no detectable levels, that is, below the lowest standard (10 pg/ml), no statistical analyses were carried out. Plasma levels of pE₃-Aβ were not detected in AD or non-AD controls.

pE₃-Aβ Levels in brain extracts

Preliminary studies showed that pE₃-Aβ levels were detectable in DEA and formic acid in AD and DS brain extracts with RabmAb 10-2-3 in ELISA (Table 3). The amount of pE₃-Aβ was about 100-fold higher in formic acid extracts than DEA extracts.

Table 3

Levels of pE₃-Aβ in cortices of AD, DS, and controls

Neuropathological Diagnosis	Cortex Extract	Age in years	μg/mg of cortex proteins
AD	DEA^a	91	0.008
AD	DEA	90	0.016
Control	DEA	92	0.0002
Control	DEA	95	0.002
DS	FA^b	59	4.77
DS	FA	47	1.52
Control	FA	59	0.009
Control	FA	68	0.009

^aDiethylamine; ^bFormic acid extract.

DISCUSSION

In this study, we generated and partially characterized a novel RabmAb clone 10-2-3 raised to a peptide corresponding to amino acid residues pE_{3 - 8}-Aβ (Table 1). Our ELISA inhibition test showed that the clone was specific to pE₃-Aβ, since Aβ₄₀ or Aβ₄₂, even at high concentrations, did not show inhibition of the binding of pE₃-Aβ and RabmAb 10-2-3. Investigators have produced rabbit polyclonal antibodies specific to pE₃-Aβ [31 –33]. However, the production of rabbit polyclonal antibodies is time consuming and the resulting antisera can differ widely in their specificities and affinities. Moreover, rabbit polyclonal antisera to C-terminal residues of Aβ which appeared to be specific in immunoblots, and ELISA often gave discrepant results in immunohistological studies [36]. To our knowledge, no other study has been published on the generation and partial characterization of RabmAb to pE₃-Aβ.

Our data showed that the isotype of RabmAb to pE₃-Aβ was found to be of the IgG class, consistent with most other rabbit monoclonal antibodies to Aβ [34]. Determination of isotype is important in characterizing the antibody, because different purification procedures for enzymatic fragmentation of the antibodies depend on the isotype or subclass of the antibody [38].

The RabmAb 10-2-3 was found to be specific for pE₃- Aβ by dot blotting method, as the antibody did not react with Aβ₄₀ and Aβ₄₂ peptides. The latter proved that the recognized epitope requires the C-terminal pyroglutamate. The epitope for the antibody was readily accessible without peptide denaturation in contrast to the epitope of 6E10, which required treatment with formic acid [45].

RabmAb 10-2-3 also detected pE₃-Aβ in brain lysates in immunoblotting. However, pE₃-Aβ was detected only in the SDS insoluble brain fraction, after extraction with formic acid. This is consistent with the described high propensity of the peptide to aggregate in vitro. A high tendency to aggregate can be also demonstrated in the brain, as the peptide is detected in formic acid extractable fraction.

Double immunostaining revealed pE₃-Aβ in the cores of almost all classical amyloid plaques, but not in a majority of diffuse plaques. These localizations are consistent with the hypothesis, that the deposition of pE₃-Aβ is an initiating event in amyloid formation [16, 17]. The reaction for pE₃-Aβ is also present in vascular amyloid deposits. The presence of immunoreactivity in the DS brain sections with RabmAb 10-2-3 in the absence of immunoreactivity with 4G8 indicates that aggregation or folding of the pE₃-Aβ has made the epitope inaccessible for 4G8 antibody, while leaving the N-terminus accessible for RabmAb 10-2-3. This explanation is supported by the results of dot-blotting using aggregated or fibrillized peptide pE₃-Aβ (Fig. 3c).

Our epitope mapping data showing that the epitope of RabmAb 10-2-3 lies within the sequence of pE₃-FRHD_nh2 is consistent with those reported in rabbit polyclonal antibody to pE₃-Aβ, and mouse monoclonal antibody to pE₃-Aβ, pE₃-FRHDSGY_nh2 [24, 33]. Since the epitope is not found in the full length Aβ, which is absent in N-amino truncated peptides, investigators have concluded that pE₃-Aβ is potentially useful target of immunotherapeutic treatment of AD [20 , 33].

The K_D for the antibody is 1 nM, which, along with its high specificity for the pE₃-FRHD sequence, suggests that this antibody should be useful for detecting pE₃-Aβ in tissues and body fluids of AD and DS. This K_D is similar in magnitude to the K_D’s determined for other high affinity Aβ antibodies that we have examined using this method [36, 37].

Employing the RabmAb 10-2-3 in a sandwich ELISA, we generated a standard curve to quantitate pE₃-Aβ levels in body fluids. Although investigators produced rabbit polyclonal antibodies to pE₃-Aβ, there was no data published on the quantitation of pE₃-Aβ in body fluids [31 –33]. Others have quantitated pE₃-Aβ levels in rodent and AD brain homogenates using a commercial kit (IBL, Japan) [14 , 48]. However, the kit is not sensitive enough to detect pE₃-Aβ levels in body fluids. In addition, there was limited or no information available about the generation or characterization of the antibody. Thus a direct comparison of our antibody with the commercial assay is difficult.

We were unable to quantitate pE₃-Aβ levels in a majority of CSF and plasma samples using chemiluminescent ELISA substrate. Our findings are consistent with previous studies where pE₃-Aβ was not detected in CSF or plasma of AD or Tg mice by using ELISA [18 , 32]. The data suggested that pE₃-Aβ is concentrated primarily in the brain parenchyma and the assay was not sensitive enough to detect pE₃-Aβ in CSF or plasma. The reason for the lack of detection of pE₃-Aβ in body fluids is not known. However, investigators have hypothesized that increased pE₃-Aβ in brain parenchyma might be due to reduced blood-brain barrier clearance or impaired secretion mechanisms [32]. The higher levels of pE₃-Aβ found in insoluble AD cortex reflects higher hydrophobicity and aggregation kinetics of pyroglutamate-modified Aβ compared with the full length unmodified Aβ [48].

Although minor or small peaks consistent with pE₃-Aβ were detected in brain extracts in mass spectrometry [27, 28], the peaks were not detected in CSF or plasma [32]. The data suggested that pE₃-Aβ is highly insoluble, and the concentration of pE₃-Aβ in body fluids is very low compared to brain extracts [23]. Thus mass spectrometry was not primarily designed to determine whether the antibody is binding to pE₃-Aβ in body fluids.

Since there has been a continuing challenge for developing sensitive methods to diagnose AD, or to measure its progression, future studies using a more sensitive platform such as single molecule assay (SIMOA, Quanterix, Lexington, MA) are warranted. The latter is about 1,000-fold more sensitive than conventional ELISA. [49, 50].

Investigators have shown depositions of pE₃-Aβ in brain homogenates from patients with AD by using ELISA kit, electrophoresis and western blotting, or immunoprecipitation and mass spectrometry [10 , 48]. The amount of pE₃-Aβ in AD brains ranged from 1% to 27% depending on the method used [10, 30]. Here we examined pE₃-Aβ levels in a small number of brain DEA soluble Aβ supernatants from AD or lysates from DS brain, and showed that pE₃-Aβ levels were detectable in ELISA using RabmAb 10-2-3. These are preliminary studies, and further detailed validation of the assay is warranted. We intend to replicate these findings using other sample sets, and to determine whether our antibody might serve to differentiate AD from healthy controls or from those with other neurodegenerative disorders. Our findings of low levels of amount of pE₃-Aβ in DEA extracts versus higher levels of pE₃-Aβ found in formic acid extracts are consistent with those reported in the literature [28 , 48].

Our rabbit mAb 10-2-3 detects pE₃-Aβ in the central portion of the classic fibrillar plaque, and in a large portion of diffuse nonfibrillar amyloid deposits in the parvocellular layer of the presubiculum, the molecular layer of the cerebellar cortex, and in vessels affected with amyloid angiopathy. This pattern is consistent with the results of numerous studies showing the presence of pE₃-Aβ in diffuse amyloid and cored plaques, and in blood vessels of postmortem brains from patients with AD, DS with AD, and elderly nondemented controls [50 –52]. Double immunostaining revealed that pE₃-Aβ is present in all cortical plaques and cortical amyloid angiopathy along with general Aβ deposits.

A number of anti-Aβ immunotherapy trials in patients with AD have failed to slow cognitive or functional decline, and led to adverse events in response to both active or passive immunization approaches [54, 55]. Since current immunotherapies may not be targeting pE₃-Aβ, recent studies have suggested that passive immunization with pE₃-Aβ may have potential for treatment of AD [19 , 26]. pE₃-Aβ is the predominant component of all N-terminal truncated Aβ peptides in AD, and DS brains. Studies with Tg mouse models for AD showed that pE₃-Aβ recognizes oligomeric forms of Aβ, and have low tendency to detect amyloid peptides [23, 56]. pE₃-Aβ also induces neuronal loss and behavioral deficits [23 , 51]. Since pE₃-Aβ appears to play a critical role in the initiation of AD, it represents a promising therapeutic target [20].

There are a number of limitations with the present study. First, measurements of pE₃-Aβ in body fluids are made close to the instrument limits of detection. Ultrasonic single molecule array immunoassay is worth developing to quantitate the low levels of pE₃-Aβ in body fluids [50, 57]. Second, our immunohistochemical studies showed the presence of pE₃-Aβ in plaques, and blood vessels in brains of DS persons with AD, and those with definite AD. However, we did not extend our findings in patients with familial AD carrying APP mutations, patients with non-AD type dementia or Tg mouse models of AD, since our aim was to generate, and partially characterize RabmAb to pE₃-Aβ. Finally, we did not evaluate our antibody for passive immunization in therapeutic studies in Tg mouse models of AD. This will be the subject of a future study.

Despite the limitations, we have reported here a novel RabmAb 10-2-3 with greatly enhanced selectivity for pE₃-Aβ with IgG class, and partially characterized using immunoassay, and immunohistochemical analyses. In addition, we have described a sensitive sandwich ELISA using a combination of RabmAb 10-2-3 and mouse mAb 6E10. One of the advantages of the production of RabmAbs when compared to mouse mAbs, is that it offers increased sensitivity with no apparent loss of specificity. Thus, RabmAb can be used in higher working dilutions (5 to 10 times on average) in ELISA or immunohistochemical studies with little or no background [58, 59]. Incorporating our antibody with quantitative proteomic techniques would generate an important tool to aid the early diagnosis of AD, in following stage of its progression or defining response to medications of AD. Since rabbit antibody is more closely related to human than mouse derived antibodies, there is a potential for developing humanized monoclonal antibodies to pE₃-Aβ as treatment for AD.

Footnotes

ACKNOWLEDGMENTS

The study was supported in part by a grant from the NIH (AG 11290), and by the NY State Office of People with Developmental Disabilities, and Epitomics, a division of Abcam.

Authors’ disclosures available online ().

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Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-β 3-42 (pE 3 -Aβ)

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Peptides and antibodies

Generation and purification of RabmAb to pE3-Aβ

Indirect ELISA

Epitope mapping by ELISA inhibition assay

Dissociation constant measurements

Detection of pE3-Aβ by dot blotting

Amyloid proteins extraction of DS and controls brain tissue

Detection of pE3-Aβ in human brains by immunoblotting

Immunofluorescence and confocal microscopy

Immunohistochemistry

Collection of CSF and plasma

Amyloid proteins extraction of soluble Aβ from AD brain tissue

Quantitation of pE3-Aβ in plasma, CSF, and brain extracts using chemiluminescent ELISA substrate

RESULTS

Specificity and affinity of RabmAb to pE3-Aβ

Epitope mapping

Detection of synthetic pE3-Aβ

Detection of pE3-Aβ in brain cortex

Immunohistochemical reactions for pE3-Aβ in brain sections

Immunocytochemistry

Development of ELISA

pE3-Aβ levels in CSF and plasma

pE3-Aβ Levels in brain extracts

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Generation and purification of RabmAb to pE₃-Aβ

Detection of pE₃-Aβ by dot blotting

Detection of pE₃-Aβ in human brains by immunoblotting

Quantitation of pE₃-Aβ in plasma, CSF, and brain extracts using chemiluminescent ELISA substrate

Specificity and affinity of RabmAb to pE₃-Aβ

Detection of synthetic pE₃-Aβ

Detection of pE₃-Aβ in brain cortex

Immunohistochemical reactions for pE₃-Aβ in brain sections

pE₃-Aβ levels in CSF and plasma

pE₃-Aβ Levels in brain extracts