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Journal of Neuroscience Methods Characterisation of two antibodies ...


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Journal of Neuroscience Methods Characterisation of two antibodies ...

  1. 1. Journal of Neuroscience Methods 176 (2009) 206–212 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: Characterisation of two antibodies to oligomeric A␤ and their use in ELISAs on human brain tissue homogenates Zoë van Helmonda,∗ , Kate Heesomb , Seth Lovea a Dementia Research Group, Institute of Clinical Neurosciences, Clinical Science at North Bristol, University of Bristol, Bristol BS16 1LE, UK b Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK a r t i c l e i n f o Article history: Received 22 February 2008 Received in revised form 28 August 2008 Accepted 1 September 2008 Keywords: Oligomeric A␤ ELISA Human brain tissue Alzheimer’s disease a b s t r a c t Oligomeric forms of A␤ are believed to be the major toxic species of this peptide in Alzheimer’s disease (AD). Although the characterisation of oligomer-specific antibodies has been reported, these have not been successfully incorporated into an enzyme-linked immunosorbent assay (ELISA), and measurement of the levels of oligomeric A␤ in brain tissue has remained problematic. We have examined the specificity of two monoclonal antibodies, 7A1a and 1G5, for synthetic oligomers of A␤1–42 and for oligomeric A␤1–42 in human brain homogenates, and the utility of these two antibodies for measuring oligomeric A␤1–42 by sandwich ELISA. Both antibodies were found to recognise a range of synthetic oligomers of A␤1–42 but to cross-react in Western blots with a 34 kDa protein, shown by two-dimensional gel electrophoresis and mass spectrometry to be tropomyosin. However, by using 7A1a and 1G5 in combination with an A␤1–42 capture antibody, we were able specifically to detect and to measure the levels of oligomeric A␤1–42 in brain homogenates by ELISA. The development of a simple ELISA for measurement of oligomeric A␤ should facilitate further studies of the role of oligomeric species of A␤ in AD. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Alzheimer’s disease (AD) is marked by progressive decline in cognitive function and distinct pathological abnormalities that include plaques of amyloid ␤ peptide (A␤) and neurofibrillary tan- gles. The accumulation of A␤ is probably central to the development of AD (Hardy and Allsop, 1991), although the precise mechanisms remain uncertain. For many years, it was assumed that fibrillar A␤ was responsible for most of the neurodegenerative changes in AD. However, the distribution and severity of neurofibrillary pathol- ogy and neuronal loss correlate poorly with the distribution and amount of A␤ in the form of plaques (Crystal et al., 1988; Davies et al., 1988; Price and Morris, 1999). Although fibrillar A␤ may contribute to the development of AD, oligomeric forms of A␤ – particularly of A␤1–42 – are now thought to be the major toxic species. Until recently, researchers lacked a ready means of directly demonstrating A␤ oligomers in brain tissue. Kayed et al. (2003) reported the characterisation of an antibody, A11, that recog- ∗ Corresponding author at: Dementia Research Group, John James Laboratories, Frenchay Hospital, Bristol BS16 1LE, UK. Tel.: +44 117 9701212x2380; fax: +44 117 9186665. E-mail address: (Z. van Helmond). nises micellar but not soluble, low molecular weight (less than approximately 40 kDa) A␤ or fibrils. A11 was raised against A␤1–40 which had been tethered at one end to prevent its aggregation into fibrils. In vitro studies, employing ELISA and dot blot tech- niques, showed that A11 recognises oligomeric species of several amyloidogenic polypeptides in addition to A␤40 (A␤42, islet amy- loid polypeptide, ␣-synuclein, polyglutamine, insulin and prion protein), suggesting that during amyloidogenesis these share a common structure (Kayed et al., 2003). On immunofluorescent labelling of sections of brain from cases of AD brain and age- matched controls, the distribution of A11-immunopositive material was spatially distinct from that of fibrillar A␤ as demonstrated by thioflavin S fluorescence (Kayed et al., 2003). On dot blot anal- ysis of lysates from AD and control brain, A11 immunolabelling was detected only in AD cases and controls with “mild Braak changes”. Addition of A11 inhibited in vitro toxicity induced by oligomers of several types of amyloidogenic peptide (Kayed et al., 2004). A11 antibody was subsequently used by other groups to eluci- date the role of oligomers in various amyloidopathies: to determine whether extracellular protofibrillar A␤ species cause synaptic dysfunction in a triple-transgenic mouse harbouring PS1M146V, APPSwe and tauP301L mutations (Oddo et al., 2003); to establish that oligomers of ␣B-crystallin cause desmin-related cardiomy- opathy (Sanbe et al., 2004); and to clarify the role of heat-shock 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.09.002
  2. 2. Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 207 protein 104 (Hsp104) in the formation and elimination of self- replicating Sup35 NM [PSI+] yeast prions (Shorter and Lindquist, 2004). Post-embedding A11 immunoelectron microscopy was also used to investigate the ultrastructural distribution of oligomeric A␤ (Kokubo et al., 2005). A recent study suggested that A11 detects the toxic oligomers only of ␣-synuclein and A␤1–42, and not non-toxic aggregates (Ehrnhoefer et al., 2008). The production of two further antibodies (M93 and M94) raised against synthetic oligomeric A␤ was described by Lambert et al. (2001). These were used for immunofluores- cence and immunoperoxidase detection of oligomers (Lacor et al., 2004) and for immunoblots that demonstrated SDS-stable oligomers in AD brain tissue (Gong et al., 2003; Lacor et al., 2004). Rapid, sensitive measurement of the levels of A␤1–42 oligomers in brain tissue would be greatly facilitated by the development of a specific enzyme-linked immunosorbent assay (ELISA). As far as we are aware no such assay has been reported. Our initial attempts at developing an assay that used A11 were unsuccess- ful. Recently, two further oligomer-specific A␤ antibodies have become available from New England Rare Reagents, Portland, Maine: mouse monoclonal antibodies (clones 7A1a and 1G5) raised against synthetic oligomers of A␤40. The immunogen had a pro- prietary synthetic peptide structure that was designed to mimic the three-dimensional structure thought to form within aggregated A␤ (New England Rare Reagents, personal communication) and the antibodies have been reported to detect low molecular weight oligomers and protofibrils of A␤. Our aims in the present study were to examine the specificity of 7A1a and 1G5 for synthetic oligomers of A␤1–42 and for oligomeric A␤1–42 in human brain homogenates, and to develop a sandwich ELISA for measuring oligomeric A␤1–42. 2. Materials and methods 2.1. Study cohort Brain tissue from 15 cases of neuropathologically confirmed AD (CERAD classification ‘definite AD’, Braak tangle stage V or VI) (Braak and Braak, 1991) and 13 neuropathologically normal con- trols without AD were studied by Western blot. These same cases together with a further 94 AD and 29 control cases were sub- sequently analysed by ELISA. The cases were selected from the South West Dementia Brain Bank (SWDBB), University of Bristol. The study was conducted with the approval of Frenchay Research Ethics Committee. The AD cases ranged from 47 to 98 years in age (mean = 79.3, S.D. = 9.6) and comprised 70 females and 39 males. The post-mortem delays were between 4 and 101.5 h (mean = 44.8, S.D. = 24.5). The controls were similarly aged, from 43 to 95 years (mean = 76.6, S.D. = 10.3) and comprised 16 females and 26 males. The post-mortem delays were between 3 and 106 h (mean = 41.4, S.D. = 28.6). 2.2. Oligomerisation of synthetic Aˇ1–42 Lyophilised A␤1–42 peptide (Anaspec, CA) was dissolved in 35% acetonitrile (Ryu et al., 2004) to a final stock concentration of 1.6 mM. 10 ␮l aliquots were stored at −20 ◦C until required. Stock A␤1–42 peptide was diluted to 300 ␮M in either phosphate-buffered saline (PBS; 0.15 M sodium chloride, 1.9 mM sodium dihydrogen orthophosphate 1-hydrate, 7.5 mM di-sodium hydrogen orthophosphate 12-hydrate, pH 7.1) or Dulbecco’s Mod- ified Eagle Medium (DMEM; Gibco, Invitrogen, UK) and incubated overnight at 37 ◦C. 2.3. Thioflavin S assay This assay was adapted from a standard thioflavin T protocol (LeVine, 1993; Maezawa et al., 2008). Briefly, 50 ␮l of oligomerised A␤1–42 peptide was mixed with 150 ␮l thioflavin S solution (5 ␮M in 50 mM glycine-NaOH, pH 8.5) and the fluorescence measured using a FLUOstar OPTIMA multidetection microplate reader (BMG Labtech GmbH, Germany) with excitation at 450 nm and emission at 490 nm. Fluorescence was measured immediately after mixing and then every 2 h for 30 h, with incubation at 37 ◦C. 2.4. Brain homogenates For each case, approximately 200 mg of frontal neocortex (Brod- mann area 6) was homogenised in 1 ml lysis buffer (1% SDS, 10 mM Tris, pH 6, 0.1 mM sodium chloride, 1 ␮M phenylmethylsulphonyl fluoride, 1 ␮g/ml aprotinin). Brain tissue was homogenised in a Bertin Technologies Precellys 24 mechanical homogeniser (Stretton Scientific, Stretton, UK) at 6000 rpm for 2 s × 30 s with approxi- mately ten 2.3 mm Biospec Products zirconia-silica beads (Stratech Scientific, Newmarket, UK) in a screw cap 2 ml centrifuge tube. Samples were cooled on ice after homogenisation. Total protein was measured using Total Protein kit (Sigma–Aldrich, Gillingham, UK) and 20 ␮l aliquots were stored at −80 ◦C until required. 2.5. Western blot 5 ␮l of the 300 ␮M synthetic A␤1–42 that had been incu- bated overnight at 37 ◦C in either PBS or DMEM were diluted to 30 ␮l in sample buffer (0.5 M Tris–HCl, pH 6.8, 0.25% glycerol, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue) containing 5% ␤- mercaptoethanol. Brain tissue homogenates containing 30 ␮g of total protein were diluted to 30 ␮l in sample buffer containing 5% ␤-mercaptoethanol. 1.25 ␮g tropomyosin-3 (TPM3) recombinant protein (Abnova, TW) was, similarly, diluted to 30 ␮l in sample buffer containing 5% ␤-mercaptoethanol. Diluted samples were loaded onto a 4–20% Tris–HCl pre-cast gel (Bio-Rad, Hemel Hemp- stead, UK) and electrophoresed at 150 mV for 1 h. Proteins were subsequently transferred to nitrocellulose membrane overnight at 30 mV. Membranes were blocked for 1 h at room temperature in 10% non-fat milk in Tris-buffered saline/Tween 20 (TBS-T; 20 mM Tris base, 0.5 M NaCl, 0.05% Tween 20), incubated for 1 h at room temperature in 5% non-fat milk in TBS-T with either 1G5 or 7A1a antibodies (both 1:250), washed 3 min × 10 min in TBS-T and then incubated for 1 h at room temperature with secondary antibody horseradish-peroxidase (HRP)-conjugated horse anti-mouse IgG (1:5000, Vector Laboratories, CA) in 5% non-fat milk in TBS-T. The membrane was then washed 3 × 10 min in TBS-T and immunoreac- tive proteins were detected by enhanced chemiluminescence (GE Healthcare, NJ). 2.6. Two-dimensional gel electrophoresis/mass spectrometry Two-dimensional gel electrophoresis followed by mass spec- trometry (2DGE/MS) was applied to crude homogenate of frontal neocortex from a single AD case that had previously been anal- ysed for oligomeric A␤ levels by Western blot. The sample was precipitated using 2D Clean-Up Kit according to the manufac- turer’s instructions (GE Healthcare, NJ). The resulting protein pellet was resuspended in 2 ␮l × 200 ␮l of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 0.5% IPG buffer, 0.002% bro- mophenol blue), sonicated for 15 min, left at room temperature for 1 h and then loaded onto 2 cm × 11 cm Immobiline DryStrip First Dimension IPG strips (GE Healthcare, NJ) by passive rehy- dration. Following overnight rehydration, isoelectric focussing was
  3. 3. 208 Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 performed using the Ettan IPGphor (GE Healthcare, NJ) accord- ing to the manufacturer’s instructions (500 V for 1 h, 1000 V for 1 h and 8000 V for 2 h). The focussed IPG strips were then incu- bated for 15 min in 10 ml SDS equilibration buffer (50 mM Tris–HCl, pH8.8, 6 M urea, 30% (v/v) glycerol, 2% SDS, 0.002% bromophenol blue) containing 100 mg DTT, and for a further 15 min in 10 ml equilibration buffer containing 250 mg iodoacetamide. The equi- librated strips were applied to the surface of 12.5% polyacrylamide gels and the proteins separated in the second dimension in the Ettan DALT ll separation unit (GE Healthcare, NJ) at 0.2 W/gel for 1 h, 0.4 W/gel for 1 h and then 20 W/gel until completion. Gels were then either fixed for an hour in 50% methanol, 10% acetic acid and stained overnight using Sypro Ruby total protein stain (Invitrogen, UK) or transferred to PVDF membrane for Western blotting. Gels were destained in 10% methanol, 7% acetic acid and imaged using a Typhoon 9200 scanner (GE Healthcare, NJ). Western blots were stained with silver post-immunodetection to give a total protein image which allowed matching of the spot patterns between gel and blot. Immunopositive spots identified on the Western blot were matched to those in the gel and the relevant spots excised from the gel using the ProPic automated spot picker and then digested with trypsin in the ProGest auto- mated digestion unit (both from PerkinElmer Life Sciences, MA). The resulting peptides were analysed by tandem mass spectrom- etry in an Applied Biosystems 4700 mass spectrometer to give a peptide mass fingerprint and MSMS peptide sequence data; these were used to identify the protein, in searches against the MSDB database, performed with the Mascot search engine from Matrix Science. 2.7. Oligomeric Aˇ1–42 ELISA A Maxisorp ELISA plate (Nunc, NY) was coated with 5 ␮g/ml of rabbit polyclonal A␤1–42 antibody (Millipore, MA) (50 ␮l/well) in coating buffer (10 mM sodium carbonate, 30 mM sodium bicar- bonate, pH 9.6) overnight at 4 ◦C. The plate was washed five times with wash buffer (0.05% Tween 20, PBS) and incubated with blocking buffer (5% Tween 20, 5% non-fat milk, 1% bovine serum albumin (BSA)) for 2 h at room temperature. An 8-point standard curve (0–125 ␮g/ml) was prepared in duplicate using oligomerised A␤1–42 peptide. Sample homogenates were spun for 10 min at 20,000 × g and, from the supernatant, 15 ␮g total protein were diluted in reagent diluent (1% BSA, 0.2% Tween 20, PBS) to a final volume of 50 ␮l per well. Samples were tested in triplicate. To some wells we added duplicate samples of TPM3 recombinant protein (31.25 ␮g/ml), either immediately after dilution or after having been left overnight under the same conditions that were used for polymerisation of synthetic A␤1–42 peptide (see above). The plate was washed five times with wash buffer before sam- ples, TPM3 and standards were added. Controls included wells from which we omitted capture antibody, sample or detection antibody. The plate was incubated at room temperature for 2 h, with gen- tle shaking. The plate was washed five times with wash buffer, and either 7A1a or 1G5 diluted to 10 ␮g/ml in reagent diluent (100 ␮l/well) was added and incubated for 1.5 h at room tem- perature with gentle shaking. The plate was washed five times in wash buffer and HRP-conjugated horse anti-mouse IgG (1:500 in reagent diluent) was added (100 ␮l/well) and incubated for 30 min at room temperature with gentle shaking. The plate was washed five times with wash buffer and 100 ␮l of ABTS sub- strate (Vector Laboratories, CA) was added to each well. Protected from light, the plate was incubated for 20–25 min at RT, and then read at 450 nm. Reproducibility tests, run as two separate experiments, were conducted on seven cases (four AD and three controls). 2.8. Oligomeric Aˇ1–40 ELISA This assay followed the protocol for the oligomeric A␤1–42 ELISA but used a rabbit polyclonal A␤1–40 antibody (Alpha Diagnostic International, TX) as the capture antibody (5 ␮g/ml). 7A1a was used as the detection antibody. An 8-point standard curve (0–10 ␮g/ml) was prepared with a commercially available oligomeric A␤40 standard (Maine Biotechnology Services, ME). The manufacturer’s instructions were followed to prepare this standard. The assay was performed in duplicate to test reproducibility. 20 AD and 14 control cases were selected and analysed in duplicate for each assay run. 2.9. Statistical analyses Inspection of the levels of oligomeric A␤1–42/A␤1–40 measured showed that the distributions were skewed to the right. How- ever, the data could be normalised by logarithmic transformation. Oligomeric A␤1–42/A␤1–40 levels were analysed with respect to diagnosis by use of an independent samples t-test, with the help of Statistical Package for Social Science software (version 12.0.1 for Windows). Standard linear regression and both parametric and non-parametric correlation analyses were used to assess (i) the relationship between the levels of oligomeric A␤1–42 measured by sandwich ELISA using either 7A1a or 1G5 as the detection antibody; (ii) reproducibility of measurements of oligomeric A␤1–42/A␤1–40. Values of p < 0.05 were considered to be statistically significant. 3. Results 3.1. 7A1a and 1G5 antibodies detect multimeric Aˇ1–42 species Western blot analysis revealed that both antibodies to oligomeric A␤ (7A1a and 1G5) detected a range of A␤1–42 oligomers of synthetic A␤1–42 that had been allowed to polymerise overnight (Fig. 1). Similar results were found after preparing the peptide in either PBS or DMEM: a single band at approximately 10 kDa prob- ably represents SDS-stable dimeric states, a double band in the range of 15 kDa probably includes trimeric and tetrameric A␤1–42, and high molecular weight species ranging from about 50 kDa to greater than 150 kDa. A small amount of polymerised A␤1–42 did not enter the gel; this amount was consistently higher after overnight polymerisation in DMEM than in PBS. 3.2. Confirmation of Aˇ1–42 oligomerisation by thioflavin S assay Thioflavin analogues have previously been shown to detect oligomerisation of A␤ (Maezawa et al., 2008). Here, an increase in thioflavin S fluorescence was observed for A␤1–42 peptide that was incubated in either PBS or DMEM (Fig. 2), indicating that oligomeri- sation was occurring. Thioflavin S fluorescence was found to peak at approximately 12 h when using PBS, and 18 h for A␤1–42 incu- bated in DMEM. Thioflavin S fluorescence subsequently declined as fibrillar A␤1–42 precipitated out of solution. Fibrillisation occurred more quickly in DMEM, as evidenced by the earlier and more rapid decline in ThS fluorescence after it peaked at 18 h; the insoluble A␤1–42 could be seen on the floor of the microplate wells. 3.3. Cross-reactivity with tropomyosin in human brain homogenates Western blot analysis of homogenates from control and AD cases yielded an unexpected dense band of apparent molecular weight ∼34 kDa (Fig. 3a; left panel). The labelling intensity of this band did not vary considerably from case to case and did not differ
  4. 4. Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 209 Fig. 1. Western blot of synthetic A␤1–42 that had been allowed to polymerise overnight. Anti-oligomeric A␤ antibody 7A1a (lanes 1 and 2) and 1G5 (lanes 4 and 5) used to probe the blot detected a range of multimeric A␤1–42 species. Similar results were found when preparing the peptide in either PBS (lanes 1 and 4) or DMEM (lanes 2 and 5). A single band at approximately 10 kDa probably represents SDS-stable dimeric states. A double band in the range of 15 kDa probably includes trimeric and tetrameric A␤1–42. High molecular weight species were also present, ranging from about 50 kDa to greater than 150 kDa. Fig. 2. Thioflavin S assay confirms A␤ oligomerisation. 50 ␮l of oligomerised A␤1–42 peptide was mixed with 150 ␮l thioflavin S solution (5 ␮M in 50 mM glycine-NaOH, pH 8.5) and the fluorescence measured. An increase in thioflavin S fluorescence was observed for A␤1–42 peptide that was incubated in both PBS and DMEM, indicating that oligomerisation was occurring. Thioflavin S fluorescence was found to peak at approximately 12 h when using PBS, and 18 h for A␤1–42 incubated in DMEM. Thioflavin S fluorescence subsequently declined as fibrillar A␤1–42 precipitated out of solution. Fig. 3. Cross-reactivity with tropomyosin in human brain homogenates. (a) West- ern blot of homogenates of frontal neocortex from several brains, probed with 7A1a (probing with 1G5 yielded identical results). The dominant species detected had an apparent molecular weight of approximately 34 kDa (left panel). Over exposure revealed a range of other bands (right panel). (b) Two-dimensional gel electrophore- sis of a representative homogenate from a case of AD. 7A1a was used here. The large immunospot (arrow) represents the 34 kDa species detected by Western blot. Mass spectroscopy showed this to be tropomyosin-3 (TPM-3). (c) Western blot of TPM3 recombinant protein probed with rabbit anti-TPM3 antibody (lane 1) or 7A1a (lane 2).
  5. 5. 210 Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 Fig. 4. Results of oligomeric A␤1–42 ELISAs. Box-and-whiskers plots show the range, interquartile range and median values in homogenates of frontal neocortex from control and AD brains. Oligomeric A␤1–42 levels were found to be significantly higher in AD than control cases (p = 0.041, independent samples t-test). The mea- sured levels of oligomeric A␤1–42 differed approximately ten-fold between the two detection antibodies, 7A1a giving higher values (0.12–28.40 ␮g/ml) than 1G5 (0.13–3.16 ␮g/ml). significantly between the AD and control groups. Longer expo- sure of the membranes revealed a range of other bands (Fig. 3a; right panel), including a broad band of apparent molecular weight 50–60 kDa. For further investigation of the unexpected finding of a dominant ∼34 kDa band, we performed two-dimensional gel elec- trophoresis followed by mass spectrometry on one of the AD brain homogenates (Fig. 3b). Results revealed that the 7A1a and 1G5 antibodies were cross-reacting with tropomyosins, particu- larly tropomyosin-3 (TPM3), producing the predominant band at ∼34 kDa. Western blot analysis confirmed that TPM3 recombinant protein was detected by both of the anti-oligomeric A␤1–42 anti- bodies (Fig. 3c) (The molecular weight of the recombinant TPM3 is shown to have a molecular weight of ∼60 kDa in Fig. 3c. This is due to the recombinant protein (249 amino acids) including a glutathione-S-transferase tag (total length: 482 amino acids). 3.4. Use of ELISA to measure oligomeric Aˇ1–42 levels in AD and control brains ELISAs using A␤1–42 capture antibody and either 7A1a or 1G5 detection antibody yielded signal above background in all sam- ples of AD and control brain tissue (Fig. 4). TPM3 recombinant protein, added either directly after dilution or only after being left at 37 ◦C overnight, was not detected by this assay. Although case-to-case variation was considerable, oligomeric A␤1–42 levels were significantly higher in AD cases than controls (p = 0.041, inde- pendent samples t-test). Repetition of the ELISAs on a subset of samples showed good reproducibility of the results (p < 0.001, Pear- son correlation test, r2 = 0.984; data not shown). The amount of oligomeric A␤1–42 detected by the two antibodies differed approxi- Fig. 5. Scatterplot of results of ELISAs incorporating either 7A1a or 1G5 as the detec- tion antibody. The best-fit linear regression line (solid line) and 95% confidence band of the regression line (interrupted lines) are also shown. The results of the assays with the two antibodies showed good correlation (p < 0.0001, r2 = 0.51). mately ten-fold, 7A1a giving higher values (0.12–28.40 ␮g/ml) than 1G5 (0.13–3.16 ␮g/ml). However, the results of the assays with the two antibodies showed good correlation (p < 0.001, r2 = 0.51) (Fig. 5). 3.5. Use of ELISA to measure oligomeric Aˇ1–40 levels in AD and control brains Results confirmed the feasibility of using specific A␤1–40 cap- ture antibody and 7A1a detection antibody to measure oligomeric A␤1–40 by ELISA, which yielded signal above background in all of the samples analysed. Levels of oligomeric A␤1–40 mea- sured were found not to be significantly different in AD brains (0.26–5.21 ␮g/ml) compared to control brains (0.13–3.60 ␮g/ml). Repetition of the assay measurements showed good reproducibility (p < 0.001, r2 = 0.789; data not shown). 4. Discussion We have demonstrated that antibodies 7A1a and 1G5 detect oligomeric A␤1–42 but that they also bind to tropomyosin which limits their utility for analysing oligomeric A␤ by dot blot. However, combining of antibody to A␤1–42 with either 7A1a or 1G5 allows measurement by sandwich ELISA of the levels of A␤1–42 oligomers in homogenates of brain tissue. In this study, we have used the term A␤ oligomers to include all soluble A␤ species with the exception of monomers. This corresponds to the range of forms of A␤ that we detected by Western blot with 7A1a and 1G5. Although there was little direct evidence of labelling of insoluble A␤ in our stud- ies, it is likely that these antibodies will detect, at least to a limited degree, insoluble aggregated forms of A␤: A␤ readily binds to other proteins and it is probable that soluble oligomers bind to insoluble aggregated A␤ species. Oligomers of A␤ are potent neurotoxins in vitro (Dahlgren et al., 2002; Hartley et al., 1999; Lambert et al., 1998; Walsh et al., 2002). Their role in AD is still under scrutiny and our development and
  6. 6. Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 211 validation of a simple and sensitive assay to quantitate the levels of oligomeric A␤ in human brain tissue should prove very valu- able. Stenh et al. (2005) demonstrated that oligomerisation of A␤ results in underestimation of total A␤ levels by ELISA. Because the C-terminal part of the A␤ peptide is thought to be hidden inside the hydrophobic core of the aggregate (Roher et al., 2000), the immunological properties of monomeric and oligomeric A␤ dif- fer. Stenh et al. (2005) and Roher et al. (2000) suggested that the levels of A␤ detected by ELISA depend on the conformation speci- ficity of the antibodies used and recommended either denaturing of samples prior to assay or the use of novel conformation-dependent immunoassays. In vitro, 7A1a and 1G5 label a range of potentially toxic oligomeric species of A␤. A␤ oligomers bind to synapses and affect synaptic function. Synaptic A␤ ligands were shown to compose of oligomers of between 50 and 100 kDa (Lacor et al., 2004, 2007). This is consistent with the ∼55 kDa oligomer (A␤*56) that was reported to cause cognitive impairment in vivo (Cleary et al., 2005) and with a prominent oligomer found in AD brain tissue extract (Gong et al., 2003). Smaller naturally secreted oligomers, particularly trimers, were also shown to be toxic (Townsend et al., 2006; Walsh et al., 2002). Monomeric A␤ (Mr 4.5 kDa) is not detected by either 7A1a or 1G5. When used to probe Western blots of human brain homogenate, both antibodies cross-reacted with tropomyosin. However, other specific bands were detected by these antibodies, in particu- lar a band with an approximate molecular weight of 55 kDa, as found in other studies (Cleary et al., 2005; Gong et al., 2003). A␤ peptide and tropomyosin-3 both include the N-terminal amino acid sequence DAEFR, but this region of the A␤ peptide was not part of the immunogen used to raise 7A1a or 1G5 (New England Rare Reagents, personal communication). Denaturation of oligomers by SDS prior to polyacrylamide gel electrophoresis could theoretically cause some loss of oligomer-specific confor- mation and therefore reduce labelling of Western blots. This was not, however, observed in Western blots of synthetic A␤1–42 pep- tide. To overcome the cross-reactivity issues using these antibodies, a sandwich ELISA was designed using a pan A␤1–42 (for solu- ble and insoluble A␤1–42 species) to capture target peptides, with either 7A1a or 1G5 to detect oligomeric-specific conformations. Both 7A1a and 1G5 were effective within this assay, but the lev- els of oligomeric A␤ detected using these two antibodies differed by about ten-fold. Perhaps 1G5 preferentially detects a narrower range of specific oligomeric species. There was a suggestion from our Western blot analysis that 1G5 is not as sensitive to low molecu- lar weight oligomers as 7A1a (Fig. 1). However, the reasonably good correlation between the results obtained with these two antibodies suggests that both are likely to be useful for making comparative measurements, e.g. between AD and control brains, or between different regions of the same brain. Adaptability of the oligomeric A␤1–42 ELISA to measure oligomeric A␤1–40 has also been demon- strated. In the past, sequential extraction techniques with ultracentrifu- gation were used to isolate oligomeric A␤ from human brain tissue (Kuo et al., 1996; Lue et al., 1999; McLean et al., 1999; Pitschke et al., 1998; Wang et al., 1999). The isolated oligomeric A␤ species were subsequently analysed by a number of methods, with varying results. Kuo et al. (1996) reported that AD brains contain six-fold more oligomeric A␤ (mostly A␤N–42 species) than controls do. In contrast, Wang et al. (1999) found oligomeric A␤1–40 and A␤1–42 species to constitute the largest fractions of total A␤ in normal brain but the smallest in AD. Increased levels of oligomeric A␤, particu- larly A␤1–40, were noted by Lue et al. (1999) in AD compared to control brains. The discrepancies within these studies probably reflect the small numbers of subjects analysed, and variations in sampling, prepara- tion and assay techniques. Our development of a simple sandwich ELISA method, requiring neither sequential extraction nor ultracen- trifugation to isolate and assay oligomeric A␤ species, should help us to resolve these discrepancies by analysing a much larger num- ber of brains than has previously been practicable. Additionally, this assay is readily adaptable for measuring levels of other oligomeric A␤ species such as truncated forms of A␤. Acknowledgements This work was supported by a grant from Alzheimer’s Research Trust. 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