The specificity of OC antisera was examined by ELISA and dot blot analysis of monomeric, prefibrillar oligomeric and fibrillar Aβ and other types of amyloids (Fig. 1). ELISA analysis indicates that OC is conformation dependent and fibril specific because it recognizes Aβ fibrils, but not Aβ prefibrillar oligomers or Aβ monomer (Fig. 1A). OC recognizes a generic epitope that is associated with the fibrillar amyloid state regardless of the sequence because it reacts equally well with α-synuclein fibrils and islet amyloid polypeptide (IAPP) fibrils. Dot blot analysis confirms that the fibril epitope of both Aβ and poly Q (Q36) fibrils is recognized by OC and is complementary and mutually exclusive with the generic prefibrillar epitope of both Aβ and polyQ oligomers that is recognized by A11. A11 does not recognize the fibrillar samples that stain with OC, while OC does not stain the prefibrillar samples recognized by A11 (Fig. 1B). The size distribution of Aβ aggregates that are recognized by OC and A11 was determined by western blotting of fibrillar and prefibrillar oligomer Aβ samples (Fig. 2). Aβ42 fibrils contain 6E10 and 4G8 reactive bands at the positions of 4.5 kDa monomer, dimer, tetramer and a broad smear of aggregates up to the top of the gel. Prefibrillar oligomer Aβ42 samples contain A11 positive aggregates of the same approximate size range as fibrils that are stained by 4G8, but the prefibrillar oligomers are not stained by 6E10. We have previously reported that 6E10 does not stain prefibrillar oligomers prepared by dilution of a stock solution of Aβ42 in 100 mM NaOH in PBS, but it does recognize prefibrillar oligomers formed in water at pH 2.5 and 6E10 recognizes random coil monomer and amyloid fibrils 22. These results indicate that prefibrillar Aβ oligomers are polymorphic at the 6E10 epitope and that the Aβ monomer in the prefibrillar oligomer preparation has a stable conformation that is not reactive with 6E10. OC anti-fibril antiserum stains the Aβ42 dimer band and higher aggregates from fibrillar samples, but does not stain Aβ monomer (Fig. 2). However, OC does not stain the prefibrillar oligomer aggregates that are stained by A11. A11 stains bands corresponding to tetramer and a broad distribution of aggregates centred on a molecular weight of approximately 60 kDa, indicating that fibrillar oligomers are conformationally distinct from prefibrillar oligomers even though their sizes are broadly overlapping. The size of the bands detected by OC and A11 depend on the time of aggregation with the size increasing with incubation time (data not shown).

The fact that low MW species are recognized on western blots by the OC anti fibril antibody, which are conformationally distinct from the prefibrillar oligomers that react with A11, suggests that at least two distinct types of oligomers exist: fibrillar oligomers and prefibrillar oligomers. We characterized the solubility and size distribution of OC positive oligomers under non-denaturing conditions by ultracentrifugation and size exclusion chromatography. Both fibril preparations prepared in HFIP/water 19 and ADDL preparations prepared in DMSO and cold F12 medium 23 contain a substantial amount of OC positive 100,000 × G soluble material, suggesting that they contain fibrillar oligomers (Fig. 3A). Samples prepared by dilution of DMSO stock solutions of Aβ42 were also fractionated by size exclusion chromatography and the resulting fractions probed with OC. The results indicate that a broad distribution of oligomer sizes from approximately 8 kDa to 200 kDa is recognized by OC (Fig. 3B). These results are consistent with the sizes identified by western blots (Fig. 2). The data suggest that these fibrillar oligomers have Aβ peptides arranged in the same conformation as in fibrils suggesting that they may represent small pieces of fibrils or fibril nuclei.

Since both A11 and OC recognize generic epitopes that do not depend on a particular amino acid sequence, we tested whether the immune response to other types of prefibrillar oligomers and fibrils is also specific for generic conformation dependent epitopes. We synthesized an oligomer mimic of islet amyloid polypeptide (IAPP) by coupling IAPP carboxy-terminal thioester to colloidal gold particles as previously described 19. We used IAPP oligomer mimics and IAPP fibrils to immunize rabbits and we characterized the specificity of the immune response by dot blot. Immunization of rabbits with IAPP oligomer mimics gives rise to a prefibrillar oligomer-specific immune response that is indistinguishable from that obtained with Aβ prefibrillar oligomer mimics (Fig. 4). We call this serum I11 to indicate its derivation from IAPP antigen and its similarity to A11. Like A11, I11 recognizes prefibrillar oligomers derived from Aβ, α-synuclein and IAPP, but not monomers or fibrils on dot blots (Fig. 4A). Western blots of prefibrillar oligomer samples probed with I11 give the same staining pattern as A11 (Fig. 4B). Similarly, rabbits vaccinated with IAPP fibrils give rise to a fibril specific immune response that recognizes Aβ fibrils and does not recognize monomer or prefibrillar oligomers (Fig. 5). The resulting serum is called LOC (Like OC). Taken together these results demonstrate that the immune response to oligomer mimics and fibrils is predominantly conformation dependent and sequence independent.

We also investigated the distribution of OC and LOC staining in human AD brain using two AD cases with Aβ and NFT pathology. Figure 6 shows significant OC immunolabeling in the hippocampus and temporal cortex. All types of Aβ deposits stain with OC, including diffuse, neuritic and cored plaques (Fig. 6) and cerebrovascular amyloid deposits (data not shown). Therefore, OC staining of diffuse amyloid deposits indicates that they share the fibrillar immunological structure of other amyloid deposits even though they do not stain with "fibril specific" dyes, such as thioflavin S. Intracellular NFTs do not stain with OC, even though it has been suggested that they contain local β-sheet character 24. Immunolabeling was virtually abolished when the antibody was incubated with a 100-fold concentration of the fibrillar antigen prior to incubation with the tissue (Fig. 6E, F). We next compared OC and LOC immunoreactivity by confocal microscopy as both antibodies recognize similar assembly states of Aβ. The staining patterns of the two antibodies overlap significantly within plaques of an AD case since most of the fluorescent signals are coincident in the merged image (Fig. 6G–I). Since LOC is raised against IAPP, this indicates that the plaque staining is conformation dependent and not due to a small amount of linear Aβ sequence specific antibodies that may contaminate OC.

Lysates of human AD brain were also analyzed biochemically with OC. OC immunoreactivity was found in the Triton X-100 100,000 × G insoluble fraction of all AD patients examined (Fig. 7). This appeared as very high MW material that sticks at the top of the gel or in the stacking gel after boiling in SDS sample buffer. After stripping and reprobing, this high MW material also stains with 4G8 (anti-Aβ17–28) antibody. No OC staining was observed in the Triton X-100 soluble fraction, indicating that OC does not recognize APP and that the level of fibrillar oligomers in this fraction is below the limit of detection by this method (data not shown). The insoluble fraction was also dissolved in formic acid and dried prior to SDS gel electrophoresis. After formic acid treatment, OC staining is abolished although 4G8 staining is retained after stripping and reprobing, indicating that the staining is conformation dependent. The 4G8 staining reveals that formic acid treatment has a small effect on breaking down the high MW material with more immunoreactivity at the top of the running gel and a small amount of low MW Aβ immunoreactivity. Interestingly, the OC immunoreactivity is insensitive to formic acid treatment, typically used to enhance Aβ immunostaining, after fixation of human AD brain tissue with paraformaldehyde. The immunoreactivity is insensitive to pretreatment of sections with up to 90% formic acid (data not shown).

The broad specificity of OC for different types of amyloid fibrils suggests that it may have broad utility in staining different types of amyloid deposits. We also examined the OC staining of islet amyloid deposits in the pancreas of human IAPP transgenic mouse model of type II diabetes 25. OC stains intra and extracellular IAPP amyloid deposits, indicating that the IAPP deposits display the same fibril related generic epitope in vivo that they display in vitro (Fig. 8). Together, these results indicate that the broad fibril specific immunoreactivity of OC may be useful for identifying a number of different types of amyloid deposits in human disease.

Fibril antigens were prepared by stirring 2 mg/ml Aβ42 peptide in 50% HFIP/H2O, 0.02% sodium azide for 7 days. Afterwards, the HFIP was evaporated under a stream of nitrogen and the sample was stirred for an additional 7 days and dialyzed against PBS (molecular weight cut off 10,000 Da). The resulting fibrils were checked by EM and the purity was confirmed by the absence of oligomers using anti-oligomer antibody. The same protocol was used to prepare IAPP fibrils. Aβ and IAPP oligomer mimics were prepared as previously described 27 The synthesis of IAPP C-terminal thioester analog lacking cysteine residues (KANTATAATQRLANFLVHSSNNFGAILSSTNVGSNTY-SR), was also carried out according to the methods described 27. The antigens were each used to immunize two New Zealand white rabbits (Pacific Immunology Corp., Ramona, CA, 92065) according to protocols approved by IACUC. Each rabbit immunized with 500 μl of antigen in complete Freund's adjuvant (CFA), and then boosted twice at four week intervals with 500 μl of antigen in Incomplete Freund's Adjuvant (IFA).

Aβ fibrils and fibrillar oligomers were prepared by dissolving 0.3 mg of lyophilized Aβ42 in 150 ul of hexafluoro-2-propanol (HFIP) for 10–20 minutes at room temperature. The resulting Aβ solution was added to DD H₂O in a siliconized Eppendorf tube to 80 uM concentration. After 10–20 min incubation at room temperature, the samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N₂ for 10 min to evaporate the HFIP. The sample was then stirred at 500 RPM using a Teflon coated micro stir bar for 24 hours at 22°C. This method was originally reported for preparing A11 positive prefibrillar oligomers 19, but more recent work indicates that it also produces fibrillar oligomers that are OC positive. Fibrils were separated from fibrillar Aβ42 oligomers by centrifuged at 100,000 × G for 1 hour at 4°C. The supernatant containing fibrillar oligomers and pellet fraction containing fibrils were separated and collected. The pellets were resuspended in an equal volume of H₂O. Alternatively, 1 mg of lyophilized Aβ42 was dissolved in 200 μl of DMSO and incubated at room temperature for 10–15 minutes to form fibrillar oligomers. The fibrillar oligomers in DMSO were fractionated according to size using a TSK-GEL SuperSW2000 column (Tosoh Bioscience LLC) in 10 mM Phosphate, pH 7.4 at a flow rate of 0.3 ml/min.

Prefibrillar oligomers that are OC negative were prepared as previously described 22. Aβ42 stock solutions (2 mM) were prepared by dissolving the lyophilized peptide in 100 mM NaOH followed by water bath sonication for 30 s. The oligomerization reaction was initiated by diluting the stock solution in phosphate buffered saline (PBS), pH = 7.4, 0.02% sodium azide (45 μM final Aβ₄₂ concentration, final pH = 7.4) and incubated at room temperature for up to 15 days. Oligomer formation was monitored by dot blot with A11 and OC polyclonals.

ADDLs were prepared according to Lambert et al 23. Aβ42 peptide was initially incubated in HFIP at room temperature for 1 hour. The HFIP was evaporated under a gentle stream of N₂. The remaining Aβ42 was dissolved in anhydrous DMSO at 5 mM, diluted into cold phenol red-free F12 medium and aged at 4°C for 24 hours. The final peptide concentration was 80 μM.

Q36 fibrils were prepared by dissolving Q36 in 10 mM HEPES/100 mM NaCl at a concentration of 45 μM and incubating with stirring at 25 C for 6 days. Fibril formation was confirmed by EM and ThT. Q36 oligomers were prepared by dissolving Q36 in 1:1 (vol:vol) TFA:HFIP at 2 mM and diluting it to 45 μM in PBS and incubating with stirring for 6 days. Oligomer formation was confirmed with A11 antibody.

ELISA, dot blot and western blots assays were done as previously described 27. Brain tissue from autopsy cases was provided by the University of California Alzheimer's Disease Research Center and the Institute for Brain Aging & Dementia Brain Tissue Resource. For single-labeling experiments, 50 μm 4% paraformaldehyde-fixed sections containing the hippocampus and entorhinal cortex or the midfrontal cortex from an AD case (Case #1 – 65 year old female, 7.4 hour post mortem interval, MMSE = 21 15 months prior to death with a Braak & Braak stage of VI) were washed with 0.1 M Tris-buffered saline (TBS), pH 7.5, and then pretreated with 3% hydrogen peroxide in 10% methanol to block endogenous peroxidase activity. Sections were subsequently washed in TBS with 0.1% Triton X-100 (TBS-A) and then blocked for thirty minutes in TBS-A with 3% bovine serum albumin (TBS-B). In a series of preliminary studies, the effects of different concentrations of formic acid during a pretreatment phase was determined to have little effect on the extent or intensity of OC labeling of fixed tissue (data not shown) and thus this step was eliminated from the protocol. Sections were incubated overnight at room temperature in OC at a dilution of 1:10,000. Following two washes with TBS-A and a wash in TBS-B, sections were incubated in either goat anti-mouse or goat anti-rabbit biotinylated anti-IgG and then in avidin biotin complex (ABC) (Vector Laboratories, Burlingame, CA, USA). Antibodies were visualized using 3,3'-diaminobenzidine (DAB, Vector Laboratories). For quantification experiments, the frontal cortex from all cases used for quantification was immunostained in single experiments to reduce variability in the image analysis procedures and all immunohistochemical procedures were identical for the OC antibody. To determine the specificity of the OC antibody, IgG purified serum at a concentration of 1:10,000 was preincubated in 10× and 100× concentration of OC peptide for 2 hours at RT. Subsequently, a section from the midfrontal cortex was incubated in the OC antibody/peptide solution and processed for immunohistochemistry. Sections were counterstained with cresyl violet.

Hemizygous human IAPP transgenic mice (FVB-tg(IAPP)6Jdm/-A^vy/A) were characterized in details elsewhere 28. For this study we used 24 wk old mice. Before collection of mouse pancreata, the heart was perfused with 10 ml of 4% paraformaldehyde. The pancreas was dissected in cold PBS, fat and lymph nodes trimmed, the pancreas weighed, fixed in 4% paraformaldehyde at 4°C for 24 h then frozen in OCT.

An additional AD case was used for confocal studies. Case #2 was a 95 year old female with a post mortem interval of 5.5 hours and a MMSE score of 12, 6.3 months prior to death. Sections containing the midfrontal cortex were incubated in OC overnight (1: 5,000) and positive labeling was detected using anti-rabbit IgG Alexa Fluor 568 (Molecular Probes, Eugene, OR; 1:200). Alexa Fluoro 488 (Molecular Probes, Eugene, OR; 1:200) was subsequently used to visualize LOC (1:5,000). The order of the primary antibodies was also reversed in additional experiments and the results were similar. Because both antibodies were raised in the same species (rabbit) an intervening formaldehyde treatment was used between the first and second immunolabel 29. Sections were allowed to dry on slides prior to rehydration and coverslipping with VectaShield (Vector Laboratories, Burlingame, CA, USA). Confocal images were collected on an Olympus IX70 inverted microscope and a BioRad Radiance 2000 Laser Scanning System using a 40, 60 or 100× objective for image analysis. Each antibody label was excited and scanned using lambda strobing and barrier filters at 510 and 480 nm. A z-series scan at either 0.5 or 1 μm intervals was captured to determine the spatial colocalization characteristics of OC with LOC.