Murine and human AA amyloid extracts were isolated from amyloidotic tissues by a serial flotation procedure, as described previously (Pras et al., 1968). Human AA was kindly provided by Dr. Per Westermark (University of Uppsala, Sweden). All procedures using human-derived materials were approved by the University of Tennessee Institutional Review Board. AA amyloidosis was induced in 8-week-old transgenic H2-L^d-huIL-6 Tg Balb/c (Wall et al., 2008) mice by injection into the lateral tail vein of 100 μl PBS containing 100 μg of a spleen-derived AA extract.

All animal experiments were performed in accordance with Institutional Animal Care and Use Committee-approved protocols.

Six micrometer-thick sections, cut from formalin-fixed, paraffin-embedded tissues, were subjected to antigen retrieval using the High pH Target Retrieval^™ system (Dako). After a 30-min incubation at 90°C, followed by 20 min at room temperature, the tissues were immunostained overnight at 4°C with a 3-μg/ml solution of biotinylated anti-AA amyloid-specific mAb in 0.05% Tween/PBS. The reaction was developed using the Vectastain ABC kit (Vector Labs). The presence of amyloid was confirmed by staining with a 0.1% alkaline Congo red solution, where the characteristic green birefringence was seen upon examination using a Leica DM500 light microscope fitted with cross-polarizing filters. Digital microscopic images were acquired with a cooled CCD camera (SPOT, Diagnostic Instruments).

Wells of NUNC Maxisorb plates (eBioscience) were coated with extracts of murine AA amyloid fibrils (8 μg/ml) or the two synthetic peptides (3 μg/ml) and incubated overnight at 37°C. The dried wells were washed ×1 with assay buffer (PBS containing 0.05% Tween 20) and blocked with 200 μl of 1% BSA in PBS at 37°C for 1 h, followed by addition of the anti-AA amyloid-specific and control mAbs titrated in triplicate over the range of 1 M × 10⁻¹¹ M–1 M × 10⁻⁶ M (to determine background values, wells were filled only with antibody). After a 1-h incubation at 37°C, the wells were washed ×2, filled with 100 μl of a 1:5000 dilution of goat anti-mouse HRP conjugate and, following another 1-h incubation at 37°C and washes, 100 μl of ABTS in 0.06% H₂O₂ were added. The absorbance at 405 nm was measured 30 min later with a Wallac Victor3 plate reader (PerkinElmer) and the data analyzed using SigmaPlot (SPSS Inc.).

Quadruplicate rows of NUNC Maxisorb wells were coated with 50 μl of murine or human renal or splenic AA fibrils suspended in PBS (8 μg/ml), the plates dried overnight at 37°C, and the wells blocked for 1 h at 37°C with 1% BSA/PBS (200 μl/well). The AA samples used for competition were suspended in assay buffer containing 1% BSA to make a 2× stock solution (800 μg/ml) and then serially diluted to a final concentration of 1 × 10⁻¹¹ M. Anti-AA amyloid-specific and control mAbs were diluted with assay buffer to form a 2× stock solution and added to the human and murine AA fibril preparations to yield a final concentration of 50 nM and a starting 400 μg titer of competitor. Next, the competitor-mAb solutions were transferred to the AA fibril-coated plates and left for 1 h at 37°C [to establish maximum binding, one row of wells contained mAb alone (no competitor) and non-specific (background) binding was measured in those with antibody, but no fibrils]. After two washes, 100 μl of a 1:5000 dilution of goat anti-mouse HRP-conjugated secondary antibody was added and the plates incubated for 1 h at 37°C, washed, and the reaction developed by addition of 100 μl of ABTS in 0.06% H₂O₂. After 30 min, the absorbance was measured at 405 nm with the microplate reader. Data were analyzed using Excel and SigmaPlot, with each point representing the mean of triplicate values corrected for non-specific binding.

Forty nanomolar of anti-AA and control mAbs were labeled with 2 mCi of reductant-free ¹²⁵I (Perkin Elmer), using limiting amounts of chloramine T (Wall et al., 2006b). The reaction was quenched with a two-fold molar excess of sodium metabisulfite and the labeled reagents were then suspended in PBS containing 5 mg/ml BSA; unbound isotope and protein aggregates were removed by size-exclusion liquid chromatography via an Ultrogel AcA34 column (Amersham Pharmacia). Fractions containing the IgG monomer were pooled for imaging experiments. Radiolabeling efficiency was qualitatively assessed by Cyclone phosphor imaging (Packard Instruments) of ¹²⁵I-labeled mAb in 10% SDS-PAGE gels, under native and reducing conditions.

The biodistribution of the radiolabeled mAbs was determined in groups composed of three control or amyloidotic mice. Each animal was injected with 6 μg of ¹²⁵I-labeled mAb (∼150 μCi) in the lateral tail vein and, after 48 or 72 h, given a 200-μl i.v. dose of Fenestra VC^™ (Advanced Research Technologies). Thirty minutes later, the mice were euthanized by isoflurane overdose and SPECT/CT images acquired.

Single photon emission computed tomography data were collected with a microCAT II + SPECT dual modality imaging apparatus equipped with a 1-mm-pore diameter pinhole collimator (Siemens Preclinical Solutions). For imaging, the two detectors (composed of a 50-mm-diameter Hamamatsu R2486-02 multi-anode photo-multiplier tube coupled to a 1-mm × 1-mm × 8-mm CsI (Tl) crystal array arranged on a 1.2-mm² grid) were positioned ∼45 mm from the center of rotation. Each SPECT dataset comprised 45 projections collected over 360° during the course of ∼50 min. Images were reconstructed using an implementation of the expectation maximization–maximum likelihood (EM–ML) algorithm.

After acquisition of SPECT data, high-resolution CT images were obtained with the microCAT II scanner that had a circular orbit cone beam geometry and was equipped with a 20- to 80-kVp microfocus X-ray source that captured a 90-mm × 60-mm field of view using a 2048 × 3072 CCD array detector. Each CT dataset, composed of 360 projections at 1° azimuths, was procured over 8 min. Images were reconstructed in real-time on isotropic 77-μm voxels using an implementation of the Feldkamp backprojection algorithm. To facilitate co-registration of the reconstructed SPECT and CT images, Co-57-sealed sources were placed on the imaging bed and datasets were visualized and co-registered manually with a 3-D image analysis software package (Amira, Version 3.1: Mercury Computer Systems).

Tissue samples harvested from AA amyloid-bearing and control mice injected with the ¹²⁵I-labeled mAbs were placed into tared vials, weighed, and the radioactivity measured. The primary index values were expressed as % injected dose/g tissue (% ID/g).

Six micrometer-thick sections cut from formalin-fixed, paraffin-embedded blocks of tissue obtained from mice sacrificed 48 h post-injection of ¹²⁵I-labeled mAb were placed on Probond microscopic slides (ThermoFisher), dipped in NTB-2 emulsion (Eastman Kodak), stored in the dark, and developed after a 96-h exposure. The sections were counterstained with hematoxylin and eosin, cover-slipped with Permount (ThermoFisher), and examined by light microscopy. In addition, consecutively cut sections were stained with Congo red and viewed under cross-polarized illumination or immunostained using the AA-reactive mAbs as primary reagents. Digital photographs were taken and evaluated with the Image Pro Plus software package (MediaCybernetics).

The primary structures of mouse, human, and three other mammalian AA proteins are provided in Figure 1. To generate mAbs specific for the AA amyloid fibril, a 7-amino acid peptide containing the 5 carboxy-terminal residues (GHEDT) of the cleaved murine SAA that forms AA amyloid fibrils was used as the immunogen and the resultant mAbs were screened for reactivity against this component. Three monoclonal antibodies, designated 2A4, 7D8, and 8G9 (all of the IgG2 isotype), exhibited saturable binding over a molarity of 10⁻⁷–10⁻¹¹ M, with EC₅₀ values of 3.4, 1.84, and 4.0 nM, respectively (Figure 2A). In contrast, there was no affinity observed, even at 10⁻⁷ M, when the mAbs were titrated over the same concentration range using, as substrate, the longer peptide, GHEDTMADQE, that contained an additional five residues not present in the AA fibrils. To determine if the interaction of the mAbs with AA amyloid was similar to that of the immunizing peptide, mouse and human AA amyloid extracts were adsorbed onto microplate wells and antibody reactivity measured by ELISA (Figures 2B,C). In both cases, the three reagents bound with saturation values of <10⁻⁷ M; however, there was greater affinity for mouse AA, with EC₅₀s comparable to that for the GHEDT peptide (Table 1). Similar binding signal amplitudes were observed for mouse AA and the GHEDTMADQE molecule, indicating an equivalent epitope density. For human AA amyloid, the EC₅₀s were ∼10–30 nM. Notably, irrespective of the substrate (GHEDT peptide, mouse or human AA), the binding of the three mAbs varied, i.e., 7D8 > 2A4 > 8G9, with 7D8 having a three-fold lower EC₅₀ than 8G9.

To ensure that the interaction of the mAbs with AA fibrils did not result from an “artificial” neo-epitope formed as a consequence of adsorbing the material onto the microplate well, competitive binding studies were performed using soluble mouse and human AA extracts (Figures 2D–I). In these studies, although both were capable of inhibiting mAb binding to the heterologous AA amyloid on the microplate, the murine AA was more effective, as evidenced by the finding that the IC₅₀ for 7D8 binding to human AA, when mouse AA was used as a competitor, was 27 versus 15 and 18 μg/ml for mAbs 2A4 and 8G9, respectively. Similarly, as compared to 2A4 and 8G9, 7D8 required four- and seven-fold more soluble human AA extract to achieve 50% inhibition of binding. These data were consistent with the stronger affinity of mAb 7D8 to the GHEDT peptide, as well as to murine and human AA amyloid.

Although the mAbs were generated using the murine SAA GHEDT peptide as the immunogen, all showed high avidity for mouse and human AA amyloid with slightly higher affinity for the murine (HEDT) versus human (AEDS) sequences. To better understand the nature of the antibody binding sites, we performed studies using alanine (Ala)-substituted peptides as substrates in the ELISA (Figure 3). mAb 2A4 bound AEDT and HEDA peptides equally well, with an EC₅₀ equivalent to that of the unmodified HEDT molecule. In contrast, substitution of Ala for Glu in the HADT sequence resulted in a shift in the EC₅₀ from ∼3 to 100 nM (Figure 3). Furthermore, reactivity was completely abrogated by replacement of Ala for the Asp residue (HEAT). Disruption of the binding to both HADT and HEAT peptides also occurred using mAbs 7D8 and 8G9. Although the presence of glutamyl and aspartyl residues (E73 and D74) was required for optimal antibody binding, the presence of D74 was absolutely necessary to maintain a high-affinity interaction.

The ex vivo reactivity of mAbs 2A4, 7D8, and 8G9 with amyloid was demonstrated immunohistochemically, as seen when the congophilic birefringent deposits present in the liver, spleen, kidney, and pancreas of AA mice, as well as human AA amyloid-containing tissues, were immunostained with these reagents. In all instances, the 2A4 antibody exhibited the most intense binding that, notably, was abolished by absorption with the GHEDT-containing peptide used for immunization (Figure 4).

The capability of mAbs 2A4, 7D8, and 8G9 to bind AA amyloid in vivo also was assessed using radioiodinated derivatives of the three reagents which, when labeled with I-125, had a radiochemical yield of ∼50% and a specific activity of ∼25 μCi/μg. H2-L^d-huIL-6 mice with extensive systemic AA amyloidosis were given 200 μl tail vein injections of a solution containing ∼10 μg of radiolabeled antibody (∼250 μCi) in PBS and imaged 48 h later. As seen by high-resolution SPECT, there was marked uptake of the radiotracers in the liver, spleen (Figure 5) and, to a lesser extent, other amyloid-containing areas, e.g., kidneys and intestine. In contrast, there was no visualization of the amyloid in transgenic mice that received the control radioiodinated antibody (since the animals were not pre-treated with Lugol’s solution, there was thyroidal uptake of free radioiodide liberated during antibody catabolism). Further, the radiolabeled mAbs did not bind to amyloid-free organs or tissues from healthy mice (Figure 6).

The specific localization of the ¹²⁵I-labeled mAbs within amyloid was evidenced autoradiographically, where dense black deposits were seen around hepatic vasculature and sinusoids, splenic follicles, and renal papilla (Figure 7), as well as other anatomic sites deemed to contain this pathologic material based on Congo red staining. The control radioiodinated mAb did not label the amyloid deposits and was evident only within renal tubules. These results were confirmed by biodistribution measurements that revealed the liver and spleen of animals injected with ¹²⁵I-labeled mAbs 2A4, 7D8, and 8G9 to contain ∼25 and ∼45% ID/g of mAb, respectively, as compared to <1% ID/g with the control (Table 2). The ratio of tissue to muscle uptake in these two organs also was significantly greater in mice that received the three mAbs (∼100:1 and 200:1, respectively; Table 3). In the case of the pancreas and small intestine, the ratios were two- to three-fold higher, as well.