Mod Rheumatol (2000) 10:216–219
© The Japan Rheumatism Association and Springer-Verlag Tokyo 2000
ORIGINAL ARTICLE
Toshiyuki Yamada · Kouichi Itoh · Jun Igari
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
Localization of injected anti-serum amyloid A antibody to the lesions in murine reactive amyloidosis
Received: November 30, 1999 / Accepted: June 5, 2000
Abstract This paper examines whether an antibody to serum amyloid A (SAA) protein is incorporated into amyloid deposits in vivo in murine reactive amyloidosis. First, monoclonal anti-SAA antibody was injected into amyloid-laden mice, and localization of the antibody was immunohistochemically assessed. Following the injection, plasma clearance of the antibody was analyzed. The antibody was detected in the amyloid deposits in spleen, liver, and kidney from mice killed on days 0–3 postinjection. The injected antibody disappeared more rapidly from the circulation of amyloidotic animals than from nonamyloidotic animals. The anti-SAA antibody can associate with amyloid deposits in vivo. Further applications, particularly amyloid imaging, should be investigated. Key words Amyloidosis · Serum amyloid A · Monoclonal antibody · Mouse
Introduction Reactive (AA) amyloidosis is a form of generalized amyloidosis,1 a disease which involves chronic inflammatory disorders. Nowadays, rheumatoid arthritis (RA) is the most common of the underlying diseases. Amyloid deposits are found in the gastroduedonal tracts of approximately 10% of Japanese RA patients examined by endoscopic biopsy.2 Although currently it is not known what percentage of these patients developed a functional failure of vital organs such as kidney or heart, AA amyloidosis is believed to affect the prognosis of RA. The nature of amyloid deposits is aggregated proteins; in AA-amyloidosis, serum amyloid A protein (SAA) or
T. Yamada (*) · J. Igari Department of Clinical Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan K. Itoh Department of Clinical Laboratory Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan
its degraded product (AA) is a main constituent.3,4 How SAA/AA forms fibrils, or why such fibrils are not eliminated, are largely unknown. At a minimum, the prolonged high production of SAA is an essential factor leading to this disease. Synthesis of this protein, mainly in the liver, is dramatically induced under the stimulation of inflammation-related cytokines such as tumor necrosis factor-α, interleukin-1, and interleukin-6.5 Thus, the practical way to prevent the production of amyloid deposits is a reduction of inflammatory activity which, in turn, would reduce SAA synthesis. The diagnosis of amyloidosis is essentially based on histological findings in biopsy specimens. The noninvasive method scintigraphy, using radiolabeled serum amyloid P component (SAP), has been described by a group in Europe.6,7 This was based on the nature of SAP, since it binds preferentially to amyloid fibrils regardless of the amyloidosis type.8 In spite of its practical utility,9 the method is not yet available in areas outside Europe. Antibodies can reach the corresponding antigens in vivo, as proved in immune targeting for cancer. This suggests that antibodies recognizing substances in amyloid deposits could be used as an alternative to SAP in systemic amyloidosis. This preliminary study examined whether a monoclonal anti-SAA antibody would be incorporated into amyloid deposits in vivo using a mouse model of AA-amyloidosis.
Materials and methods Monoclonal antibody A rat–mouse hybrid monoclonal antibody to mouse SAA was generated as described elsewhere.10 In that study, the antibody clone M10 (IgG2b class) was shown to react with the second (carboxyl terminal) portion of SAA. Ascites was generated in Balb/c nude mice (SLC, Hamamatsu, Japan) by innoculating the M10 hybridoma, and were subsequently fractionated to immunoglobulins by treatment with 35% ammonium sulfate. The antibody preparation was adjusted
217
to a concentration of 1 mg/ml. The antibody was also biotinylated, as previously described.11 Induction of amyloidosis
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
The amyloid-enhancing factor, which was prepared according to Axelrad’s method,12 was injected intraperitoneally into female 10-week-old Icr mice (SLC). The following day, 0.5 ml Freund complete adjuvant was injected intraperitoneally. After 2 weeks, when amyloid deposits were expected to appear in the spleen and liver, the animals were deemed to be ready for the experiments. Administration and detection of the antibody The monoclonal antibody (MoAb) (50–100 µl) was injected into the amyloidotic mice via the tail vein. Nonimmune immunoglobulins obtained from normal rats were also injected in the same manner. On the designated day, the animals were killed, and their spleen, liver, and kidney were removed. These were fixed in 10% formalin and paraffinized. Sliced specimens, after deparaffinization, were subjected to immunohistochemical analysis to detect rat immunoglobulins. Rabbit antirat immunoglobulins (Dako, Carpinteria, CA, USA) were used as the first antibody, and the avidin–biotin complex immunostaining kit Vectastain (Vector Laboratories, Burlingum, CA, USA) was used, as described elsewhere.13 To investigate plasma clearance of the injected antibody, 10 µg of the biotinylated antibody were given to three amyloidotic animals and three age-matched control animals, and blood was drawn retro-orbitally under anesthesia at 4 min, 1 h, and 4 h postinjection. The plasma concentration
Fig. 1. Immunohistochemistry detecting injected antimouse SAA monoclonal antibody. Positive staining was shown in amyloid deposits in the spleen (left), liver (middle), and kidney (right) obtained from
of the biotinylated antibody was␣ measured by an enzyme immunoassay using a sandwich with immobilized avidins and peroxidase-conjugated avidins. Briefly, the 96-well microtiter plate was coated with 10 µg/ml avidin and then blocked with 1% bovine serum albumin. The plates were incubated first with the plasma samples and then with peroxidase–avidin. Color was developed using a substrate solution, and then quantified. The assay was calibrated using the injected biotinylated antibody as the standard. The results were evaluated as a percentage of the antibody concentration at 4 min. Student’s t-test was used to evaluate the variations in the results.
Results Association of injected antibodies with amyloid deposits The injected antibodies were immunohistochemically detected as rat immunoglobulins in the amyloid deposits in the spleens, kidneys, and livers of the animals killed on days 0 (4 h), 1, 2, and 3 (Fig. 1). When nonimmune rat immunoglobulins were given to the amyloidotic mice, positive staining was seen in some populations of hepatocytes from animals killed on days 0 and 1, but not from those killed on days 2 and 3 (data not shown). When the antibody M10 was given to nonamyloidotic animals, no positive staining was observed in any area. Plasma clearance of injected antibodies The injected biotin-labeled antibody disappeared more rapidly from plasma in the amyloidotic mice than from that
a mouse killed 2 days after the antibody injection. Original magnification: 332
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
218
Fig. 2. Plasma disappearance of the injected antibody in three amyloidotic mice (open circles, broken line) and three nonamyloidotic mice (closed circles). The results are shown as a percentage of the antibody concentration at 4 min
in the nonamyloidotic mice (Fig. 2). The antibody remaining at the end of the first hour was 55.3 6 2.9% (mean 6 SD) in nonamyloidotic animals compared with 45.0 6 3.3% in amyloidotic animals (P , 0.05). At the end of 4 h, the amounts had dropped to 44.0 6 3.6% in the nonamyloidotic animals and 39.9 6 6.7% in the amyloidotic animals (P , 0.05).
Discussion To our knowledge, this is the first study to use antiamyloid antibodies in vivo. Mice were chosen as the subjects since their use in AA-amyloidosis has been well studied and validated. Recently established monoclonal antibody clone 10 reinforced this choice. There may be several advantages in the in vivo use of antiamyloid antibodies compared with serum amyloid P component (SAP). A theoretical problem with the latter is that the injected SAP may compete with endogenous plasma SAP or with that which is already bound to the amyloid fibrils. A practical problem, on the other hand, is the difficulty of preparing a large amount of SAP. We know of no factory-prepared recombinant SAP which is ready to use. In contrast, monoclonal antibodies have long been available. Furthermore, once bound to the fibrils, the SAP molecules may remain in that combination for several days,6,7 during which time no beneficial effects seem to occur. Instead, SAP may stabilize the amyloid deposits at those sites. In contrast, antibodies can trigger inflammatory responses, which may degrade amyloid fibrils. One harmful
effect of the antibody is the generation of antianimal antibodies. To avoid this build-up, a modification of the antibody molecules will be necessary. The aim of this study was to confirm earlier suspicions that injected anti-SAA antibody binds to amyloid deposits. The binding was confirmed by immunohistochemical detection of rat immunoglobulins in the amyloid deposits. The positive staining of nonimmune rat immunoglobulins in the liver soon after injection may be due to the handling of foreign substances by hepatocytes.14 Since this diminishes in the late phase after injection, the presence of the antibody at this stage definitely indicates an association between antibodies and amyloid deposits. According to a previous report,15 the injected SAP is rapidly cleared from the circulation of patients who have amyloid deposits, probably because of an absorption by the amyloid deposits. This was also assessed in the present investigation, and the same tendency was observed. However, the difference in the clearance rates of the injected antibody in mice with and without amyloid deposits does not seem to be striking enough to clearly indicate the presence or absence of amyloid deposits. Changes in the amounts of antibody or in the modulation of the antibody molecule should be assessed to clarify this issue. In conclusion, the specific localization of the antibody to the amyloid deposits was confirmed. The antibody injection method may have a similar value to the SAP method. The next step should be the use of radioisotopic labeling for scintigraphic imaging of amyloid deposits, as performed in the SAP study.6
References 1. Benson MD. Amyloidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle DV, editors. The metabolic basis of inherited disease. 7th ed. New York: McGraw-Hill; 1995. pp. 4159–91. 2. Okuda Y, Takasugi K. Secondary amyloidosis in rheumatoid arthritis: diagnostic and prognostic study of 198 biopsy proven cases. Arthritis Rheum 1997;40:S115. 3. Husby G, Marhaug G, Dowton B, Sletten K, Sipe JD. Serum amyloid A (SAA): biochemistry, genetics and the pathogenesis of AA amyloidosis. Amyloid 1994;1:119–37. 4. Yamada T. Serum amyloid A (SAA): a concise review of biology, assay methods and clinical usefulness. Clin Chem Lab Med 1999;37:381–8. 5. Baumann H, Gauldie J. The acute phase response. Immunol Today 1994;15:74–80. 6. Hawkins PN, Myers MJ, Epenetos AA, Caspi D, Pepys MB. Specific localization and imaging of amyloid deposits in vivo using 123 I-labeled serum amyloid P component. J Exp Med 1988;167:903– 13. 7. Hawkins PN, Myers MJ, Lavender JP, Pepys MB. Diagnostic radionuclide imaging of amyloid: biological targeting by circulating human serum amyloid P component. Lancet 1988;i(8600):1413– 8. 8. Pepys MB, Booth DR, Hutchinson WL, Gallimore JR, Collins PM, Hohenester E. Amyloid P component. A critical review. Amyloid 1997;4:274–95. 9. Hawkins PN, Richardson S, Vigushin DM, David J, Kelsey CR, Gray RE, et al. Serum amyloid P component scintigraphy and turnover studies for diagnosis and quantitative monitoring of AA amyloidosis in juvenile rheumatoid arthritis. Arthritis Rheum 1993;36:842–51.
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Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
10. Yamada T, Fukuda T, Wada A, Itoh Y. Monoclonal antibodybased sensitive enzyme-linked immunosorbent assay for murine serum amyloid A. J Immunoassay 1999;20:223–35. 11. Yamada T, Uchiyama K, Yakata M, Gejyo F. Sandwich enzyme immunoassay for serum amyloid A protein (SAA). Clin Chim Acta 1989;178:169–76. 12. Axelrad M, Kisilevsky R, Willmer J, Chen SJ, Skinner M. Further characterization of amyloid-enhancing factor. Lab Invest 1982;47: 139–46. 13. Yamada T, Liepnieks JJ, Benson MD, Kluve-Beckerman B. Accelerated amyloid deposition in mice treated with the aspartic protease inhibitor, pepstatin. J Immunol 1996;157:901–7.
14. Meek RL, Eriksen N, Benditt EP. Serum amyloid A in the mouse. Sites of uptake and mRNA expression. Am J Pathol 1989;135:411– 9. 15. Jager PL, Hazenberg BP, Franssen EJF, Limburg PC, van Rijswijk MH, Piers DA. Kinetic studies with iodine-123-labeled serum amyloid P component in patients with systemic AA and AL amyloidosis and assessment of clinical value. J Nucl Med 1998;39: 699–706.
© The Japan Rheumatism Association and Springer-Verlag Tokyo 2000
ORIGINAL ARTICLE
Toshiyuki Yamada · Kouichi Itoh · Jun Igari
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
Localization of injected anti-serum amyloid A antibody to the lesions in murine reactive amyloidosis
Received: November 30, 1999 / Accepted: June 5, 2000
Abstract This paper examines whether an antibody to serum amyloid A (SAA) protein is incorporated into amyloid deposits in vivo in murine reactive amyloidosis. First, monoclonal anti-SAA antibody was injected into amyloid-laden mice, and localization of the antibody was immunohistochemically assessed. Following the injection, plasma clearance of the antibody was analyzed. The antibody was detected in the amyloid deposits in spleen, liver, and kidney from mice killed on days 0–3 postinjection. The injected antibody disappeared more rapidly from the circulation of amyloidotic animals than from nonamyloidotic animals. The anti-SAA antibody can associate with amyloid deposits in vivo. Further applications, particularly amyloid imaging, should be investigated. Key words Amyloidosis · Serum amyloid A · Monoclonal antibody · Mouse
Introduction Reactive (AA) amyloidosis is a form of generalized amyloidosis,1 a disease which involves chronic inflammatory disorders. Nowadays, rheumatoid arthritis (RA) is the most common of the underlying diseases. Amyloid deposits are found in the gastroduedonal tracts of approximately 10% of Japanese RA patients examined by endoscopic biopsy.2 Although currently it is not known what percentage of these patients developed a functional failure of vital organs such as kidney or heart, AA amyloidosis is believed to affect the prognosis of RA. The nature of amyloid deposits is aggregated proteins; in AA-amyloidosis, serum amyloid A protein (SAA) or
T. Yamada (*) · J. Igari Department of Clinical Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan K. Itoh Department of Clinical Laboratory Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan
its degraded product (AA) is a main constituent.3,4 How SAA/AA forms fibrils, or why such fibrils are not eliminated, are largely unknown. At a minimum, the prolonged high production of SAA is an essential factor leading to this disease. Synthesis of this protein, mainly in the liver, is dramatically induced under the stimulation of inflammation-related cytokines such as tumor necrosis factor-α, interleukin-1, and interleukin-6.5 Thus, the practical way to prevent the production of amyloid deposits is a reduction of inflammatory activity which, in turn, would reduce SAA synthesis. The diagnosis of amyloidosis is essentially based on histological findings in biopsy specimens. The noninvasive method scintigraphy, using radiolabeled serum amyloid P component (SAP), has been described by a group in Europe.6,7 This was based on the nature of SAP, since it binds preferentially to amyloid fibrils regardless of the amyloidosis type.8 In spite of its practical utility,9 the method is not yet available in areas outside Europe. Antibodies can reach the corresponding antigens in vivo, as proved in immune targeting for cancer. This suggests that antibodies recognizing substances in amyloid deposits could be used as an alternative to SAP in systemic amyloidosis. This preliminary study examined whether a monoclonal anti-SAA antibody would be incorporated into amyloid deposits in vivo using a mouse model of AA-amyloidosis.
Materials and methods Monoclonal antibody A rat–mouse hybrid monoclonal antibody to mouse SAA was generated as described elsewhere.10 In that study, the antibody clone M10 (IgG2b class) was shown to react with the second (carboxyl terminal) portion of SAA. Ascites was generated in Balb/c nude mice (SLC, Hamamatsu, Japan) by innoculating the M10 hybridoma, and were subsequently fractionated to immunoglobulins by treatment with 35% ammonium sulfate. The antibody preparation was adjusted
217
to a concentration of 1 mg/ml. The antibody was also biotinylated, as previously described.11 Induction of amyloidosis
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
The amyloid-enhancing factor, which was prepared according to Axelrad’s method,12 was injected intraperitoneally into female 10-week-old Icr mice (SLC). The following day, 0.5 ml Freund complete adjuvant was injected intraperitoneally. After 2 weeks, when amyloid deposits were expected to appear in the spleen and liver, the animals were deemed to be ready for the experiments. Administration and detection of the antibody The monoclonal antibody (MoAb) (50–100 µl) was injected into the amyloidotic mice via the tail vein. Nonimmune immunoglobulins obtained from normal rats were also injected in the same manner. On the designated day, the animals were killed, and their spleen, liver, and kidney were removed. These were fixed in 10% formalin and paraffinized. Sliced specimens, after deparaffinization, were subjected to immunohistochemical analysis to detect rat immunoglobulins. Rabbit antirat immunoglobulins (Dako, Carpinteria, CA, USA) were used as the first antibody, and the avidin–biotin complex immunostaining kit Vectastain (Vector Laboratories, Burlingum, CA, USA) was used, as described elsewhere.13 To investigate plasma clearance of the injected antibody, 10 µg of the biotinylated antibody were given to three amyloidotic animals and three age-matched control animals, and blood was drawn retro-orbitally under anesthesia at 4 min, 1 h, and 4 h postinjection. The plasma concentration
Fig. 1. Immunohistochemistry detecting injected antimouse SAA monoclonal antibody. Positive staining was shown in amyloid deposits in the spleen (left), liver (middle), and kidney (right) obtained from
of the biotinylated antibody was␣ measured by an enzyme immunoassay using a sandwich with immobilized avidins and peroxidase-conjugated avidins. Briefly, the 96-well microtiter plate was coated with 10 µg/ml avidin and then blocked with 1% bovine serum albumin. The plates were incubated first with the plasma samples and then with peroxidase–avidin. Color was developed using a substrate solution, and then quantified. The assay was calibrated using the injected biotinylated antibody as the standard. The results were evaluated as a percentage of the antibody concentration at 4 min. Student’s t-test was used to evaluate the variations in the results.
Results Association of injected antibodies with amyloid deposits The injected antibodies were immunohistochemically detected as rat immunoglobulins in the amyloid deposits in the spleens, kidneys, and livers of the animals killed on days 0 (4 h), 1, 2, and 3 (Fig. 1). When nonimmune rat immunoglobulins were given to the amyloidotic mice, positive staining was seen in some populations of hepatocytes from animals killed on days 0 and 1, but not from those killed on days 2 and 3 (data not shown). When the antibody M10 was given to nonamyloidotic animals, no positive staining was observed in any area. Plasma clearance of injected antibodies The injected biotin-labeled antibody disappeared more rapidly from plasma in the amyloidotic mice than from that
a mouse killed 2 days after the antibody injection. Original magnification: 332
Mod Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
218
Fig. 2. Plasma disappearance of the injected antibody in three amyloidotic mice (open circles, broken line) and three nonamyloidotic mice (closed circles). The results are shown as a percentage of the antibody concentration at 4 min
in the nonamyloidotic mice (Fig. 2). The antibody remaining at the end of the first hour was 55.3 6 2.9% (mean 6 SD) in nonamyloidotic animals compared with 45.0 6 3.3% in amyloidotic animals (P , 0.05). At the end of 4 h, the amounts had dropped to 44.0 6 3.6% in the nonamyloidotic animals and 39.9 6 6.7% in the amyloidotic animals (P , 0.05).
Discussion To our knowledge, this is the first study to use antiamyloid antibodies in vivo. Mice were chosen as the subjects since their use in AA-amyloidosis has been well studied and validated. Recently established monoclonal antibody clone 10 reinforced this choice. There may be several advantages in the in vivo use of antiamyloid antibodies compared with serum amyloid P component (SAP). A theoretical problem with the latter is that the injected SAP may compete with endogenous plasma SAP or with that which is already bound to the amyloid fibrils. A practical problem, on the other hand, is the difficulty of preparing a large amount of SAP. We know of no factory-prepared recombinant SAP which is ready to use. In contrast, monoclonal antibodies have long been available. Furthermore, once bound to the fibrils, the SAP molecules may remain in that combination for several days,6,7 during which time no beneficial effects seem to occur. Instead, SAP may stabilize the amyloid deposits at those sites. In contrast, antibodies can trigger inflammatory responses, which may degrade amyloid fibrils. One harmful
effect of the antibody is the generation of antianimal antibodies. To avoid this build-up, a modification of the antibody molecules will be necessary. The aim of this study was to confirm earlier suspicions that injected anti-SAA antibody binds to amyloid deposits. The binding was confirmed by immunohistochemical detection of rat immunoglobulins in the amyloid deposits. The positive staining of nonimmune rat immunoglobulins in the liver soon after injection may be due to the handling of foreign substances by hepatocytes.14 Since this diminishes in the late phase after injection, the presence of the antibody at this stage definitely indicates an association between antibodies and amyloid deposits. According to a previous report,15 the injected SAP is rapidly cleared from the circulation of patients who have amyloid deposits, probably because of an absorption by the amyloid deposits. This was also assessed in the present investigation, and the same tendency was observed. However, the difference in the clearance rates of the injected antibody in mice with and without amyloid deposits does not seem to be striking enough to clearly indicate the presence or absence of amyloid deposits. Changes in the amounts of antibody or in the modulation of the antibody molecule should be assessed to clarify this issue. In conclusion, the specific localization of the antibody to the amyloid deposits was confirmed. The antibody injection method may have a similar value to the SAP method. The next step should be the use of radioisotopic labeling for scintigraphic imaging of amyloid deposits, as performed in the SAP study.6
References 1. Benson MD. Amyloidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle DV, editors. The metabolic basis of inherited disease. 7th ed. New York: McGraw-Hill; 1995. pp. 4159–91. 2. Okuda Y, Takasugi K. Secondary amyloidosis in rheumatoid arthritis: diagnostic and prognostic study of 198 biopsy proven cases. Arthritis Rheum 1997;40:S115. 3. Husby G, Marhaug G, Dowton B, Sletten K, Sipe JD. Serum amyloid A (SAA): biochemistry, genetics and the pathogenesis of AA amyloidosis. Amyloid 1994;1:119–37. 4. Yamada T. Serum amyloid A (SAA): a concise review of biology, assay methods and clinical usefulness. Clin Chem Lab Med 1999;37:381–8. 5. Baumann H, Gauldie J. The acute phase response. Immunol Today 1994;15:74–80. 6. Hawkins PN, Myers MJ, Epenetos AA, Caspi D, Pepys MB. Specific localization and imaging of amyloid deposits in vivo using 123 I-labeled serum amyloid P component. J Exp Med 1988;167:903– 13. 7. Hawkins PN, Myers MJ, Lavender JP, Pepys MB. Diagnostic radionuclide imaging of amyloid: biological targeting by circulating human serum amyloid P component. Lancet 1988;i(8600):1413– 8. 8. Pepys MB, Booth DR, Hutchinson WL, Gallimore JR, Collins PM, Hohenester E. Amyloid P component. A critical review. Amyloid 1997;4:274–95. 9. Hawkins PN, Richardson S, Vigushin DM, David J, Kelsey CR, Gray RE, et al. Serum amyloid P component scintigraphy and turnover studies for diagnosis and quantitative monitoring of AA amyloidosis in juvenile rheumatoid arthritis. Arthritis Rheum 1993;36:842–51.
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10. Yamada T, Fukuda T, Wada A, Itoh Y. Monoclonal antibodybased sensitive enzyme-linked immunosorbent assay for murine serum amyloid A. J Immunoassay 1999;20:223–35. 11. Yamada T, Uchiyama K, Yakata M, Gejyo F. Sandwich enzyme immunoassay for serum amyloid A protein (SAA). Clin Chim Acta 1989;178:169–76. 12. Axelrad M, Kisilevsky R, Willmer J, Chen SJ, Skinner M. Further characterization of amyloid-enhancing factor. Lab Invest 1982;47: 139–46. 13. Yamada T, Liepnieks JJ, Benson MD, Kluve-Beckerman B. Accelerated amyloid deposition in mice treated with the aspartic protease inhibitor, pepstatin. J Immunol 1996;157:901–7.
14. Meek RL, Eriksen N, Benditt EP. Serum amyloid A in the mouse. Sites of uptake and mRNA expression. Am J Pathol 1989;135:411– 9. 15. Jager PL, Hazenberg BP, Franssen EJF, Limburg PC, van Rijswijk MH, Piers DA. Kinetic studies with iodine-123-labeled serum amyloid P component in patients with systemic AA and AL amyloidosis and assessment of clinical value. J Nucl Med 1998;39: 699–706.
