Immunoradiometric assay for human serum amyloid P component

Human serum amyloid P component (SAP) is of increasing interest for its possible pathogenic role in amyloidosis and Alzheimer's disease, and as a therapeutic target in these conditions. We have developed and validated a robust and reproducible immunoradiometric assay (IRMA) for human SAP in serum, plasma and cerebrospinal fluid, and characterized the notable stability of human SAP immunoreactivity during storage of undiluted serum at 4°C and 37°C as well as frozen at -30°C. SAP values were also stable after repeated freeze thawing of highly diluted serum samples. The 100 fold dynamic range of the assay, 0.5-50 μg/L, encompassed all values seen in blood and cerebrospinal fluid, when tested at suitable dilutions, from both normal healthy individuals and patients, including subjects receiving the SAP-depleting drug, CPHPC. Furthermore by comparing the IRMA values in the presence and absence of calcium, the new assay revealed interference due to the binding of CPHPC by SAP, which was markedly enhanced in heparinized plasma. It is therefore essential that SAP assays in samples from patients on CPHPC be conducted in the absence of free calcium, in order to completely abrogate interference and determine the actual total SAP concentration. Estimates by the IRMA of SAP concentration in 49 serum samples from amyloidosis patients corresponded closely with those obtained by the established standard electro-immunoassay method and by a newly developed commercial ELISA kit (Hycult Biotechnology).     

血清淀粉样P成分的DNA结合特性

目的 检测血清淀粉样P成分(SAP)与不同DNA的结合活性并研究SAP与DNA的结合是否有利于此类抗原的清除,探讨它在SLE发生发展中的作用及意义。方法 用亲和层析和凝胶过滤方法自小鼠和人血清中纯化SAP,分别提取来自细菌、质粒、酵母、小鼠淋巴细胞等不同DNA,用DOT-EIA方法检测SAP与它们的结合活性,用巨噬细胞吞噬实验观察SAP与DNA结合后对DNA清除的影响。结果 人和小鼠SAP均与细菌DNA结合最强,其次为质粒、酵母DNA,与淋巴细胞DNA和小牛胸腺DNA的结合较弱,与单链λDNA结合最弱;与来源于活化状态小鼠淋巴细胞DNA的结合力高于非活化状态;SAP与活化淋巴细胞DNA结合后可促进巨噬细胞对DNA的吞噬。结论 SAP与不同DNA结合活性各异。     

Effects of serum amyloid P component on human lymphocytes

The P component of amyloid is a normal serum protein designated SAP. SAP has substantial homology with C-reactive protein (CRP). Recent studies have established the structure, tissue distribution and binding reactivities of SAP; however, as yet, very minimal insight into its function has been achieved. Recent studies, though somewhat controversial, have suggested a regulatory role for CRP on the immune response. In view of these studies, we wanted to evaluate the in vitro effects of SAP on several immunological properties of human lymphocytes. We found that SAP had a marked inhibitory effect on the proliferative response of lymphocytes to a variety of T-dependent mitogens. In addition, SAP markedly enhanced the formation of active E rosettes, a marker of activated T cells.     

Isolation of human C-reactive protein and serum amyloid P component

Procedures are described for the isolation in high yield of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP). CRP was obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled to cyanogen bromide-activated Sepharose. It was then gel filtered on Ultrogel AcA44 (acrylamideagarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity for agarose. Residual trace contaminants were removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and/or by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300 35–44% of the initial CRP was recovered in pure from according to biochemical and immunochemical criteria. SAP was isolated from normal serum by calcium-dependent affinity chromatography on unsubstituted Sepharose beads, followed by solid-phase immunoabsorption of contaminants and finally gel filtration on Sephacryl S-300. At least 50% of the SAP in the starting material was recovered in pure form according to biochemical and immuunochemical criteria. Ready availability of such preparations facilitates biochemical, biophysical and particularly biological studies of these plasma proteins.     

Evidence for an extended interacting surface between beta-amyloid and serum amyloid P component

Studying the interaction between serum amyloid P component (SAP) and beta-amyloid (Abeta) a new Abeta binding site was identified on the SAP near the known binding site at the two bound calcium ions. SAP stabilizes deposits in neurodegenerative diseases, which is manifested via Abeta-binding. Because the inhibition of this interaction is a potential therapeutic target in neurodegeneration, the structural basis of SAP-Abeta binding was studied. The chymotryptic digestion of SAP resulted in a 18,223Da product identified by mass spectrometry. This cleavage was inhibited by Abeta revealing that this cleaving site between Tyr-140 and Gly-141 is involved in the interaction between SAP and Abeta These results suggest that the Abeta-binding site on SAP is larger than it was recently assumed.     

Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins

Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure that has morphological and immunochemical identity to the amyloid P component found in normal tissue and amyloid deposits. In the presence of calcium, SAP binds to certain complex polysaccharides, including agarose and zymosan. While the binding of SAP to agarose involves interaction with a galactose pyruvate acetal, the ligand in zymosan has not been defined. In the present study we determined that SAP binds to ligand(s) in a soluble extract of zymosan prepared by alkaline hydrolysis, which contains the mannose oligosaccharide sequences alpha DMan1----3DMan and alpha DMan1----6DMan. SAP did not bind to the alkali-insoluble fraction of zymosan, which is predominantly a glucan polymer, and its binding to zymosan extract which had been absorbed with concanavalin A was markedly reduced, suggesting that mannose residues are involved in the binding of SAP to zymosan. We also demonstrated that SAP binds to the glycoproteins ovalbumin, thyroglobulin, beta-glucuronidase and C3bi, which contain mannose-terminated sequences, while it did not bind to native and desialized preparations of ovomucoid, alpha 1-acid glycoprotein and glycophorin, which lack terminal mannose residues. SAP did not bind to pneumococcal C polysaccharide or to N-acetylglucosamine oligosaccharides covalently linked to a protein carrier. The binding of SAP to ligand(s) in zymosan extract or ovalbumin was inhibited by the preincubation of SAP with either zymosan extract or ovalbumin glycopeptides, both of which share similar mannose oligosaccharide sequences. All of the SAP binding reactions required calcium, were maximal at approximately 1 mM calcium, and gave similar results whether purified SAP or SAP in serum was used. These findings indicate that mannose-terminated oligosaccharides of polysaccharides and glycoproteins represent a new class of ligands for SAP and suggest that SAP may function as a mannose-binding protein.     

Circulating serum amyloid P component is the precursor of amyloid P component in tissue amyloid deposits.

Intravenous administration of 125I-labelled isolated mouse serum amyloid P component (SAP) to mice with systemic amyloidosis was followed by specific deposition of the labelled protein in amyloidotic organs. Although only a small proportion of the total injected dose became localized in this way, the amount correlated with the quantity of amyloid present in different organs and was greatest in the spleen. No such localization was detected in the organs of control, untreated mice or animals which had received inflammatory stimuli but did not have amyloidosis. The labelled SAP was found by autoradiography to be present in the same distribution within the tissues as the Congophilic amyloid deposits. These observations establish directly, for the first time, that circulating SAP is the precursor of the amyloid P component (AP) in systemic amyloidosis. They were confirmed by the further finding that intravenous injection into amyloidotic mice of human SAP, either in whole human serum or in isolated pure form, was followed by appearance of the human SAP in the mouse amyloid deposits. In addition to elucidating one aspect of the pathogenesis of amyloid deposition and strengthening the homology of functional behaviour between SAP of different species, the present results suggest a means for selective targeting of diagnostic tracers and/or effector agents to amyloid deposits in vivo.     

Evidence for a serum inhibitor of Clq

Clq was isolated from fresh serum and chromatographed on a column of insolubilized Concanavalin A. Two fractions were obtained; one before (fraction A) and one after elutiion (fraction B) with α-methyl-glucopyrannoside. Fraction B contained Clq and precipitated upon double diffusion in agarose against fraction A. The anionic nature of fraction A was demonstrated by electrophoresis in agarose. Fraction B Clq was found to be more soluble in low ionic strength buffers and to have an increased hemolytic activity. Fraction A inhibited Clq hemolytic activity in a dose dependent fashion. This effect could not be eliminated by DNA-ase digestion.     

Human serum amyloid P component binds to peripheral blood monocytes

The binding of human serum amyloid P component (SAP) to blood cells and monocyte-derived dendritic cells was investigated by flow cytometry. Monocytes bound biotinylated SAP with avidity in a dose-dependent and saturable manner. By contrast, the binding of SAP to monocyte-derived dendritic cells was weak. No binding to erythrocytes, NK cells, T lymphocytes or B lymphocytes could be detected. The SAP-monocyte interaction was calcium-independent and readily inhibited by C1q. SAP was nonmitogenic for human mononuclear cells and had no apparent influence on lymphocyte proliferation induced by mitogenic lectins. We speculate that binding of SAP by monocytes could be of physiological relevance at extravascular sites by influencing complement regulation.     

Identification of multiple forms of the P component of amyloid isolated from human serum

We examined isolated serum amyloid P component (SAP), the circulating counterpart of the amyloid P component, and established a previously unreported heterogeneity for SAP by immunological and biochemical methods. Highly purified SAP had a gel filtration Mr of 255,000, a Stokes radius of 57 A, a calculated frictional coefficient of 1.4, and 10 subunits of Mr of approximately 25,000. We present evidence suggestive of varying affinities of SAP for agarose, to which SAP is known to adsorb in the presence of calcium, by fused rocket immunoelectrophoresis. We resolved SAP subunits by isoelectric focusing into multiple species with isoelectric points of 6.08 (two forms), 6.02, and 5.95; three of these forms were delineated by high resolution two-dimensional SDS gel electrophoresis to have an Mr = 25,500, while a fourth (pI = 6.08) had an Mr = 24,500. The observed isoelectric charge heterogeneity could not be eliminated by neuraminidase treatment event though the electrophoretic migration of native desialized SAP was impeded (alpha 1 to beta) when examined by crossed immunoelectrophoresis, being consistent with the removal of anionic charges. Further, an additional neuraminidase-generated component was detected at the beta-position which could be removed by concanavalin A affinity. These data suggest SAP may exist in microheterologous or allotypic forms, the significance of which is under investigation.     

Isolation and characterization of guinea-pig serum amyloid P component.

A pentraxin was isolated from acute-phase guinea-pig serum by calcium-dependent affinity chromatography on agarose. It was immunochemically identical to guinea-pig amyloid P component and therefore has been called guinea-pig serum amyloid P component (SAP). Guinea-pig SAP has an apparent MW of between 265,000 and 300,000 by different techniques, and is composed of 10 noncovalently associated subunits arranged in two pentameric annular discs interacting face-to-face. It is apparently composed of two types of subunit, which run as a closely spaced doublet on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least one type of subunit is glycosylated. The serum concentration was 16 +/- 4 mg/l in outbred animals, rising to 25 +/- 4 mg/l in an acute-phase response. Binding to agarose correlated with the agarose pyruvate content and was completely abolished by diazomethane treatment of the agarose, which methylates the pyruvate carboxylic moiety. Binding was also inhibited in the presence of free methyl 4,6-o-(carboxyethylidine)-beta-D-galactopyranoside. No protein resembling C-reactive protein (CRP) was obtained by calcium-dependent affinity chromatography of acute-phase guinea-pig serum on phosphorylcholine (PC)-Sepharose, and it not clear whether a counterpart of CRP exists in this species.     

Quantitation of serum amyloid P component by an enzyme-linked immunoassay

A solid phase enzyme-linked immunoassay (ELISA) for the murine acute-phase reactant, serum amyloid P component (SAP), was developed. The assay is based on our finding of a calcium-dependent binding of SAP to trinitrophenyl-conjugated proteins. The wells of polystyrene microtiter plates are coated with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), then incubated with SAP-containing samples. The amount of SAP is determined by indirect ELISA, wells being sequentially incubated with rabbit anti-SAP antiserum and horseradish peroxidase-linked donkey anti-rabbit IgG conjugate. The limit of sensitivity of the assay is 0.6 ng/ml SAP. Comparison with data on sera obtained by rocket immunoelectrophoresis assay yielded a correlation coefficient of 0.89. The binding of SAP to TNP-KLH was inhibited by calcium chelators and low concentrations of non-ionic detergents. Chromatography of serum on TNP-Sepharose provided a efficient and simple way of purifying SAP. The assay was also adapted for the quantitation of human SAP. The use of the assay in studying the binding specifities of SAP and its physiological role is discussed.     

Measurement of murine serum amyloid P component by rate nephelometry

Murine serum amyloid P component (SAP) is an acute-phase protein that is increased 2–10-fold in concentration following appropriate inflammatory or infections stimuli. Previous studies of the acute-phase SAP response have employed quantitative immunoelectrophoresis or radioimmunoassay to measure SAP concentration. A rate nephelometric procedure has been developed which measures SAP concentration rapidly and with equivalent or greater precision than the previously applied techniques. This simple method will facilitate experimental and clinical studies of the acute-phase response.     

Localization of human serum amyloid P component and heparan sulfate proteoglycan in in vitro-formed Abeta fibrils

Ultrastructural studies of the localization of serum amyloid P component (SAP) in amyloid fibrils have given divergent results. We here report for the first time that electron microscopy of SAP coincubated with Abeta1-42 peptides or with mature Abeta1-42 fibrils, revealed SAP molecules coating the surface of the mature fibrils and that protofibrils of Abeta1-42 did not bind SAP. Also when incubated with extracted amyloid light chain (AL)-fibrils the SAP molecules aligned on the fibril surface. Heparan sulfate proteoglycan bound to the surface of the Abeta fibrils with a spacing of about 50 nm. We conclude that SAP does not bind to protofibrils but to the surface of mature Abeta fibrils and that it may stabilize and protect the fibrils.     

The major acute phase reactants: C-reactive protein,serum amyloid P component and serum amyloid A protein

Following an acute phase stimulus, such as infection or physical injury, many liver-derived plasma proteins are increased in concentration. These provide enhanced protection against invading micro-organisms, limit tissue damage and promote a rapid return to homeostasis. Diana Steel and Alexander Whitehead discuss the gene structure, regulation and possible clinical significance of the most dramatically induced acute phase reactants.     

Binding of human serum amyloid P componentto L-selectin

Serum concentrations of soluble L-selectin by far exceed those of other soluble adhesion molecules, and serum soluble L-selectin concentrations are remarkably stable upon prolonged storage. We present evidence for Ca(2+)-dependent binding interactions between human serum amyloid P (SAP), a proteolysis-resistant pentraxin glycoprotein, and L-selectin, as shown by surface plasmon resonance measurements, protein band shift assays in a native PAGE system, and after SDS-PAGE and membrane transfer. Monoclonal antibodies to L-selectin strongly reduced binding of biotinylated SAP to L-selectin-IgG chimeras immobilized on microtiter plates. As binding was reduced by prior glycopeptidase F treatment of L-selectin but not of SAP, it appears to be based on SAP lectin domain interactions with N-linked L-selectin carbohydrates. In freshly prepared human lymphocytes, SAP incubation induced expression of a beta2 integrin neoepitope associated with high-affinity binding. This was partially blocked by pre-incubation with Fab fragments of two anti-L-selectin antibodies. In flow chamber experiments, SAP inhibited the adherence of human neutrophils to activated endothelium under shear stress. Thus, SAP binds to human L-selectin and affects L-selectin-dependent leukocyte-endothelial interactions.     

Age-related deposition of amyloid P component in normal human testis

The distribution of amyloid P component in normal human testes from fetal life to old age was studied by a direct immunofluorescent technique on frozen sections. Amyloid P is readily and invariably detected in association with elastic fibres around seminiferous tubules and in blood vessels from the age of 30 years upwards. The same is true for most cases during the twenties, but in no case below the age of 18 was its presence demonstrable.     

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1β or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.     

Competition between serum amyloid protein and apoprotein E for binding with human serum albumin

Research Center for preventive Medicine, Ministry of Health of Russian Federation, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences V. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 12, pp. 598–600, December, 1992.