The Experts below are selected from a list of 64512 Experts worldwide ranked by ideXlab platform
Toshiyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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measurement of serum amyloid a1 saa1 a major isotype of acute phase saa
Clinical Chemistry and Laboratory Medicine, 2006Co-Authors: Yuanyuan Xu, Takahiko Satoh, Toshiyuki Yamada, Yasuaki OkudaAbstract:Serum amyloid A (SAA), a plasma precursor of reactive amyloid deposits, is a multigene product. SAA1 and SAA2, with primary structures that are 93% identical (98 of 104 amino acids), behave as acute phase proteins, as demonstrated by their increasing levels in plasma. Heretofore, it has been understood that SAA1 predominates and functions as an isotype in plasma. However, accurate measurements differentiating the two isotypes have not been reported. In this study, using monoclonal antibodies specific for SAA1, we developed an enzyme-linked immunosorbent assay (ELISA) for SAA1. The levels and ratios of SAA1 in total SAA (TSAA) were investigated in healthy subjects and patients with rheumatoid arthritis (RA). The SAA1/TSAA ratio was 74±12% and 77±12% in healthy subjects and RA patients, respectively. In RA patients, the ratios were not influenced by SAA1 genotype, which has been proposed to affect plasma SAA values. The kinetics of SAA1 in inflamed patients undergoing hemodialysis was found to be parallel with total SAA and C-reactive protein. Finally, this study confirmed that SAA1 is a major isotype of acute phase SAA and may determine total SAA values. This specific assay could be used in the evaluation of SAA behavior in several clinical conditions.
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an allele of serum amyloid a1 associated with amyloidosis in both japanese and caucasians
Amyloid, 2003Co-Authors: Toshiyuki Yamada, Kiyoshi Takasugi, Lishan Wang, Duane Marks, Yasuaki Okuda, Merrill D Benson, Barbara KluvebeckerrnanAbstract:Serum amyloid A1 (SAAI), one of the two isotypes of acute phase SAA, is the predominant precursor to amyloid A (AA) protein, the chief constituent of fibrillar deposits in reactive (AA) amyloidosis. Prolonged hyperexpression of SAA protein accompanying chronic inflammation is critical to, but seems not to be sufficient for, the development of AA amyloidosis. Several previous studies have investigated the possibility of linkage between SAAI exon 3 polymorphisms and susceptibility to amyloidosis. While the SAAI. I allele was found to have a negative association with amylodosis in Japanese subjects, it showed a positive association in Caucasians. Moriguchi and colleagues recently showed that a single nucleotide polymorphism (SNP) at position -13 in the SAAl S' flanking region was more strongly associated with amyloidosis than was the exon 3 polymorphism. To test whether this SNP may be an amyloidogenic factor common to Japanese and Caucasians, we have analyzed the SAA l gene in amyloid and non-amyloid patien...
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Serum amyloid A1 alleles and plasma concentrations of serum amyloid A
Amyloid, 1999Co-Authors: Toshiyuki Yamada, Atsufumi Wada, Yoshihisa Itoh, Kouichi ItohAbstract:Serum amyloid A1 (SAAI), the predominant isotype of acute phase SAA in plasma and the predominant precursor offibrillar deposits in reactive amyloidosis, is encoded by a gene, for which six allelic variants have been described Recent studies proposed that the allele SAA1.3 was positively correlated with the development of reactive amyloidosis in Japanese. This study examined whether the plasma concentration of total SAA is injluenced by specific SAA1 alleles. Two hundred and eighty healthy Japanese subjects were examined to determine the allelic distribution of SAA1 and SAA2 genes by the PCR-RFLP method, and to measure the total plasma SAA concentrations. SAA concentrations were significantly higher (P
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serum amyloid a saa a concise review of biology assay methods and clinical usefulness
Clinical Chemistry and Laboratory Medicine, 1999Co-Authors: Toshiyuki YamadaAbstract:Serum amyloid A (SAA) is a family of proteins encoded in a multigene complex. Acute phase isotypes SAA1 and SAA2 are synthesized in response to inflammatory cytokines. SAA and C-reactive protein (CRP) are now the most sensitive indicators for assessing inflammatory activity. In viral infection and kidney allograft rejection, SAA proved more useful than CRP. Development of convenient assay methods for SAA will facilitate its use in clinical laboratories.
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serum amyloid a1 alleles and plasma concentrations of serum amyloid a
Amyloid, 1999Co-Authors: Toshiyuki Yamada, Atsufumi Wada, Yoshihisa Itoh, Kouichi ItohAbstract:Serum amyloid A1 (SAAI), the predominant isotype of acute phase SAA in plasma and the predominant precursor offibrillar deposits in reactive amyloidosis, is encoded by a gene, for which six allelic variants have been described Recent studies proposed that the allele SAA1.3 was positively correlated with the development of reactive amyloidosis in Japanese. This study examined whether the plasma concentration of total SAA is injluenced by specific SAA1 alleles. Two hundred and eighty healthy Japanese subjects were examined to determine the allelic distribution of SAA1 and SAA2 genes by the PCR-RFLP method, and to measure the total plasma SAA concentrations. SAA concentrations were significantly higher (P<0. 001) in subjects with the allele SAA 1.5 than those without it, suggesting that SAA 1.5 may have a distinctive feature in the process of synthesis or catabolism. Subjects with the allele SAA1.3 had lower SAA concentrations, though not statistically significant, than those with SAA1.1. There was not signi...
Merrill D Benson - One of the best experts on this subject based on the ideXlab platform.
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an allele of serum amyloid a1 associated with amyloidosis in both japanese and caucasians
Amyloid, 2003Co-Authors: Toshiyuki Yamada, Kiyoshi Takasugi, Lishan Wang, Duane Marks, Yasuaki Okuda, Merrill D Benson, Barbara KluvebeckerrnanAbstract:Serum amyloid A1 (SAAI), one of the two isotypes of acute phase SAA, is the predominant precursor to amyloid A (AA) protein, the chief constituent of fibrillar deposits in reactive (AA) amyloidosis. Prolonged hyperexpression of SAA protein accompanying chronic inflammation is critical to, but seems not to be sufficient for, the development of AA amyloidosis. Several previous studies have investigated the possibility of linkage between SAAI exon 3 polymorphisms and susceptibility to amyloidosis. While the SAAI. I allele was found to have a negative association with amylodosis in Japanese subjects, it showed a positive association in Caucasians. Moriguchi and colleagues recently showed that a single nucleotide polymorphism (SNP) at position -13 in the SAAl S' flanking region was more strongly associated with amyloidosis than was the exon 3 polymorphism. To test whether this SNP may be an amyloidogenic factor common to Japanese and Caucasians, we have analyzed the SAA l gene in amyloid and non-amyloid patien...
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characterization of amyloid a protein in human secondary amyloidosis the predominant deposition of serum amyloid a1
Biochimica et Biophysica Acta, 1995Co-Authors: Juris J Liepnieks, Barbara Kluvebeckerman, Merrill D BensonAbstract:Abstract Serum amyloid A protein (SAA) is the plasma precursor for amyloid A protein (AA), the subunit protein in amyloid deposits of secondary or reactive amyloidosis. Several forms of acute phase SAA have been identified in human plasma. To elucidate whether one of these forms of SAA predominates in the formation of AA amyloid deposits, the amino acid sequence of the subunit protein in six cases of reactive amyloidosis was investigated. Minimal heterogeneity was present at the N-terminus as all samples started with residue 1, 2, or 3 of SAA. The C-terminus, however, was more heterogeneous with the AA protein in each case terminating at multiple sites from residue 58 to 84 of SAA. Since less than 20% of the AA protein in each case contained sequence past residue 67 of SAA, the sequence and recovery of tryptic peptides containing residues 52, 57, and 60 where human SAA1 and 2 differ was used to determine the relative amounts of SAA1 and 2 present. One sample contained only SAA1 sequence, four contained approx. 11% or less of SAA2 sequence, and the sixth contained 24–33% of SAA2 sequence. Thus, while five of the six AA samples contained both SAA1 and 2, the predominant form in all cases was SAA1. In three of the six cases, the protein defensin was isolated along with the AA protein from the fibrils. This may suggest neutrophil involvement in SAA processing to AA fibrils.
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Fibril formation from recombinant human serum amyloid A
Biochimica et biophysica acta, 1994Co-Authors: Toshiyuki Yamada, Juris J Liepnieks, Barbara Kluve-beckerman, Merrill D BensonAbstract:Three isotypes of human serum amyloid A (SAA), SAA1, SAA2 beta, and SAA4 were expressed at high levels in Escherichia coli (E. coli) using a pET vector expression system. Each SAA cDNA was ligated to the vector pET-21a(+) and transformed into E. coli, strain BL21(DE3)pLysS. Expression conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to total E. coli proteins. Each recombinant SAA (rSAA) was purified by molecular sieve chromatography followed by chromatofocusing or hydrophobic interaction chromatography. The yield of purified protein was 5-10 mg per 11 of culture. When subjected to in vitro fibril forming conditions, rSAA1 formed amyloid-like fibrils confirmed by Congo red staining and electron microscopy. In contrast, rSAA2 beta and rSAA4 showed negative Congo red staining and curvilinear or flattened fibrillar structures on electron microscopy. This suggests that SAA1 has greater potential for forming amyloid fibrils than either SAA2 beta or SAA4.
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Differential Regulation of Human Serum Amyloid a Isoforms
Amyloid and Amyloidosis 1990, 1991Co-Authors: Barbara Kluve-beckerman, Juris J Liepnieks, Merrill D BensonAbstract:Significantly more SAA1 than SAA2 is routinely purified from the plasma of patients studied by this laboratory. Investigating the reason for this disparity, we have compared mRNA levels for SAAl and SAA2 in a patient whose ratio of purified SAA1/SAA2 was approximately six. Relative levels of SAA1 and SAA2 mRNA were determined by Northern analysis using isotype-specific oligonucleotide probes. Scanning densitometry of the resulting autoradiographs revealed a 2-fold predominance of SAA1 over SAA2 mRNA. Thus, a higher steady state level of SAA1 mRNA explains in part the predominance of the SAA1 isotype.
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Nonexpression of the human serum amyloid A three (SAA3) gene.
DNA and cell biology, 1991Co-Authors: Barbara Kluve-beckerman, Mitchell L. Drumm, Merrill D BensonAbstract:Serum amyloid A (SAA) is a major acute-phase plasma protein synthesized by the liver. In addition to the two major plasma isoforms described in humans (SAA1 and SAA2), a third form (SAA3) has been demonstrated in several other species and is distinguished by predominant extrahepatic expression. Two clones, Ch11g5-1-1 and HDg1-1, containing the human SAA3 gene are described in this report. The human SAA3 gene is comparable in organization to the SAA1 and SAA2 genes and shares with them 87% nucleotide identity in the region spanning exon 3 through exon 4. Sequences 5' to exon 3, however, are strikingly different from those in the SAA1 and SAA2 genes. For instance, the sequence deduced for amino acids 1-12 (exon 2) has only 25% identity with human SAA1 and SAA2; it most closely resembles that of rabbit SAA3 isolated from synovial fibroblast cultures (75% identity). Although rabbit SAA3 induces collagenase production in an autocrine fashion the human SAA3 gene is not expressed. This is shown by: (i) a single base insertion in the sequence corresponding to codon 31, (ii) the inability of a 918-bp fragment immediately upstream from SAA3 exon sequences to direct transcription of a chloramphenicol acetyltransferase reporter gene, and (iii) the absence of detectable human SAA3 in mRNA.
C L Chen - One of the best experts on this subject based on the ideXlab platform.
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influence of genotypes at saa1 and SAA2 loci on the development and the length of latent period of secondary aa amyloidosis in patients with rheumatoid arthritis
Human Genetics, 1999Co-Authors: M Moriguchi, Chihiro Terai, Y Koseki, M Uesato, Atsuo Nakajima, S Inada, M Nishinarita, S Uchida, Seong Yoon Kim, C L ChenAbstract:To examine whether polymorphism at the SAA loci is associated with the development of amyloid protein A (AA)-amyloidosis, we determined the genotypes at the SAA1 and SAA2 loci in 43 AA-amyloidosis patients (amyloidosis population) and 77 patients with rheumatoid arthritis (RA) who had been ill for less than 5 years (early RA population). We also compared the frequencies of the genotypes at the SAA1 locus among 90 Korean, 95 Taiwanese, and 103 Japanese healthy subjects. The frequencies of the γ/γ genotype and γ alleles at the SAA1 locus were significantly higher in the amyloidosis population than in the early RA population (34.9% versus 7.8%, and 58.1% versus 33.8%, χ2 test P=0.0001). The frequencies of the γ allele at the SAA1 locus in Koreans, Taiwanese, and Japanese were 41.6%, 35.6%, and 37.4%, respectively. The length of the latent period of AA-amyloidosis was significantly longer in the patients with smaller numbers of the γ allele at the SAA1 locus (Spearman's correlation coefficient: –0.42, P<0.05). On the other hand, the mean C-reactive protein (CRP) level during 2 years prior to the diagnosis of AA-amyloidosis was significantly higher in the patients with larger numbers of the γ allele at the SAA1 locus (Spearman's correlation coefficient: 0.34, P<0.05). No significant association was found between amyloidosis and polymorphism at the SAA2 locus. We postulate that the allele SAA1 γ renders an RA patient susceptible to amyloidosis, possibly by affecting the severity of inflammation in RA.
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Influence of genotypes at SAA1 and SAA2 loci on the development and the length of latent period of secondary AA-amyloidosis in patients with rheumatoid arthritis
Human genetics, 1999Co-Authors: M Moriguchi, Chihiro Terai, Y Koseki, M Uesato, Atsuo Nakajima, S Inada, M Nishinarita, S Uchida, Seong Yoon Kim, C L ChenAbstract:To examine whether polymorphism at the SAA loci is associated with the development of amyloid protein A (AA)-amyloidosis, we determined the genotypes at the SAA1 and SAA2 loci in 43 AA-amyloidosis patients (amyloidosis population) and 77 patients with rheumatoid arthritis (RA) who had been ill for less than 5 years (early RA population). We also compared the frequencies of the genotypes at the SAA1 locus among 90 Korean, 95 Taiwanese, and 103 Japanese healthy subjects. The frequencies of the γ/γ genotype and γ alleles at the SAA1 locus were significantly higher in the amyloidosis population than in the early RA population (34.9% versus 7.8%, and 58.1% versus 33.8%, χ2 test P=0.0001). The frequencies of the γ allele at the SAA1 locus in Koreans, Taiwanese, and Japanese were 41.6%, 35.6%, and 37.4%, respectively. The length of the latent period of AA-amyloidosis was significantly longer in the patients with smaller numbers of the γ allele at the SAA1 locus (Spearman's correlation coefficient: –0.42, P
Bruno Domon - One of the best experts on this subject based on the ideXlab platform.
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quantification of saa1 and SAA2 in lung cancer plasma using the isotype specific prm assays
Proteomics, 2015Co-Authors: Yeoun Jin Kim, Sebastien Gallien, Panchali Goswami, Katriina Sertamo, Marc Schlesser, Guy Berchem, Victoria Elkhoury, Bruno DomonAbstract:The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
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Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype‐specific PRM assays
Proteomics, 2015Co-Authors: Yeoun Jin Kim, Sebastien Gallien, Victoria El-khoury, Panchali Goswami, Katriina Sertamo, Marc Schlesser, Guy Berchem, Bruno DomonAbstract:The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
Alexander S. Whitehead - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the human acute phase serum amyloid A genes by tumour necrosis factor-alpha, interleukin-6 and glucocorticoids in hepatic and epithelial cell lines.
Scandinavian journal of immunology, 2004Co-Authors: Caroline F. Thorn, Alexander S. WhiteheadAbstract:The major acute-phase protein serum amyloid A, A-SAA, is upregulated by a variety of inflammatory stimuli, including cytokines and glucocorticoids (GCs). Elevated systemic concentrations of both A-SAA and tumour necrosis factor (TNF)-α are a feature of inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Here, we examine the roles of TNF-α, interleukin-6 (IL-6) and GCs on the transcriptional regulation of the two human A-SAA genes (SAA1 and SAA2) and show that these stimuli have different effects on the SAA1 and SAA2 promoters in HepG2 hepatoma and KB epithelial cell lines. Both genes are induced modestly by TNF-α and IL-6 alone and synergistically by TNF-α plus IL-6. The TNF-driven induction of SAA1, but not that of SAA2, can be enhanced by GCs in both cell lines, whereas GCs alone can upregulate SAA1 only in epithelial cells. The upregulation of both genes by cytokines, and of SAA1 by GCs, is more rapid in epithelial cells than hepatoma cells. We established that the order in which either cell line was treated with TNF-α and IL-6 influenced A-SAA promoter transcriptional activation. Treatment with TNF-α followed by IL-6 resulted in a much greater induction of both A-SAA genes than treatment with IL-6 followed by TNF-α.
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Tissue-specific regulation of the human acute-phase serum amyloid A genes, SAA1 and SAA2, by glucocorticoids in hepatic and epithelial cells.
European journal of immunology, 2003Co-Authors: Caroline F. Thorn, Alexander S. WhiteheadAbstract:The human acute-phase protein serum amyloid A (A-SAA), encoded by the SAA1 and SAA2 genes, is dramatically induced by pro-inflammatory mediators during the acute-phase response to infection or injury. Circulating A-SAA is predominantly synthesized by the liver. However, other tissues are the source of locally produced A-SAA. Here, we establish that the qualitative and kinetic aspects of SAA1 and SAA2 transcription following treatment of HepG2 hepatoma cells and KB epithelial cells with glucocorticoids and cytokines are quite distinct. Untreated HepG2 cells do not express A-SAA mRNA and glucocorticoids, when administered alone, fail to induce either SAA1 or SAA2. In contrast, untreated KB cells constitutively express SAA1 mRNA. Following cytokine stimulation, both A-SAA genes are rapidly up-regulated to similar extents. As in the hepatoma cell line, co-stimulation of KB cells with glucocorticoids places SAA1 at a transcriptional advantage over SAA2. Interestingly, SAA1 can be significantly induced by glucocorticoids alone in KB cells. The effects of glucocorticoids on SAA1 in both cell lines is glucocorticoid receptor-dependent. Differential regulation of A-SAA expression in these cell lines may reflect different temporal and spatial requirements for A-SAA synthesis in response to different inflammatory challenges.
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Differential glucocorticoid enhancement of the cytokine-driven transcriptional activation of the human acute phase serum amyloid A genes, SAA1 and SAA2.
Journal of immunology (Baltimore Md. : 1950), 2002Co-Authors: Caroline F. Thorn, Alexander S. WhiteheadAbstract:The human acute phase serum amyloid A (A-SAA) genes, SAA1 and SAA2 , have a high degree of sequence identity that extends ∼450 bp upstream of their transcription start sites. Each promoter contains analogously positioned functional binding sites for the transcription factors NF-κB and NF-IL6. In human HepG2 hepatoma cells transfected with SAA promoter luciferase reporter constructs, administration of IL-1 and IL-6, singly or in combination, induced SAA1 and SAA2 transcriptional readouts that were qualitatively indistinguishable. However, under induced conditions, the SAA2 promoter had a significant quantitative transcriptional advantage over the SAA1 promoter. The application of the synthetic glucocorticoid dexamethasone in the context of cytokine stimulation enhanced the transcriptional activity of the SAA1 , but not the SAA2 , promoter such that readout from the former became equivalent to that from the latter. A putative glucocorticoid response element (GRE) is present (between residues −208 and −194) only in the SAA1 gene; a similar sequence in the corresponding region of the SAA2 gene is disrupted by a nine-residue insertion. The SAA1 GRE was shown to be functionally active and the SAA2 disrupted GRE was shown to be functionally inactive in experiments using reporter constructs carrying SAA1 and SAA2 promoters that had been modified by site-specific mutagenesis. Quantitative analysis of transcript-specific RT-PCR products, derived from SAA1 and SAA2 mRNAs after treatment of HepG2 cells with cytokines in the presence or absence of dexamethasone, confirmed that the endogenous SAA1 gene has a cytokine-driven transcriptional disadvantage that is superseded by a marginal transcriptional advantage when glucocorticoids are present.
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Posttranscriptional Regulation of Acute Phase Serum Amyloid A2 Expression by the 5′- and 3′-Untranslated Regions of Its mRNA
Journal of immunology (Baltimore Md. : 1950), 1999Co-Authors: Daniel B. Longley, Diana M. Steel, Alexander S. WhiteheadAbstract:Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body’s early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2 , which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5′- and 3′-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5′-UTR and/or 3′-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1β and IL-6, the SAA2 5′- and 3′-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5′-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3′-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5′- and 3′-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.
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Generation of soluble recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use in development of a A-SAA specific ELISA
Journal of immunological methods, 1996Co-Authors: Christine C. Mccormack, Audrey Hobson, Sean Doyle, John Jackson, Cormac G. Kilty, Alexander S. WhiteheadAbstract:Human acute phase serum amyloid A (the A-SAA2 isoform) was expressed at high levels using the pGEX bacterial expression system. A-SAA2 protein was expressed in E. coli NM544 as part of a fusion protein facilitating rapid purification. A-SAA2 was cleaved from the fusion moiety in the presence of a non-ionic detergent (Triton X-100) to release a soluble A-SAA2. Further purification using ion exchange chromatography yielded a pure A-SAA2 (3 mg per litre of culture). Antibodies generated against recombinant A-SAA2 were specific for the acute phase SAAs, A-SAA1 and A-SAA2 and showed no cross-reactivity with the constitutively expressed SAA (C-SAA). These antibodies were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) specific for the measurement of A-SAA in serum.
