A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration
2005; Elsevier BV; Volume: 46; Issue: 9 Linguagem: Inglês
10.1194/jlr.d500018-jlr200
ISSN1539-7262
AutoresMitsuaki Ishihara, Takeshi Kujiraoka, Tadao Iwasaki, Makoto Nagano, Mayumi Takano, Jun Ishii, Masahiro Tsuji, Hajime Ide, Irina P. Miller, N.E. Miller, Hiroaki Hattori,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoApolipoprotein A-V (apoA-V) is a recently discovered apolipoprotein that appears to have a role in plasma triglyceride (TG) transport. We have developed an ELISA for apoA-V using monoclonal antibodies that has a lower limit of detection of 0.3 ng/ml and linearity up to 20 ng/ml. The ELISA was then used to quantify plasma apoA-V in 196 healthy subjects and 106 patients with insulin-resistant diabetes mellitus. In the healthy subjects, total apoA-V concentration was 179.2 ± 74.8 ng/ml, and it was greater in females than in males (P < 0.005). It was correlated positively with the plasma HDL cholesterol (r = 0.32, P < 0.0001), apoA-I (r = 0.27, P = 0.0001), and apoE (r = 0.18, P = 0.011) concentrations and negatively with plasma TG concentration (r = −0.22, P = 0.021). In relation to single nucleotide polymorphism 3 (−1131C/T) of the apoA-V gene, apoA-V concentration was higher in the T/T type than in the C/C type (P < 0.01). Plasma TG concentration was lower in the T/T type than in the C/C or C/T type (P < 0.05).ApoA-V concentration was lower in the diabetic patients (69.4 ± 44.3 ng/ml; P < 0.01) than in the healthy controls. Apolipoprotein A-V (apoA-V) is a recently discovered apolipoprotein that appears to have a role in plasma triglyceride (TG) transport. We have developed an ELISA for apoA-V using monoclonal antibodies that has a lower limit of detection of 0.3 ng/ml and linearity up to 20 ng/ml. The ELISA was then used to quantify plasma apoA-V in 196 healthy subjects and 106 patients with insulin-resistant diabetes mellitus. In the healthy subjects, total apoA-V concentration was 179.2 ± 74.8 ng/ml, and it was greater in females than in males (P < 0.005). It was correlated positively with the plasma HDL cholesterol (r = 0.32, P < 0.0001), apoA-I (r = 0.27, P = 0.0001), and apoE (r = 0.18, P = 0.011) concentrations and negatively with plasma TG concentration (r = −0.22, P = 0.021). In relation to single nucleotide polymorphism 3 (−1131C/T) of the apoA-V gene, apoA-V concentration was higher in the T/T type than in the C/C type (P < 0.01). Plasma TG concentration was lower in the T/T type than in the C/C or C/T type (P < 0.05). ApoA-V concentration was lower in the diabetic patients (69.4 ± 44.3 ng/ml; P < 0.01) than in the healthy controls. Plasma triglyceride (TG) levels are influenced by both genetic and environmental factors and are a major independent risk factor for coronary heart disease (1Sharrett A.R. Ballantyne C.M. Coady S.A. Heiss G. Sorlie P.D. Catellier D. Patsch W. Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions. The Atherosclerosis Risk in Communities (ARIC) Study.Circulation. 2001; 104: 1108-1113Google Scholar, 2Hokanson J.E. 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Rader D.J. Tall A.R. Cholesteryl ester transfer protein: a novel target for raising HDL and inhibiting atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 160-167Google Scholar, 11van Tol A. Phospholipid transfer protein.Curr. Opin. Lipidol. 2002; 13: 135-139Google Scholar). These factors and their associated gene-environment interactions are of importance in the pathogenesis of coronary heart disease. Apolipoprotein A-V (apoA-V) has recently been identified by comparative sequencing of human and mouse DNA and is located ∼27 kb distal to the apoA-IV gene in the APOA1/C3/A4 gene cluster on chromosome 11q23 (12Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in human and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Google Scholar). ApoA-V, shown to be expressed mostly in liver and independently named regeneration-associated protein 3, is upregulated after the early phase of liver regeneration after hepatectomy in rat (13van der Vliet H.N. Sammelsm M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.J. Biol. Chem. 2001; 276: 44512-44520Google Scholar). In mice overexpressing the human apoA-V gene, TG concentrations decreased by 50–70%, and in apoA-V gene knockout mice, plasma TG concentrations increased ∼4-fold (12Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in human and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Google Scholar, 13van der Vliet H.N. Sammelsm M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.J. Biol. Chem. 2001; 276: 44512-44520Google Scholar, 14van der Vliet H.N. Schaap F.G. Levels J.H. Ottenhoff R. Looije N. Wesseling J.G. Groen A.K. Chamuleau R.A. Adenoviral overexpression of apolipoprotein A-V reduces serum levels of triglycerides and cholesterol in mice.Biochem. Biophys. Res. Commun. 2002; 295: 1156-1159Google Scholar). These results suggest that apoA-V expression may strongly influence, and be negatively associated with, plasma TG concentrations. ApoA-V both enhances lipoprotein lipase-mediated hydrolysis of plasma TG and inhibits hepatic VLDL-TG production (15Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis.J. Biol. Chem. 2004; 279: 27941-27947Google Scholar). ApoA-V also stimulates the efflux of cholesterol from cells by a mechanism independent on the ABCA1 protein, as do other exchangeable apolipoproteins, such as apoA-I and apoA-IV (16Beckstead J.A. Oda M.N. Martin D.D. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9414-9423Google Scholar). It was recently described that apoA-V mRNA is regulated by peroxisome proliferator-activated receptor α agonists (17Vu-Dac N. Gervois P. Jakel H. Nowak M. Bauge E. Dehondt H. Staels B. Pennacchio L.A. Rubin E.M. Fruchart-Najib J. et al.Apolipoprotein A5, a crucial determinant of plasma triglyceride levels, is highly responsive to peroxisome proliferator-activated receptor alpha activators.J. Biol. Chem. 2003; 278: 17982-17985Google Scholar, 18Prieur X. Coste H. Rodriguez J.C. The human apolipoprotein AV gene is regulated by peroxisome proliferator-activated receptor-alpha and contains a novel farnesoid X-activated receptor response element.J. Biol. Chem. 2003; 278: 25468-25480Google Scholar) and that the liver X receptor ligand T0901317 decreases apoA-V mRNA through the activation of sterol-regulatory element binding protein 1c (SREBP-1c) (19Jakel H. Nowak M. Moitrot E. Dehondt H. Hum D.W. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. The liver X receptor ligand T0901317 down-regulates APOA5 gene expression through activation of SREBP-1c.J. Biol. Chem. 2004; 279: 45462-45469Google Scholar). These results raise the possibility that some TG-lowering agents, such as fenofibrate, may act by altering the expression of apoA-V. In addition, associations have been identified between plasma TG concentrations and several apoA-V polymorphisms, including −1131T/C, −3A/G, S19W, and 1259T/C (12Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in human and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Google Scholar, 20Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides.Hum. Mol. Genet. 2002; 11: 3039-3046Google Scholar, 21Pennacchio L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. Two independent apolipoprotein A5 haplotypes influence human plasma triglyceride levels.Hum. Mol. Genet. 2002; 11: 3031-3038Google Scholar, 22Eichenbaum-Voline S. Olivier M. Jones E.L. Naoumova R.P. Jones B. Gau B. Patel H.N. Seed M. Betteridge D.J. Galton D.J. et al.Linkage and association between distinct variants of the APO A1/C3/A5 gene cluster and familial combined hyperlipidemia.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 2070-2077Google Scholar, 23Evans D. Buchwald A. Beil F.U. The single nucleotide polymorphism −1131T>C in the apolipoprotein A5 (APO A5) gene is associated with elevated triglycerides in patients with hyperlipidemia.J. Mol. Med. 2003; 81: 645-654Google Scholar, 24Kao J.T. Wen H.C. Chien K.L. Hsu H.C. Lin S.W. A novel genetic variant in the apolipoprotein A5 gene is associated with hypertriglyceridemia.Hum. Mol. Genet. 2003; 12: 2533-2539Google Scholar, 25Vrablik M. Horinek A. Ceska R. Adamkova V. Poledne R. Hubacek J.A. Ser19-Trp polymorphism within the apolipoprotein AV gene in hypertriglyceridaemic people.J. Med. Genet. 2003; 40 (http://www.jmedgenet.com/cgi/content/full/40/8/e105): e105Google Scholar, 26Lai C.Q. Demissie S. Cupples L.A. Zhu Y. Adiconis X. Parnell L.D. Corella D. Ordvas J.M. Influence of the APOA5 locus on plasma triglyceride, remnant-like particles, lipoprotein subclasses and cardiovascular disease risk in the Framingham Heart Study.J. Lipid Res. 2004; 45: 2096-2105Google Scholar). Plasma apoA-V concentrations were recently measured in Caucasians using an ELISA procedure that uses polyclonal antibodies against the N and C termini of the protein (27O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Google Scholar). We have raised two monoclonal antibodies (MAbs) against human apoA-V and used them to develop a new sandwich ELISA. We then used the assay to study plasma total apoA-V concentrations and the distribution of apoA-V between HDLs and other lipoproteins in healthy subjects. CHAPS was purchased from Wako Pure Chemical Industries (Osaka, Japan). Protein A-Sepharose FF was from Amersham Bioscience (Uppsala, Sweden). Blood from 196 apparently healthy volunteers (105 males, 91 females) without any medication who had fasted overnight was collected at the BML Clinical Reference Laboratory (Saitama, Japan). Blood from 106 non-insulin-dependent diabetics (61 males and 45 females) was collected at the outpatient clinic of the Hokkaido Hospital for Social Insurance (Sapporo, Japan) after overnight fasting. EDTA-plasma was isolated immediately by centrifugation at 4°C and stored at −80°C until use. Subjects were not taking medications. Lipid profiles are shown in Table 1. In healthy subjects, concentrations of total and LDL cholesterol and TG were greater, and those of apoA-I and apoE were lower, in males than in females. This study was approved by the ethical committees of the Hokkaido Hospital for Social Insurance and BML. Informed consent was obtained from all subjects.TABLE 1Lipid parameters of healthy and NIDDM subjectsVariablesHealthyNIDDMNumber (male/female)196 (105/91)96 (45/51)Age (years)34.8 ± 8.258.7 ± 12.0aSignificantly different from healthy controls (P < 0.0001).Total cholesterol (mmol/l)5.0 ± 0.94.9 ± 1.4TG (mmol/l)0.8 ± 0.51.6 ± 1.0aSignificantly different from healthy controls (P < 0.0001).LDL cholesterol (mmol/l)2.8 ± 0.82.8 ± 0.9 bSignificantly different from healthy controls (P < 0.01).HDL cholesterol (mmol/l)1.9 ± 0.41.4 ± 0.4aSignificantly different from healthy controls (P < 0.0001).ApoA-I (g/l)1.5 ± 0.21.2 ± 0.2aSignificantly different from healthy controls (P < 0.0001).ApoA-II (g/l)0.9 ± 0.20.3 ± 0.1aSignificantly different from healthy controls (P < 0.0001).ApoB (g/l)0.8 ± 0.11.1 ± 0.3cSignificantly different from healthy controls (P < 0.0005).ApoE (g/l)0.04 ± 0.010.04 ± 0.03ApoA-I, apolipoprotein A-I; NIDDM, non-insulin-dependent diabetes mellitus; TG, triglyceride. Values shown are means ± SD.a Significantly different from healthy controls (P < 0.0001).b Significantly different from healthy controls (P < 0.01).c Significantly different from healthy controls (P < 0.0005). Open table in a new tab ApoA-I, apolipoprotein A-I; NIDDM, non-insulin-dependent diabetes mellitus; TG, triglyceride. Values shown are means ± SD. Human apoA-V cDNA was obtained by RT-PCR from mRNA of HepG2 cells. PCR was carried out using 5′-GACGGATCCAAAGGCTTCTGGGACTACTTCAGCC-3′ as the sense primer and 5′-GACGTCGACTCAGGGGTCCCCCAGATGGCTGTGG-3′ as the antisense primer for apoA-V cDNA1 and 5′-GACGAATTCAGCAGATAATGGCAAGCATGGCTGC-3′ as the sense primer and 5′-GACGAATTCTCAGTGATGGTGATGGTGATGGGGGTCCCCCAGATGGCTGTGGCCC-3′ as the antisense primer for apoA-V cDNA2. The apoA-V cDNAs encoded amino acids 22–363 and 1–363 for cDNA1 and cDNA2, respectively, and apoA-V cDNA2 was constructed with a 6×His tag at the C terminus. The apoA-V cDNA1 was subcloned into the pQE-30 plasmid (Qiagen) to yield the pQE-30/apoA-V1 vector. Escherichia coli JM109 (Toyobo, Tokyo, Japan) bearing the pQE-30/apoA-V1 plasmid was cultured in Terrific Broth medium containing 50 mg/l ampicillin at 37°C. Expression was induced with 1 mM isopropyl thiogalactopyranoside, and after 5 h, the cells were harvested by centrifugation. The cells suspended in phosphate buffer (50 mM sodium phosphate and 0.5 M NaCl, pH 8.0) were disrupted by sonication. The insoluble fraction was pelleted by centrifugation at 30,000 g for 30 min at 4°C, and the pellet was dissolved in the phosphate buffer (pH 8.0) containing 7 M urea and 10 mM imidazole, followed by sonication. Then, the urea-solubilized fraction was centrifuged at 30,000 g for 30 min at 4°C, and the supernatant was loaded onto a nickel-nitrilotriacetic acid agarose column (Qiagen). The recombinant protein was eluted with acetate buffer (50 mM sodium acetate and 0.5 M NaCl, pH 4.5) containing 7 M urea. The purity of purified recombinant human apoA-V (rhapoA-V), subjected to SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining, was determined by gel scanning using the Intelligent Quantifier system (BioImage) as described previously (28Kujiraoka T. Iwasaki T. Ishihara M. Ito M. Nagano M. Kawaguchi A. Takahashi S. Ishii J. Tsuji M. Egashira T. et al.Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus.J. Lipid Res. 2003; 44: 2006-2014Google Scholar). The apoA-V cDNA2 was subcloned into the pEF321 mammalian expression vector (28Kujiraoka T. Iwasaki T. Ishihara M. Ito M. Nagano M. Kawaguchi A. Takahashi S. Ishii J. Tsuji M. Egashira T. et al.Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus.J. Lipid Res. 2003; 44: 2006-2014Google Scholar) to yield the pEF321/apoA-V vector. CHO-K1 cells stably transfected with pEF321/apoA-V vector were cultured in serum-free medium CHO-S-SFM II (Invitrogen), and the culture medium was collected. RhapoA-V was partially purified by metal affinity column chromatography using Talon® metal affinity resin (Clontech). The purity of rhapoA-V was confirmed by SDS-PAGE followed by CBB staining or immunoblotting. For immunoblotting, rhapoA-V was detected with Tetra-His antibody (Qiagen) as the primary antibody and horseradish peroxidase-conjugated anti-mouse IgG (Zymed Laboratories) as the secondary antibody. Bound antibodies were detected with an enhanced chemiluminescence kit (Perkin-Elmer Life Sciences). MAbs against apoA-V were obtained by the method of DNA-based immunization (29Tascon R.E. Colston M.J. Ragno S. Stavropoulos E. Gregory D. Lowrie D.B. Vaccination against tuberculosis by DNA injection.Nat. Med. 1996; 2: 888-892Google Scholar, 30Ogawa K. Tanaka K. Ishii A. Nakamura Y. Kondo S. Suganuma K. Takano S. Nakamura M. Nagata K. A novel serum protein that is selectively produced by cytotoxic lymphocytes.J. Immunol. 2001; 166: 6404-6412Google Scholar). In brief, Balb/c mice were injected subcutaneously with 50 μg plasmids of apoA-V cDNA2 inserted into pcDNA3.1(+) vector (Invitrogen) six times every 2 weeks. The final immunization was done intraperitoneally with 2.5 μg of rhapoA-V from CHO-K1 cells, and spleen cells from the mice were fused with Sp2/0 cells (31Gefter M.L. Margulies D.H. Scharff M.D. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells.Somatic Cell Genet. 1977; 3: 231-236Google Scholar). The supernatants of hybridoma cells were screened by ELISA using plates coated with partially purified rhapoA-V (50 ng/well) and by immunoblotting. Positive hybridoma cells were cloned at least three times by limiting dilution and injected intraperitoneally into pristane-primed Balb/c mice. The IgG fraction was isolated from ascitic fluid using protein A-Sepharose FF as described previously (28Kujiraoka T. Iwasaki T. Ishihara M. Ito M. Nagano M. Kawaguchi A. Takahashi S. Ishii J. Tsuji M. Egashira T. et al.Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus.J. Lipid Res. 2003; 44: 2006-2014Google Scholar), dialyzed at 4°C against PBS, and stored at −80°C. The specificities of MAbs B10E and E8E were confirmed by ELISA and immunoblotting against purified HDL and rhapoA-V. MAb isotype was characterized using the IsoStrip mouse MAb isotyping kit (Roche Diagnostics, Basel, Switzerland) and was IgG2a and IgG1 for MAbs B10E and E8E, respectively. MAb B10E (100 μl of 5 μg/ml solution in PBS) was coated onto a microtiter plate (Nunc Immunoplate II) by incubation at 4°C overnight. The wells were then blocked with 200 μl of PBS containing 30 g/l BSA for 2 h at room temperature. After the plate had been washed with 300 μl of PBS containing 1 g/l Tween 20, 100 μl of the calibrator solution and plasma samples (1:50) diluted with PBS containing 5 g/l CHAPS and 3 g/l BSA was added and incubated for 2 h at room temperature. After the plate had been washed five times, 100 μl of 0.5 μg/ml biotinylated MAb E8E was added to each well, and the mixture was incubated for 2 h at room temperature. After the plate had been washed five more times, 100 μl of 0.05 μg/ml horseradish peroxidase-conjugated streptavidin (Vector Laboratories) was added, and the mixture was incubated for 1 h at room temperature. After the plate had been washed, 100 μl of substrate solution (50 mM citrate-phosphate buffer, pH 5.0) containing 0.4 g/l o-phenylenediamine dihydrochloride and 0.15 ml/l H2O2 was added to each well. After 0.5 h, the reaction was stopped by the addition of 50 μl of 4 mol/l H2SO4. The absorbance was measured at 492 nm with a microplate reader. Purified bacterial rhapoA-V and pooled culture medium from CHO-K1 cells served as primary and secondary calibrators, respectively. When purified rhapoA-V was added to samples of plasma (n = 3) in sufficient amounts to increase the total apoA-V concentration by 100–400 ng/ml, the final concentrations given by the ELISA averaged 100.7% (85.6–111.7%) of those predicted. The intra-assay and interassay coefficients of variation of the ELISA were 2.2∼3.8% (n = 10) and 5.5∼8.7% (n = 5), respectively. No interference with the ELISA was observed with hemoglobin (5.0 g/l), bilirubin (0.3 g/l), or triacylglycerol (4.25 g/l). Storage of plasma and serum samples for 14 days did not affect the apoA-V concentration as determined by the ELISA (data not shown). The single nucleotide polymorphism (SNP) 1,131 bp upstream of the translation start site (T-1131C; SNP3) of the apoA-V gene was detected by the Invader assay as described previously (28Kujiraoka T. Iwasaki T. Ishihara M. Ito M. Nagano M. Kawaguchi A. Takahashi S. Ishii J. Tsuji M. Egashira T. et al.Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus.J. Lipid Res. 2003; 44: 2006-2014Google Scholar, 32Nagano M. Yamashita S. Hirano K. Ito M. Maruyama T. Ishihara M. Sagehashi Y. Oka T. Kujiraoka T. Hattori H. et al.Two novel missense mutations in the CETP gene in Japanese hyperalphalipoproteinemic subjects: high-throughput assay by Invader assay.J. Lipid Res. 2002; 43: 1011-1018Google Scholar). Primary probes and Invader oligonucleotides for each mutation were designed with Invader Creator software to have theoretic annealing temperatures of 63°C and 77°C, respectively, using a nearest neighbor algorithm on the basis of final probe and target concentrations. The primary probes and Invader oligonucleotides used are shown in Table 2. Genotyping was performed by calculation, using the ratios of net counts with wild primary probe to net counts with mutant primary probe. The accuracy of each genotyping was 100%, determined by comparison with results obtained previously by PCR-restriction fragment-length polymorphism analysis and direct sequencing.TABLE 2Oligonucleotide sequences of major allele, minor allele, and Invader probes for apoA-V SNP3 detectionSNPNucleotide ChangeTargetProbesSequencesDyeSNP3Major allele5′-ACGGACGCGGAGCACTTTCGCTCCAGTTV-3′REDT-1131CT to CSenseMinor allele5′-CGCGCCGAGGTACTTTCGCTCCAGTTCV-3′FAMInvader5′-GTGGAGTTCAGCTTTTCCTCATGGGGCAAATCTA-3′SNP, single nucleotide polymorphism; V (in sequences), amino blocking group. The flap sequences of primary probes are shown in boldface. Open table in a new tab SNP, single nucleotide polymorphism; V (in sequences), amino blocking group. The flap sequences of primary probes are shown in boldface. Measurements of plasma total cholesterol, triacylglycerol, and HDL cholesterol concentrations were performed with a Hitachi 7450 automated analyzer using commercial kits (Daiichi Pure Chemicals, Tokyo, Japan). HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with a commercial reagent containing dextran sulfate, phosphotungstate, and magnesium chloride (Daiichi Pure Chemicals) (28Kujiraoka T. Iwasaki T. Ishihara M. Ito M. Nagano M. Kawaguchi A. Takahashi S. Ishii J. Tsuji M. Egashira T. et al.Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus.J. Lipid Res. 2003; 44: 2006-2014Google Scholar). LDL cholesterol concentration was calculated according to Friedewald, Levy, and Fredrickson (33Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Google Scholar). Protein content was determined by the BCA protein assay kit (Pierce) using BSA as a calibrator. SDS-PAGE was performed by the Laemmli method (34Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Google Scholar), and immunoblotting was as described by Towbin, Staehelin, and Gordon (35Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. USA. 1979; 76: 4350-4354Google Scholar). Results are expressed as means ± SD. ANOVA was used for group comparisons. Correlations were analyzed by Spearman's rank correlation coefficient. P < 0.05 was considered statistically significant. The bacterial rhapoA-V purified from the lysate of E. coli showed a major band of ∼40 kDa (Fig. 1A). This represented >95% of the total protein after scanning the gel. Mice were first immunized by DNA injection, followed by partially purified rhapoA-V from CHO culture medium. Two MAbs specific for apoA-V were established: MAb B10E and MAb E8E. When rhapoA-V and human plasma were subjected to SDS-PAGE, both MAbs reacted with a single protein (Fig. 1B), the molecular mass of which (40 kDa, Fig. 1B, lanes 1 and 2) was similar to that previously reported for human plasma apoA-V (16Beckstead J.A. Oda M.N. Martin D.D. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9414-9423Google Scholar). The molecular weight of bacterial rhapoA-V (amino acids 22–363) appeared to be greater than those in plasma and culture medium, suggesting that plasma apoA-V may be secreted with more processing. By agarose electrophoresis and Western blotting, apoA-V was detected in the portion corresponding to α-lipoproteins, presumably because of the lower content of apoA-V in the latter (data not shown). There was no evidence of recognition of other plasma proteins. Both MAbs reacted similarly with rhapoA-V from E. coli or CHO cells coated on a microtiter plate (Fig. 2).Fig. 2Reactivity of MAbs against purified rhapoA-V. Purified rhapoA-V (100 ng/well; closed symbols) or partially purified rhapoA-V (100 μl; open symbols) from CHO-K1 cells was coated onto a microtiter plate. ELISA was carried out as described in Methods. Circles, E8E; squares, B10E.View Large Image Figure ViewerDownload (PPT) A sandwich ELISA for plasma apoA-V was established using MAb B10E for capture and biotinylated MAb E8E for detection. The system showed a dose-dependent response to purified bacterial rhapoA-V, to CHO culture medium expressing rhapoA-V, and to plasma, and the reactivity was equal with both bacterial and mammalian rhapoA-V (Fig. 3). For calibration of the ELISA, purified bacterial rhapoA-V was used as the primary calibrator. When subjected to SDS-PAGE and visualized by CBB staining, the purified bacterial rhapoA-V showed a single major 40 kDa band (Fig. 1), which represented >95% of the total protein in the preparation (as determined by gel scanning using the Intelligent Quantifier system). The protein concentration of this primary rhapoA-V calibrator, assayed using a bicinchoninic acid protein kit with BSA as calibrator, was typically 1.94 mg/ml. To obtain a calibration curve for the ELISA, dilutions of the primary calibrator were made in PBS containing 5 g/l CHAPS to provide 0.031–2.0 ng of rhapoA-V protein per well (15.6–1,000 ng/ml). When the rhapoA-V culture medium, as a secondary calibrator, was diluted in PBS containing 5 g/l CHAPS to cover the apoA-V concentration range 0.3125–20.0 ng/ml, the curve was identical to that obtained with the primary calibrator (Fig. 4). The ELISA was linear up to 1,000 ng/ml and suitable for quantifying apoA-V concentrations as low as 15.6 ng/ml. The linearity was also confirmed with serially diluted plasma samples of several concentrations (186 to 831 ng/ml) (data not shown). To avoid potential nonlinearity caused by very low or high absorbance, the apoA-V concentrations in plasma samples were measured using several dilutions (1:2 to 1:2,048). At the lowest dilutions of 1:2 to 1:16, results obtained with plasma were not identical to those obtained with the recombinant proteins (Fig. 3). Fifty-fold dilution of plasma, in which the diluted aliquot gave an absorbance between 0.5 and 1.2, was chosen for routine use. The detergent CHAPS was included in the diluent to avoid any effects of differences between samples in their lipid or apolipoprotein compositions. We examined several detergents for sample dilution, including Tween 20, Triton X-100, Nonidet P-40, SDS, CHAPS, CHAPSO, BIGCHAP, deoxy-BIGCHAP, n-octyl-β-d-glucoside, n-heptyl-β-d-thioglucoside, n-octyl-β-d-thioglucoside, n-dodecyl-β-d-maltoside, MEGA-8, MEGA-9, MEGA-10, sucrose monocaprate, sodium cholate, and digitonin (Detergent Starter Kit II; Wako Pure Chemical Industries). Plasma samples diluted (10-fold) and the rhapoA-V culture medium diluted (20-fold) with PBS gave similar absorbance with each detergent, but most detergents gave higher absorbance in the background (b
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