Title: A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin
Abstract: Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 ± 5.5 μg/ml (mean ± SD) in healthy women and 6.2 ± 3.6 μg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin). Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 ± 5.5 μg/ml (mean ± SD) in healthy women and 6.2 ± 3.6 μg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin). Adiponectin is an adipocyte-specific secretory protein that is highly and specifically expressed in adipose tissue (1Maeda K. Okubo K. Shimomura I. Funahashi T. Matsuzawa Y. Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1).Biochem. Biophys. Res. Commun. 1996; 221: 286-289Crossref PubMed Scopus (1849) Google Scholar, 2Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. A novel serum protein similar to C1q, produced exclusively in adipocytes.J. Biol. Chem. 1995; 10: 26746-26749Abstract Full Text Full Text PDF Scopus (2744) Google Scholar, 3Hu E. Liang P. Spiegelman B.M. AdipoQ is a novel adipose-specific gene dysregulated in obesity.J. Biol. Chem. 1996; 271: 10697-10703Abstract Full Text Full Text PDF PubMed Scopus (1890) Google Scholar). Adiponectin includes a collagen-like domain, and in this domain, three adiponectin peptides form one stable trimer and the trimers further multimerize to form "bouquet" forms (Fig. 1). In human plasma, adiponectin was found to circulate as a trimer, a hexamer, and a high-molecular-weight (HMW) multimer, and we purified the HMW adiponectin of 420 kDa from human serum using gelatin-Cellulofine and previously reported it as the gelatin binding protein of 28 kDa (GBP28) in 1996 (4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.J. Biochem. (Tokyo). 1996; 120: 803-812Crossref PubMed Scopus (786) Google Scholar). Plasma adiponectin levels are reported to be decreased in obese individuals, to be negatively correlated with visceral fat accumulation, and to be significantly lower in type 2 diabetic patients with coronary artery disease (5Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. et al.Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4076) Google Scholar, 6Cnop M. Havel P.J. Utzschneider K.M. Carr D.B. Sinha M.K. Boyko E.J. Retzlaff B.M. Knopp R.H. Brunzell J.D. Kahn S.E. Relationship of adiponectin to body fat distribution, insulin sensitivity and plasma lipoproteins: evidence for independent roles of age and sex.Diabetologia. 2003; 46: 459-469Crossref PubMed Scopus (1187) Google Scholar, 7Hotta K. Funahashi T. Arita Y. Takahashi M. Matsuda M. Okamoto Y. Iwahashi H. Kuriyama H. Ouchi N. Maeda K. et al.Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1595-1599Crossref PubMed Scopus (2742) Google Scholar). Adiponectin mRNA levels are significantly reduced in omental adipose tissue of obese patients with type 2 diabetes compared with lean and obese normoglycemic subjects, and although less pronounced, the levels are also reduced in subcutaneous adipose tissue of type 2 diabetic patients (8Statnick M.A. Beavers L.S. Conner L.J. Corominola H. Johnson D. Hammond C.D. Rafaeloff-Phail R. Seng T. Suter T.M. Sluka J.P. et al.Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes.Int. J. Exp. Diabetes Res. 2000; 1: 81-88Crossref PubMed Google Scholar). Plasma adiponectin concentrations in patients with acute coronary syndrome, both acute myocardial infarction and unstable angina pectoris, are significantly lower than those in patients with stable angina pectoris and in controls, and a low adiponectin concentration is correlated independently with the development of an acute coronary disease (9Nakamura Y. Shimada K. Fukuda D. Shimada Y. Ehara S. Hirose M. Kataoka T. Kamimori K. Shimodozono S. Kobayashi Y. et al.Implications of plasma concentrations of adiponectin in patients with coronary artery disease.Heart. 2004; 90: 528-533Crossref PubMed Scopus (229) Google Scholar). Plasma adiponectin levels are an inverse predictor of the cardiovascular outcome in patients with end-stage renal disease (10Zoccali C. Mallamaci F. Tripepi G. Benedetto F.A. Cutrupi S. Parlongo S. Malatino L.S. Bonanno G. Seminara G. Rapisarda F. et al.Adiponectin, metabolic risk factors, and cardiovascular events among patients with end-stage renal disease.J. Am. Soc. Nephrol. 2002; 13: 134-141Crossref PubMed Google Scholar). Tietge et al. (11Tietge U.J. Boker K.H. Manns M.P. Bahr M.J. Elevated circulating adiponectin levels in liver cirrhosis are associated with reduced liver function and altered hepatic hemodynamics.Am. J. Physiol. Endocrinol. Metab. 2004; 287: E82-E89Crossref PubMed Scopus (145) Google Scholar) reported that plasma adiponectin levels in cirrhosis are increased significantly, because the liver is a major source of adiponectin extraction and the adiponectin levels in cirrhosis do not correlate with parameters of body composition or metabolism but exclusively with reduced liver function and altered hepatic hemodynamics. As for physiological function, adiponectin was reported to inhibit processes that could be related to atherosclerotic plaque formation and suggested to act as an antiatherogenic factor (12Ouchi N. Kihara S. Arita Y. Maeda K. Kuriyama H. Okamoto Y. Hotta K. Nishida M. Takahashi M. Nakamura T. et al.Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin.Circulation. 1999; 100: 2473-2476Crossref PubMed Scopus (1947) Google Scholar, 13Yokota T. Oritani K. Takahashi I. Ishikawa J. Matsuyama A. Ouchi N. Kihara S. Funahashi T. Tenner A.J. Tomiyama Y. et al.Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages.Blood. 2000; 96: 1723-1732Crossref PubMed Google Scholar, 14Ouchi N. Kihara S. Arita Y. Okamoto Y. Maeda K. Kuriyama H. Hotta K. Nishida M. Takahashi M. 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Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.Proc. Natl. Acad. Sci. USA. 2001; 98: 2005-2010Crossref PubMed Scopus (1757) Google Scholar) reported that adiponectin increased fatty acid oxidation in muscle and caused weight loss in mice in 2001, adiponectin was reported to have a number of antidiabetic activities (16Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. et al.Disruption of adiponectin causes insulin resistance and neointimal formation.J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 20Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.Nat. 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Chem. 2003; 278: 50810-50817Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar), and in 2004, the amount of HMW adiponectin, not the absolute amount of adiponectin, was reported to be important in antidiabetic activities and vascular protective activities (27Pajvani U.B. Hawkins M. Combs T.P. Rajala M.W. Doebber T. Berger J.P. Wagner J.A. Wu M. Knopps A. Xiang A.H. et al.Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity.J. Biol. Chem. 2004; 279: 12152-12162Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar, 28Kobayashi H. Ouchi N. Kihara S. Walsh K. Kumada M. Abe Y. Funahashi T. Matsuzawa Y. Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin.Circ. Res. 2004; 94: e27-e31Crossref PubMed Google Scholar). With the ELISA systems that are commercially available now, total adiponectin can be measured, and because of this, the adiponectin levels reported to date have been those of total adiponectin. Here, we describe the development and evaluation of a sandwich ELISA method for HMW adiponectin involving monoclonal antibodies raised against HMW adiponectin, GBP28. Our sandwich ELISA system is available as a kit from Fujirebio Co. (Tokyo, Japan). A HiLoad 16/60 Superdex 200 prep-grade column, a Superdex 200 HR 10/30 column, protein molecular weight markers for size-exclusion chromatography, and ECL Plus Western blotting detection reagent were purchased from Amersham Biosciences (Uppsala, Sweden). Gelatin-Cellulofine was from Seikagaku Kogyo (Tokyo, Japan). LipofectAMINE reagent was from Invitrogen. A commercial ELISA kit for human adiponectin was purchased from Otsuka Pharmaceuticals (Tokyo, Japan). A monoclonal anti-human adiponectin/Acrp30 antibody and a biotinylated anti-human adiponectin/Acrp30 antibody were obtained from R&D Systems. Alkaline phosphatase- and peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories. Horseradish peroxidase streptavidin (streptavidin-conjugated horseradish peroxidase) was from Vector Laboratories. Tetramethylbenzidine solution was purchased from Dako Japan Co. (Kyoto, Japan), and nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were from Sigma Chemical. The bicinchoninic acid protein assay kit was from Pierce. Human plasma was kindly provided by the Japan Red Cross. From human plasma, HMW adiponectin was purified by affinity to gelatin-Cellulofine as described previously (4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.J. Biochem. (Tokyo). 1996; 120: 803-812Crossref PubMed Scopus (786) Google Scholar). Briefly, pooled human plasma was applied to the gelatin-Cellulofine column, and after thorough washing with 10 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl (TBS), HMW adiponectin was eluted with 10 mM Tris-HCl, pH 7.4, containing 1 M NaCl. After dialysis against TBS, the eluate was applied to a sulfate-Cellulofine column. The flow-through fractions were pooled, concentrated, and applied to the HiLoad 16/60 Superdex 200 prep-grade column repeatedly. Mouse monoclonal antibodies against HMW adiponectin were produced by the ordinary method. Balb/c mice were immunized with human HMW adiponectin using Freund's complete adjuvant, and spleen cells were fused with mouse myeloma cell line P3U1 using polyethylene glycol 4000. For screening, an HMW adiponectin coat plate was used, and the specificity of antibodies was verified by Western blotting. Rabbit anti-sera against HMW adiponectin (anti-HMW), the C-terminal 20 amino acids of adiponectin (anti-C), and the N-terminal 20 amino acids of adiponectin (anti-N) were described previously (4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.J. Biochem. (Tokyo). 1996; 120: 803-812Crossref PubMed Scopus (786) Google Scholar, 29Yoda M. Nakano Y. Tobe T. Shioda S. Choi-Miura N.H. Tomita M. Characterization of mouse GBP28 and its induction by exposure to cold.Int. J. Obes. Relat. Metab. Disord. 2001; 25: 75-83Crossref PubMed Scopus (39) Google Scholar). One of the obtained monoclonal antibodies against HMW adiponectin, IH7 (IgG2a), was digested with pepsin, reduced with 2-mercaptoethanol, and then reacted with maleimidoyl horseradish peroxidase to prepare horseradish peroxidase-conjugated IH7 Fab′ (POD-IH7). A sandwich ELISA for adiponectin was developed using IH7 as the capture antibody and POD-IH7 as the detection antibody. A 96-well microtiter plate was coated with 100 μl/well IH7 (1 μg/ml 10 mM Tris-HCl, pH 7.4), at 4°C overnight and then blocked with TBS containing 1% BSA at 4°C overnight. After washing of the plate with TBS containing 0.05% Tween 20 (TTBS), 100 μl aliquots of HMW adiponectin standards (2–50 ng/ml; five point calibration curve) or adequately diluted serum samples (400- to 1,000-fold) with TBS containing 1% BSA were added and the plate was incubated at room temperature for 1 h. After the plate had been washed three times with TTBS, 100 μl of POD-IH7 (50 ng/ml TBS containing 1% BSA) was added to each well, followed by incubation at room temperature for 30 min. After the plate had been washed three times with TTBS, 100 μl of the tetramethylbenzidine solution was added, followed by incubation at room temperature for 30 min. After the color had developed, 50 μl of 0.36 M H2SO4 was added to stop the reaction, and then the plate was read at 450 nm with a Bio-Rad model 680 microplate reader. For HMW adiponectin analysis, serum samples from 246 subjects (age range, 25–83 years) were randomly chosen as a subsample for the Health Examination Survey study carried out in Japan. The serum samples were stored at −70°C before HMW adiponectin analysis. Human serum was diluted with the same volume of phosphate-buffered saline (10 mM sodium phosphate and 150 mM NaCl, pH 7.4). To separate 0.5 ml of serum, the HiLoad 16/60 Superdex 200 prep-grade column equilibrated with the same buffer was used, and for 75 μl of serum, the Superdex 200 HR 10/30 column was used. The full-length adiponectin cDNA was inserted into expression vector pZeoSV2(+). cDNAs of the N-terminal cysteine mutant, Cys36Ser, and the C-terminal cysteine mutant, Cys152Ser, were also prepared and inserted into pZeoSV2(+). These constructs were transfected into CHO-K1 cells by the LipofectAMINE method according to the manufacturer's instructions, and stably transfected cell lines for the wild-type adiponectin and two mutants were established. SDS-PAGE was performed by the method of Laemmli, followed by Coomassie staining or Western blotting. After treatment with each antibody and alkaline phosphatase- or peroxidase-conjugated secondary antibodies of a nitrocellulose membrane, bands were detected with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate or ECL Plus Western blotting detection reagent according to the manufacturer's instructions and quantified with a GS-800 Calibrated Densitometer (Bio-Rad). For protein size analysis, a prestained SDS-PAGE standard, Precision Plus Protein Standards Dual Color (Bio-Rad), was used. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) version 12.0J (SPSS, Chicago, IL). The values are expressed as means ± SD. Correlation analysis was performed with the Pearson correlation test using unadjusted values, and statistical significance was tested using unpaired or paired t-tests. P < 0.05 was considered significant. This study was approved by the Ethical Committees of Showa University and Keio University Ise Keio Hospital. The investigations were conducted according to the principles outlined in the Declaration of Helsinki, and all patients gave informed consent. Mice were immunized with human HMW adiponectin, GBP28, and three mouse monoclonal antibodies, IH5, IH6, and IH7, were obtained (for IH5, IH6, and IH7, contact the corresponding author by e-mail). As described previously (4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.J. Biochem. (Tokyo). 1996; 120: 803-812Crossref PubMed Scopus (786) Google Scholar), because adiponectin possesses a collagen-like domain, the protein shows a very special character on SDS-PAGE (Fig. 2). As shown in Fig. 3A, lanes 4 and 2, when HMW adiponectin was boiled in Laemmli's loading buffer, it gave a single band corresponding to 28 kDa under reducing conditions, which represents the adiponectin monomer, and a single band corresponding to 56 kDa under nonreducing conditions, which represents the disulfide-bonded adiponectin dimer. On the other hand, without boiling, under reducing conditions, HMW adiponectin gave a band corresponding to 65 kDa, which represents the adiponectin trimer, and under nonreducing conditions, it gave a band corresponding to 150 kDa and a major band at almost the top of the gel, which represent the adiponectin hexamer and the HMW multimer, respectively (Fig. 3A, lanes 3, 1). When rabbit anti-HMW serum is used for immunoblotting, it can only detect bands of adiponectin without heat denaturation (i.e., that of the trimer and multimer bands of 65 kDa, 150 kDa, and higher molecular mass). On the other hand, rabbit anti-C and anti-N sera can only detect bands of heat-denatured adiponectin (i.e., the monomer and dimer bands of 28 and 56 kDa) (Fig. 3B, lanes 6, 8). When the reactivity of the three monoclonal antibodies, IH5, IH6, and IH7, was analyzed, they showed the same characteristics as anti-HMW (i.e., they only reacted with the non-heat-denatured bands: the trimer, hexamer, and higher multimer bands) (Fig. 3B, lanes 1, 3).Fig. 3Immunoblot analysis and 12.5% Laemmli's SDS-PAGE of HMW adiponectin. A: Coomassie staining. B: Immunoblotting with IH7 (lanes 1–4) and anti-C (lanes 5–8). Lanes 1, 3, 5, and 7 were not heat-denatured, and lanes 2, 4, 6, and 8 were heat-denatured at 100°C for 3 min under nonreducing or reducing conditions. H, HMW; h, hexamer; t, trimer; d, dimer; m, monomer; 2-ME, 2-mercaptoethanol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To elucidate the recognition sites for IH5, IH6, and IH7, reactivity with the wild-type recombinant and two cysteine mutants, Cys36Ser and Cys152Ser, was analyzed. As shown in Fig. 4A, lane 1, they recognized the three bands of the wild-type recombinant (i.e., the HMW, hexamer, and trimer). When the elution profile was analyzed by gel-filtration chromatography, the wild-type recombinant was eluted as the HMW, hexamer, and trimer molecules, the ratio being almost 1:1:1, and they were all detected by IH5, IH6, and IH7 (Fig. 4B, lanes 1–3). The N-terminal cysteine, cysteine 36, was reported to be important for the formation of the HMW and hexamer, although the C-terminal cysteine, cysteine 152, after mutation to alanine, did not have any effect on multimer formation (26Tsao T.S. Tomas E. Murrey H.E. Hug C. Lee D.H. Ruderman N.B. Heuser J.E. Lodish H.F. Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity: Different oligomers activate different signal transduction pathways.J. Biol. Chem. 2003; 278: 50810-50817Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The Cys36Ser and Cys152Ser mutants were secreted into the medium far less than the wild-type recombinant, and their amounts were ∼1/30th and 1/10th that of the wild-type recombinant, respectively, on densitometry analysis. When their elution profiles were analyzed by gel-filtration chromatography, these two cysteine mutants were mostly eluted as the trimer (Fig. 4C, D, lanes 7–9). In contrast to the cysteine-to-alanine mutation, a cysteine-to-serine mutation might induce some conformational change, and the Cys36Ser and Cys152Ser mutants did not form stable multimers. Unexpectedly, IH5, IH6, and IH7 scarcely reacted with the HMW, hexamer, and trimer forms of the Cys152Ser mutant (Fig. 4C, lanes 1–3). As described previously (4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.J. Biochem. (Tokyo). 1996; 120: 803-812Crossref PubMed Scopus (786) Google Scholar), heat denaturation or acidic pH treatment of HMW adiponectin induces a conformational change of the collagen-like domain, and after these treatments, HMW adiponectin could be detected with anti-C. After acetate treatment of the same nitrocellulose membrane, the HMW, hexamer, and trimer bands of Cys152Ser could be detected with anti-C (Fig. 4C, lanes 4–6). These results suggested that, although the Cys152Ser mutant could form the trimer and higher multimers, their conformations are different from the native forms, at least with respect to the IH5, IH6, and IH7 recognition sites. The Cys36Ser mutant was secreted far less efficiently than the wild type and the Cys152Ser mutant, and as shown in Fig. 4D, lanes 7–9, almost all of the secreted molecules were proteolytically cleaved into an ∼26 kDa form similar to the Cys36Ala mutant reported by Pajvani et al. (25Pajvani U.B. Du X. Combs T.P. Berg A.H. Rajala M.W. Schulthess T. Engel J. Brownlee M. Scherer P.E. Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity.J. Biol. Chem. 2003; 278: 9073-9085Abstract Full Text Full Text PDF PubMed Scopus (936) Google Scholar). Instead of its instability, the Cys36Ser mutant could form low amounts of HMW and hexamer, which could react with IH5, IH6, and IH7 (Fig. 4D, lanes 1–3). After acetate treatment, in addition to the HMW and hexamer bands, anti-C detected monomer and dimer bands of ∼26 and ∼52 kDa, which might be formed through cross-linking of collagen-like domains. This means that the Cys36Ser mutant could not form a trimer that is stable in SDS-containing loading buffer like the native adiponectin, suggesting the importance of cysteine 36 for the formation of a stable trimer. From these results, IH5, IH6, and IH7 were suggested to react with the C-terminal globular domain of one trimer. Adiponectin contains at least two O-glycoside chains, which are attached to threonine 22 and threonine 37 in the N-terminal nonglobular domain (Fig. 1). The wild-type recombinant contains two more O-glycoside chains that are attached to threonine 20 and threonine 21, and these additional O-glycoside chains do not influence the reactivity of IH5, IH6, and IH7. Also, O-glycoside digestion of these adiponectins with neuraminidase and O-glycanase did not have any effect on the reactivity with IH5, IH6, and IH7 (data not shown). These data might further support the notion that the recognition epitopes of IH5, IH6, and IH7 are in the C-terminal globular domain of one trimer. Using an HMW adiponectin coat plate, the reactivity of three monoclonal antibodies was also analyzed. After treatment with one monoclonal antibody, the plate was treated with each of the other monoclonal antibodies, which were biotinylated, and the bound biotinylated antibody was detected with streptavidin-conjugated horseradish peroxidase. In these experiments, it was shown that IH5, IH6, and IH7 bind to same site or nearby sites to block binding of the other two antibodies. Using IH5, IH6, and IH7, we tried to develop sandwich ELISA systems and found that with any combination of two of the three antibodies and even with the same antibody, a similar sandwich ELISA system could be constructed. Finally, using IH7 as the capture antibody and POD-IH7 as the detecting antibody, a sandwich ELISA was developed. As the adiponectin standard, HMW adiponectin, GBP28, purified from human serum was used. The concentration of standard HMW adiponectin was decided based on the absorbance at 280 nm and the results of quantitative amino acid composition analysis and the bicinchoninic acid protein assay with BSA as a standard. These three analyses gave almost the same concentration for the HMW adiponectin standard, and the absorbance of 1.0 at 280 nm was about 1.0 mg/ml. From these data, the absorbance at 280 nm was chosen as the concentration of the HMW adiponectin standard. When human serum is separated by gel chromatography on a HiLoad 16/60 Superdex 200 prep-grade column, adiponectin is eluted as three peaks corresponding to apparent molecular masses of 420, 240, and 180 kDa, which represent HMW and the hexamer and trimer. Although adiponectin mole