Adiponectin

Adiponectin is an abundant protein hormone which belongs to a family of so-called adipokines. Adiponectin is expressed mostly by adipocytes and is important regulator of lipid and glucose metabolism. It is established that adiponectin is a insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties (1). Potential diagnostic usage of adiponectin was a subject of increasing interest in recent years. It was shown that decreased serum adiponectin concentration indicates insulin resistance and type 2 diabetes (2). Besides, hypoadiponectinemia was shown to be associated with coronary artery disease (3). Several authors point out that high level of circulating adiponectin reduce risk of coronary heart disease among type 2 diabetes patients (4) and is associated with reduced risk of myocardial infarction in apparently healthy men (5). So, there is growing interest among medical professionals to use adiponectin for insulin resistance diagnosis and predicting of cardiovascular complications in subjects with type 2 diabetes. Human adiponectin consists of 244 amino acid residues and has distinct domain structure: it contains both collagen-like and globular C1q-like domains. Collagenlike parts of three adiponectin molecules can interact forming triple coiled coil structure much alike to that in collagen (6). C1q-like domains form a head of adiponectin globula (Fig.1) and share a great degree of structural similarity to complement component C1q. Several oligomeric forms of native adiponectin circulating in the blood are described in literature: trimers (low-molecular weight form, LMW), hexamers (medium molecular weight form, MMW) and higher order multimers (high molecular weight form, HMW). Three monomers of adiponectin form a trimer. Trimers linked by disulfide bond form a hexamer. The exact structure of the HMW form of adiponectin is not yet known. Most likely several combined hexamers and/or trimers constitute high-molecular weight form of adiponectin. It is generally believed that disulfide bonds as well as some bonds with participation of modified amino acid residues in collagen domain of adiponectin, take part in holding subunits of HMW form of adiponectin together (Fig. 1). It is also believed that those oligomeric forms exist in the bloodstream as separate moieties and do not convert into each other. (7) It has been shown recently, that adiponectin oligomers are capable of binding Ca2+ ions which are thought to participate in maintenance of conformational stability of adiponectin (10). Concentration of total adiponectin in the blood is about 3-30 g/ml, whereas concentration of the closest structural homolog of adiponectin, C1q, is about 80-200 g/ml. It is therefore of utmost importance that antiadiponectin antibodies would have no cross-reactivity with human C1q. (8) Some authors describe significant gender differences in adiponectin level in healthy adults and these differences are believed to contribute to discrepancies in adiponectin concentration reported by various authors. It was shown, that biologic activity of adiponectin is mediated by high-molecular weight form and, not surprisingly, it has been suggested recently that concentration of HMW form of adiponectin or ratio HMW/total adiponectin (sum of three types of oligomers) in serum correlates stronger than total adiponectin with insulin resistance and other measures of type 2 diabetes (9). HyTest offers new generation of anti-human adiponectin monoclonal antibodies suitable both for research purposes (Western blotting, direct ELISA) and for the development of adiponectin-specific sandwich immunoassays.

Figure 1. Schematic representation of adiponectin oligomeric forms.

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trimer (LMW form of adiponectin)

hexamer (MMW form of adiponectin)

HMW form of adiponectin

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Anti-human adiponectin monoclonal antibodies

Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application: Mice Balb/c Sp2/0 Human adiponectin Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Adiponectin sandwich immunoassay, adiponectin immunodetection in Western blotting (Adn20, Adn23, Adn63, Adn214, Adn222 and Adn243) All antibodies were tested in direct ELISA for crossreaction with C1q, which is the most abundant adiponectin homolog in blood. None of selected MAbs showed any crossreaction with human C1q.

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized either with human recombinant adiponectin or with native human adiponectin.

Applications
1. Adiponectin sandwich immunoassay
All MAbs were tested in two-site combinations as capture or detection antibodies in sandwich ELISA with native adiponectin. Seven two-site combinations were selected for the development of sandwich immunoassays on the basis of sensitivity and specificity to different oligomeric forms of adiponectin: Adn20 Adn36 Adn94 Adn279 Adn214 Adn222 Adn305 Adn23 Adn27 Adn63 Adn94 Adn27 Adn94 Adn279
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Representative curve demonstrating detection of purified native adiponectin by assay Adn279-Adn94, is shown on Fig. 2

Figure 2: Calibration curve for sandwich adiponectin immunoassay. MAb Adn279 was used as a coating (1 g/well), MAb Adn94 was labeled with stable Eu3+ chelate and was used as a detection (0.2 g/well) antibody. Native adiponectin purified from human plasma was used as a calibrator.

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All assays were tested with serial dilutions of normal human serum to evaluate interaction of MAbs with native adiponectin in complex environment. All assays demonstrated steady decrease of signal correlating with degree of serum dilution. The representative titration curve for assay Adn94-Adn63 (capture antibody-detection antibody, respectively) is shown on Fig. 3.
Figure 3: Normal human serum titration curve in sandwich immunofluorescent assay. Adn94 MAb was used as a coating antibody (1 g/well), MAb Adn63 was used as a detection antibody (0.2 g/well). Normal human serum, serially diluted with phosphate-buffered saline (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20) was used as an antigen.

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Assays Adn36-Adn27 and Adn20-Adn23 react differently with adiponectin in serum and citrate plasma (Fig. 4). Other MAbs two-site combinations (Adn94Adn63, Adn279-Adn94, Adn214-Adn27, Adn222Adn94, Adn305-Adn279) react with antigen in serum and plasma identically.
Figure 4: Normal human serum or citrate plasma titration curves for MAb assay Adn36-Adn27. Normal human pooled serum or citrate plasma, serially diluted with phosphate-buffered saline (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20) was used as an antigen

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Recognition of adiponectin by assays Adn20-Adn23 and Adn36-Adn27 in serum is Ca2+-sensitive (Fig. 5). Chelating of Ca2+ ions by EGTA leads to the rearrangements in adiponectin structure and to changes in the interaction of one of the antibodies with the antigen. Other assays do not demonstrate Ca2+-dependence in the antigen recognition and react identically with adiponectin in serum or citrate plasma.
Figure 5: Serum titration curve for the assay Adn20-Adn23. Pooled normal human serum was serially diluted with phosphatebuffered saline with EGTA or w/o EGTA (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10 mM EGTA).

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2. Assays detecting total, HMW or LMW forms of human adiponectin

To establish an oligomer specificity of HyTest assays, serum proteins were separated according to their molecular masses by means of size-exclusion chromatography and immunoreactivity in fractions was measured. Assay Adn20-Adn23 detects two oligomeric forms of adiponectin: mostly HMW and to a lesser extent, MMW form (Fig. 6A). Assay Adn94Adn63 recognizes all three Adn oligomeric forms total adiponectin (Fig. 6B).and assay Adn214-Adn27 reacts primarily with LMW form of adiponectin (Fig. 6C).

Figure 6: Immunoreactivity in protein fractions after size-exclusion chromatography, measured by assay Adn20-Adn23 (A) and by assays Adn94-Adn63 (B), Adn214-Adn27 (C) in sandwich ELISA. 1 ml of normal human serum was applied onto column. Positions of oligomeric forms of adiponectin and molecular weight markers are depicted on the picture. Black line is optical density detected at 280 nm

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3. Western blotting
All MAbs were tested on their ability to recognize adiponectin in Western blotting. Only six out of tested antibodies, MAbs Adn20, Adn23, Adn63, Adn214, Adn222, and Adn243 reacted with adiponectin transferred onto nitrocellulose membrane after SDSPAGE in reducing conditions (Fig. 7).
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Adn dimer

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Adn monomer

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Figure 7: Immunodetection of native adiponectin with antiAdn MAbs in Western blotting after SDS-electrophoresis in reducing conditions. 40 ng of native purified adiponectin was loaded onto each track, nitrocellulose membrane was stained with 5 g/ml of various anti-adiponectin MAbs in phosphate-buffered saline, containing 5% of dry milk and 0.1% Tween-20. Lane 1 Adn20 Lane 2 Adn23 Lane 3 Adn63 Lane 4 Adn214 Lane 5 Adn222 Lane 6 Adn243 Molecular weight markers are marked by arrows.

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4. Native purified adiponectin antigen

Native adiponectin purified from normal human plasma is the best calibrator for immunoassays. Native adiponectin was isolated from normal human plasma using a combination of chromatographic methods. Its purity is about 92% as calculated by densitometry of protein bands stained with Coomassie Brilliant Blue R-250 after SDS-electrophoresis in reduced conditions (Fig. 9). Native purified adiponectin fully recovers its immunoreactivity after lyophilization and reconstitution by addition of deionized water (Fig. 10).
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Figure 10: Lyophilization does not affect immunological activity of native purified adiponectin measured by assay Adn94-Adn63.
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Purified native adiponectin contains all three oligomeric forms of Adn (Fig.11) and therefore can serve as a calibrator for all types of Adn assays: total Adn, HMW- or LMW-specific.

Figure 9: SDS-electrophoresis in reducing conditions and Western blotting of native purified adiponectin from human plasma. Lane 1 2 g of purified adiponectin loaded onto track, stained with Coomassie Brilliant Blue R-250 Lane 2 200 ng of purified adiponectin loaded onto track, stained with Adn23 MAb in Western blotting.

Figure 11: Native purified adiponectin contains all oligomeric forms. 3g of adiponectin was applied onto gel-filtration column and immunoreactivity in fractions was measured with HyTest assay Adn94-Adn63. Molecular weight markers are depicted by arrows, black curve represents optical density measured at 280 nm.

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Ordering information: Product

Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin Anti-Human adiponectin

Cat.#
2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6 2AN6

MAb
Adn20 Adn23 Adn27 Adn36 Adn63 Adn94 Adn97 Adn130 Adn214 Adn222 Adn243 Adn279 Adn305

Subclass
IgG2a IgG2a IgG2a IgG2a IgG1 IgG1 IgG1 IgG2a IgG1 IgG1 IgG2a IgG1 IgG1

Application
WB WB EIA EIA EIA, WB EIA EIA EIA EIA, WB EIA, WB EIA, WB EIA EIA

Ordering information: Product

Adiponectin, human, native

Cat.#
8AN7

Purity
>95%

Source
Pooled human plasma

References:
1. 2. 3. 4. 5. Wang, Y., et al., Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerizationdependent manner. J.B.C., 2005, 280, 18, 18341-18347. Ryo, M., et al., Adiponectin as a biomarker of the metabolic syndrome. Circ. J., 2004, 68, 975-981. Kumada, M., et al., Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler. Thromb. Vasc. Biol., 2003, 23, 85-89. Schulze, M., et al., Adiponectin and future coronary heart disease events among men with type 2 diabetes. Diabetes, 2005, 54, 534-539. Pischon, T., et. al., Plasma adiponectin levels and risk of myocardial infarction in men. JAMA, 2004, 291, 14, 1730-1737. 6. Pajvani, U., et al., Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. J.B.C., 2003, 278, 11, 9073-9085. 7. Wang, y., et al., Hydroxylation and Glycosylation of the Four Conserved Lysine Residues in the Collagenous Domain of Adiponectin. J.B.C., 2002, 277, 22, 19521-19529. 8. Wouters, D, Evaluation of classical complement pathway activation in rheumatoid arthritis. Arhtritis & Rheumatism, 2006, 54, 1143-1150. 9. Lara-Castro et al., Adiponectin multimeric complexes and the metabolic syndrome trait cluster. Diabetes, 2006, 55, 249-259. 10. Schraw T, Wang ZV, Halberg N, Hawkins M, Scherer PE. Plasma adiponectin complexes have distinct biochemical characteristics. Endocrinology, 2008, 149(5), 2270-82.

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August 2009

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