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1. INTRODUCTION Adiponectin is a protein comprised of 244 amino acids and is one of the adipocytokines secreted by the adipocyte. Adiponectin has been reported to have several physiological actions, such as protective activities against atherosclerosis1), improved insulin resistance2), and prevention of hepatic fibrosis3). It has been reported that adiponectin is known to have a multimeric structure; it has trimer, hexamer, and HMW forms in the blood4-6). An ELISA system for the selective measurement of adiponectin multimers by using protease has recently been developed7). This system has adopted a simple pretreatment method; multimeric adiponectin in the blood is converted mainly to a dimer with the addition of SDS-containing acid buffer to the serum, without boiling specimens. This kit can also correctly measure the total adiponectin concentration in human blood by adopting the same pretreatment method.

2. ASSAY PRINCIPLES The principle of the assay is shown in Figure 1. This kit operates based on the underlying principle of a sandwich enzyme-linked immunosorbent assay (ELISA) that uses two ki nds of a nti-human adiponectin monoclonal antibodies (MoAbs). The specimens are pretreated as outlined below. Multimers of adiponectin are classified into 4 fractions in this kit. 1) Total adiponectin fraction: “Total-Ad” 2) High-molecular adiponectin fraction (equivalent of dodecamer-octadecamer): “HMW-Ad” 3) Middle-molecular adiponectin fraction (equivalent of hexamer): “MMW-Ad” 4) Low-molecular adiponectin fraction (equivalent of trimer including albumin-binding adiponectin): “LMW-Ad”

Pre-treatment of Specimens Specimens are treated with SDS-containing acid buffer to convert multimeric adiponectin to mostly a dimer form. Test wells are coated with anti-human adiponectin MoAb. The standard solution and pretreated specimen are captured by the antibody in the first incubation. After the first incubation and washing to remove all of the bound material, biotin-labeled MoAb is added. After the second incubation and subsequent washes, HRP–labeled streptavidin is added. After the third incubation and subsequent washes, substrate solution is added. Next, stopreagent is added. The intensity of the color that develops is read by a microplate reader. The absorbance isproportional to the concentration of adiponectin in the sample. In this kit, normal human serum pretreated withsample pre-treatment buffer is used for the Calibrator. The adiponectin concentration of the Calibrator iscalculated by selectively purifying HMW-Ad protein from human plasma.

Ｙ

Pretreated sample for total - Ad

SH+

Human Adiponectin multimers

S-SS-S

S-SS-S

Albumin

S-S

HMW - Ad MMW - Ad

LMW - Ad

Total - Ad

S-S

Dimeric form

Sample pre-treatment buffer

(non-heating)

Anti-human Adiponectin MoAb

coated wells

Ｙ

－ BiotinＡvidin-HRP

Substrate

← Stop solution

Abs 492nm1st reaction

Pretreated sample Biotin labeled

Anti-human Adiponectin

MoAb

2nd reaction 3rd reaction

HRP labeled Avidin Enzyme

reaction

Ｙ Ｙ

Ｙ

－ Biotin

S-S

S-S

S-S

S-S

S-S

ＹS-S

S-S

Figure 1. Assay Principle

Ｙ

Pretreated sample for total - Ad

SH+

Human Adiponectin multimers

S-SS-S

S-SS-S

Albumin

S-S

HMW - Ad MMW - Ad

LMW - Ad

Total - Ad

S-S

Dimeric formS-S

Dimeric form

Sample pre-treatment buffer

(non-heating)

Anti-human Adiponectin MoAb

coated wells

Ｙ

－ＢＡvidin-HRP

Substrates

← Stop solution

Abs492nm1st reaction

Pretreated sample Biotin labeled

Anti-human Adiponectin

MoAb

2nd reaction 3rd reaction

HRP labeled Avidin Enzyme

reaction

Ｙ Ｙ

Ｙ

－Ｂ

S-S

S-S

S-S

S-S

S-S

ＹS-S

S-S

Figure 1. Assay Principle

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3. KIT COMPONENTS

Reagent Composition Quantity Preparation

Wash Buffer Concentrate Phosphate buffer (pH 7.2) 100 ml x 1 vial 10X

Sample Pretreatment Buffer Citrate buffer (pH 3.0) containing SDS 50 ml x 1 vial Ready to use

Dilution Buffer Phosphate buffer (pH 7.2) containing BSA 100 ml x 1 vial Ready to use

Monoclonal Ab Coated Plate

Anti-human adiponectin mouse monoclonal Ab coated plate 1 plate (96 wells) Ready to use

Calibrator (stock solution) Human serum stabilized in SamplePretreatment Buffer 0.25 ml x 1 vial Concentration

indicated on vial

Biotin Labeled Monoclonal Ab Biotin-conjugated anti-human adiponectin monoclonal antibody 6 ml x 1 vial Ready to use

Enzyme Labeled Streptavidin Horseradish peroxidase (HRP) labeled streptavidin 6 ml x 1 vial Ready to use

Substrate O-phenylenediamine (OPD) 2 vials Lyophilized

Substrate Buffer Citrate buffer (pH 5.0) containing H2O2 15 ml x 1 vial Ready to use

Stop Reagent 7.7% H2SO4 15 ml x 1 vial Ready to use

Buffer Solution Tris buffer (pH 8.0) 50 ml x 1 vial Ready to use

High and Low Controls Phosphate buffer containing processed human serum 2 vials Lyophilized

Serum Control Processed human serum 1 vial Lyophilized

Plate Sealers n/a 3 each Ready to use

4. MATERIALS REQUIRED BUT NOT INCLUDED • Microfuge tubes (1 ml) • Precision pipettes with disposable tips capable of dispensing 10 µl, 100 µl, 500 µl • Repeating or multi-channel pipette • Volumetric container • Volumetric pipettes • Distilled (deionized) water • Incubator or water bath (37°C) • Microplate washer or wash bottle • Microplate reader with 492 and optional 600-700 nm filter

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5. REAGENT PREPARATION AND STORAGEUse reagents after they reach room temperature.

a) Wash bufferDilute Wash buffer concentrate with 900 mL deionized water. Working Wash buffer is stored at 2-10℃. b) Sample pre-treatment bufferWhite precipitation (SDS) is completely dissolved by warming the buffer and thoroughly stirring before use. c) Dilution bufferDilution buffer is ready to use. d) MoAb coated wellsMoAb coated wells are ready to use. The unused strips should be returned to the laminate bag and stored at 2-10℃.e) Working Calibrator Note: There may be a precipitate in the tube. Allow the solution to stand at room temperature and stir thoroughly before using.Just prior to use, dilute the Calibrator 1: 101 with Dilution buffer. The serial dilution series should be preparedas described below to construct a calibration curve.The remaining Calibrator should be stored at 2-10℃, and the same procedure should be repeated when it is to be used again.* Since precipitation is temporarily caused when the Calibrator is added to the Dilution buffer, stir thoroughly.* The working calibrator should be prepared at the same time as the dilution of the samples (after specimenpre-treatment). The diluted pre-treated specimen and the working calibrator are added to the MoAb coated wells in succession.

≪Example of serial dilution≫

* The concentration of a 1:101 dilution is indicated on the label of the calibrator.

f) Biotin labeled-MoAbBiotin labeled-MoAb is ready to use. g) Enzyme labeled streptavidinEnzyme labeled streptavidin solution is ready to use. h) Substrate solutionJust prior to use, reconstitute the Substrate (lyophilized) by adding 6 mL of Substrate buffer to the substrate vial. The Substrate solution should be used immediately after reconstitution and the remaining solution should be discarded.i) Stop reagent Stop reagent is ready to use.

4.8*ng/mL

2.4ng/mL

1.2ng/mL

0.6ng/mL

0.15ng/mL

0.3ng/mL

0.075ng/mL

0ng/mL

150μL

⑤Calibrator

1： 101 with Dilution buffer (for example, 10 µL Calibrator + 1000 μL Dilution buffer)

4.8*ng/mL

2.4ng/mL

1.2ng/mL

0.6ng/mL

0.15ng/mL

0.3ng/mL

0.075ng/mL

0ng/mL

4.8*ng/mL

2.4ng/mL

1.2ng/mL

0.6ng/mL

0.15ng/mL

0.3ng/mL

0.075ng/mL

0ng/mL

⑤Calibrator

150μL 150μL 150μL150μL150μL

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j) Low, High, and Serum Controls Reconstitute each Control with deionized water according to the Certificate of Analysis. Replace the rubber stopper and cap. Gently swirl the vial and let stand for 10 minutes prior to use. The contents of the vial should be in solution with no visible particulate matter. Store Controls in aliquots at -20°C. Avoid repeated freeze/thaw cycles. Reconstituted Low and High Controls are ready for use. Reconstituted Serum Control should be pretreated and diluted before use, in the same manner as the serum samples. Refer to the Certificate of Analysis for the lot specific reference ranges.

6 . A S S A Y P R O C E D U R E

1) Pre-treatment of specimens (human serum and plasma) To 10 µL of serum, plasma, or Serum Control add 100 µL of the Buffer solution and 400 µL of b) Sample pre-treatment buffer and stir thoroughly (1:51 dilution).

Pretreated samples are stable at 4 or 25°C for up to 2 days.

2) Dilution of pretreated specimens To 1.0 mL of the c) Dilution buffer add 10 µL of a pretreated specimen obtained in 1) above (1:101 dilution, final dilution: 1:5151).

Samples must be used within 2 hours of dilution (at room temperature). *Since precipitation is temporarily caused when a pretreated specimen is added to the Dilution buffer, stir thoroughly.

3) Assay method i. Take the necessary number of strips out of the laminate bag, add 50 µL each of the e) Working calibrators,

diluted samples, and controls to each test well, and incubate the covered plate for 60 min at 20-30°C.

ii. After thoroughly removing the solution from the wells, add 350-400 µL of a) Wash buffer to each well, and thoroughly remove the droplets. Repeat this cycle twice.

iii. Add 50 µL of the f) Biotin labeled-MoAb to each washed well and incubate the covered plate for 60 min at 20-30°C.

iv. Repeat step (ii). v. Add 50 µL of the g) Enzyme labeled streptavidin to each washed well and incubate the covered plate for 30

min at 20-30°C.

vi. Repeat step (ii). vii. Add 50 µL of the h) Substrate solution to each washed well, and incubate the covered plate for 10 min at

20-30°C. Then add 50 µL of the i) Stop reagent to each test well.

viii. Determine the absorbance of each well with a plate reader set at a wavelength of 492 nm within 30 min after the addition of the Stop reagent. (Set the sub-wavelength at 600-700 nm if necessary.)

4) Calculation of the concentration of adiponectin multimers Calculate the absorbance by subtracting the absorbance of the 0 ng/mL calibrator from those of the other calibrators and diluted samples. Plot the absorbance of the calibrators against the calibrator concentrations on log-log or semi-log graph paper. Draw a smooth curve through these points to construct the calibration curve. Read the concentrations of the diluted samples from the calibration curve. Calculate the concentration for the original, undiluted samples by multiplying by the dilution factor (1:5151).

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Figure 2 Summary of Assay procedure

Working Calibrator

and Diluted pre-treated sample 50 µL

MoAb coated plate

（Washing 3 times) 350 µL × 3

※ 1st reaction, 20 -30℃, 60 min

Biotin labeled MoAb solution 50 µL

（Washing 3 times) 350 µL× 3

※ 2nd reaction, 20 -30℃, 60 min

HRP labeled streptavidin solution 50 µL

（Washing 3 times) 350 µL × 3

※ 3rd reaction, 20 -30℃, 30 min

Substrate solution 50 µL

※ Enzyme reaction, 20 -30℃, 10 min

Stop reagent 50 µL

Absorbance measurement at 492 nm within 30

Human serum or plasma 10 µL

Pre – treatement for Total Adiponectin

Buffer solution100 µL

Sample pre – treatment buffer 400 μL

Calibrator and pre – treated samples were further diluted

101-fold with Dilution buffer.

Calibrator

7. PROCEDURAL NOTES1. The specimen used can be serum, EDTA-plasma, or heparinized plasma. Citrated plasma must not be used

because measurement results tend to be lower.

note - Refer to the typical data in “8. TYPICAL PERFORMANCE CHARACTERISTICS 5） Serum/Plasma

samples”.

2. A calibration curve must be run with each assay. Calibrators should be assayed in duplicate.

3. When the concentration of adiponectin in a specimen exceeds the calibration curve range, dilute

the pretreated specimen with c)Dilution buffer, and assay again.

4. Each kit can be divided and used twice. The remaining reagents should be stored as directed in the package

5. When the treatment of specimens is to be suspended for 2 hours or longer for reasons such as an overload,

preserve the specimens at room temperature after the step of the addition of the b) Sample pre-treatment

buffer, and add them to the MoAb coated wells promptly after dilution of the pretreated specimens.

note - Refer to the typical data in “8. TYPICAL PERFORMANCE CHARACTERISTICS 6） Sample stability”.

insert and used before the expiration date.

min after the addition of the Stop reagent

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8. TYPICAL PERFORMANCE CHARACTERISTICS

1) SensitivityThe absorbance is not less than 0.9 OD when the standard solution (4.8 ng/mL) is measured.

This test kit is effective in the range of 0.075 to 4.8 ng/mL when operated as directed under the ASSAY PROCEDURE.

The detection limit was 0.019 ng/mL (3SDblank).

0.01

0.1

1

10

0.01 0.1 1 10

Concentration of Adiponectin (ng/mL)

Abso

rban

ce a

t 492

nm

2) ReproducibilityThe coefficients of variation for observed values are not more than 10% when the total adiponectin levels in the same specimens are measured simultaneously 8 times.

Figure.3 Typical Standard Curve

・ Intra-Assay (Within an assay: n = 8 )

・ Inter-Assay (Between assays: n = 8 )

Humanserum 1

Humanserum 2

Mean (µg/ml) 3.98 9.22SD (µg/ml) 0.21 0.48CV (%) 5.4 5.3

Humanserum 3

Mean (µg/ml) 7.66 SD (µg/ml) 0.38 CV (%) 5.0

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0.0

0.5

1.0

1.5

2.0

2.5

3.0

11108/8/80 20 40 60 80 100 120

dilution factor

Αdi

pone

ctin

(ng/

mL)

Human Serum (1)Human Serum (2)Human Plasma (1)Human Plasma (2)

1/1 (= × 5151)1/21/4

4） Spiking RecoveryVarying amounts of the purified HMW form of human adiponectin were added to human serum samples and assayed.

Fig.4 Serial dilution of human serum or plasma.

3） Effects of DilutionPre-treated Human serum and plasma samples (5151 times diluted) were serially diluted further with Dilution buffer and assayed.

5） Serum/Plasma samplesEDTA, Heparin, and Citrate plasma were compared with serum samples obtained from 4 healthy volunteers at the same time.

HMW- AdiponectinAdded (μg/ml)

Observed(μg/ml)

Expected(μg/ml)

RecoveryO/ E (% )

0.00 7.33 7.31 1001.14 8.51 8.46 1012.29 9.88 9.60 1034.57 11.8 11.9 999.14 16.9 16.5 103

TotalAdiponectin

Sample (n=4) Mean Adiponectin(μg/ml)

MeanPlasma/Serum

Serum 5.84 100EDTA plasma 5.37 93Heparin plasma 5.37 94Citrate plasma 4.45 76

TotalAdiponectin

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6 ) Sample stability 1) Effects of Freeze/Thaw

4 human serum samples were frozen and thawed 3 times.

Recovery (%)

Sample (n = 4) Mean (μg/ml) F/T (x 1) F/T (x 2) F/T (x 3) Total Adiponectin 8.34 99 92 95

2) Stability of frozen sample Serum sample was stable for at least 1 year at -30◦C.

Recovery (%) Control Serum (μg/ml) 1.5M 4M 8M 13M

Total Adiponectin 7.75 105 102 96 95 3) Stability of pre-treated sample

Pre-treated samples (n = 8) were stable for at least 2 days at 4◦C or 25◦C. Note: There may be a precipitate in the tube when pre-treated samples are stored at 4◦C. Allow the solution to stand at room temperature and stir thoroughly before using.

Recovery (%) Sample (n = 8) Mean (μg/ml) 4◦C, 1 day 4◦C, 2 days 25◦C, 1 day 25◦C, 2 days

Total Adiponectin 7.19 95 93 98 100

4) Stability of pre-treated and diluted 1:101 sample Pre-treated samples diluted by 1:101 (n = 8) were stable for at least 2 hr at RT. Note: Detectable adiponectin levels in pre-treated samples diluted 1:101 tend to decrease with time. Therefore, these samples should be added to the plate as soon as possible after dilution.

Recovery (%) Sample (n = 8) Mean (μg/ml) 0.5 hr 1 hr 2 hr 4 hr

Total Adiponectin 7.82 97 95 96 92

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Commercial ELISA ① y = 0.663x - 0.894 (r = 0.992)

　　　　　〃　 　　　　 ② y = 0.547x + 0.412 (r = 0.991)

　　　　　〃　 　　　　 ③ y = 0.889x + 0.479 (r = 0.994)

　　　　　〃　 　　　　 ④ y = 1.031x + 0.688 (r = 0.980)

　　　　　〃　 　　　　 ⑤ y = 0.453x + 1.436 (r = 0.994)

　　　　　 　 Y X

Total Adiponectin ELISA

8) Correlation between this ELISA for total Human Adiponectin and other commercial ELISAsThis total human adiponectin ELISA was compared to other various commercial ELISAs; measurements were made on 36 (M/F = 18/18) serum samples obtained from healthy volunteers.

7) Cross reactvity• This assay recognizes the denatured dimeric or monomeric form of human Adiponectin.

• No cross-reactivity has been observed with the following other adipocytokines: human Resistin, Leptin, TNF-α, and IL-6 at 100 ng/ml.

• Among animal species, specific signal was observed with Cynomolgus monkey sera. The signal was equivalent to approx. 40 μg/ml of human adiponectin in the assay for total adiponectin.

• No signal has been obtained when the sera of the following species were measured in the assay: mouse, rat, rabbit, goat, sheep, porcine, horse, and bovine.

9. References

1) Okamoto Y, et al. Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2002;106: 2767-2770.

2) Yamauchi T, et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. 2001;7: 941-946.

3) Kamada Y, et al. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin.Gastroenterology. 2003;125:1796-1807.

4) Waki H, et al. Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J Biol Chem 2003; 278: 40352-40363.

5) Pajvani UB, et al. Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 2003; 278: 9073-9085.

6) Hada Y, et al. Selective purification and characterization of adiponectin multimer species from human plasma. Biochem Biophys Res Commun. 2007;356:487-493.

7) Ebinuma H, et al. A novel ELISA system for selective measurement of human adiponectinmultimers by using proteases. Clinica Chimica Acta 2006, 372: 47-53.

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