# US 7,608,405 B2 · Method of selectively assaying adiponectin multimers

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- Number: US 7,608,405 B2
- Document ID: US7608405B2
- Status: Granted
- Kind: B2
- Application number: 10/575,931
- Filed: 2004-10-15
- Published: 2007-02-22
- Granted: 2009-10-27
- Earliest claimed: 2003-10-15
- Assignees: Daiichi Pure Chemicals Co., Ltd.; Toudai TLO, Ltd.
- Inventors: Hiroyuki Ebinuma; Hirokazu Yago; Yuka Akimoto; Osamu Miyazaki; Takashi Kadowaki; Toshimasa Yamauchi; Kazuo Hara
- Web page: https://patentgrep.com/patent/US7608405B2

## CPC

- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- G01N 2800/042: Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- G01N 2800/32: Cardiovascular disorders
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods for selectively assaying a target adiponectin multimer in a biological sample. Such methods accurately evaluate the relationship between a disease and adiponectin through selective assay of adiponectin multimers and provide information that cannot be obtained through measurement of the total amount of adiponectin alone. A method for selectively assaying of a target adiponectin multimer in a biological sample comprising distinguishing target adiponectin multimer from the other adiponectin multimers by using a protease and/or an antibody.

## Claims

### Claim 1

A method for selectively quantifying a target adiponectin multimer in a biological sample, comprising:
(a) selectively digesting at least one type of adiponectin multimer other than the target multimer with a protease, and
(b) immunologically quantifying the amount of the target adiponectin multimer.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the target adiponectin is HMW-Ad (high molecular weight adiponectin) and wherein the method comprises:
(a) digesting ULMW-Ad (ultra low molecular weight adiponectin), LMW-Ad (low molecular weight adiponectin) and MMW-Ad (middle molecular weight adiponectin) multimers in a sample suspected of containing HMW-Ad with at least one protease; and
(b) immunologically quantifying the HMW-Ad.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the at least one protease digests ULMW-Ad, LMW-Ad and MMW-Ad to a level undetectable by PAGE (2 to 15%) separation, but does not digest HMW-Ad to a level undetectable by PAGE (polyacrylamide gel electrophoresis) (2 to 15%) separation when added to a buffer solution containing the adiponectin multimers and incubated for at least 60 mins at 37° C.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the at least one protease digests ULMW-Ad, LMW-Ad and MMW-Ad to a level undetectable by PAGE (2 to 15%) separation, but does not digest HMW-Ad to a level undetectable by PAGE (2 to 15%) separation, when the at least one protease is added to a 50 mM phosphate buffer solution having a pH of 8.0 containing adiponectin multimers, followed by incubation for 60 minutes at 37° C.

### Claim 5 (depends on 2)

The method according to claim 2, wherein the protease is derived from a microorganism.

### Claim 6 (depends on 2)

The method according to claim 2, wherein the protease is selected from proteinase K, protease P “Amano,” protease N “Amano” and Umamizyme.

### Claim 7 (depends on 2)

The method according to claim 2, wherein the at least one protease includes proteinase K.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the digesting step is performed at 4° C. to 60° C.

### Claim 9 (depends on 2)

The method according to claim 2, wherein the digesting step is performed from 5 minutes to 24 hours.

### Claim 10 (depends on 2)

The method according to claim 2, wherein HMW-Ad is immunologically quantified by sandwich ELISA (enzyme linked immunosorbent assay).

### Claim 11 (depends on 10)

The method according to claim 10, wherein the sandwich ELISA comprises:
(i) pretreating a sample with protease which is capable of digesting ULMW-Ad, LMW-Ad and MMW-Ad to a level undetectable by PAGE (2 to 15%) separation, but not capable of digesting HMW-Ad to a level undetectable by PAGE (2 to 15%) separation;
(ii) adding the pretreated sample to anti-Ad monoclonal antibody-coated plate and incubating at room temperature;
(iii) adding a biotin labeled antibody solution and incubating at room temperature;
(iv) adding a HRP labeled streptoavidin solution and incubating at room temperature;
(v) adding a substrate solution and incubating at room temperature;
(vi) adding a stop solution; and
(vii) measuring absorbance.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the biological sample is obtained from a human.

### Claim 13 (depends on 2)

The method of claim 2, wherein said biological sample is obtained from a subject suspected of having a disease or pathological condition, and wherein said method further comprises:
evaluating a disease or pathological condition based on the amount of HMW-Ad in the sample.

### Claim 14 (depends on 13)

The method as described in claim 13, wherein the evaluation is performed on the basis of change in the amount of HMW-Ad.

### Claim 15 (depends on 13)

The method as described in claim 13, wherein the evaluation is performed through calculating the ratio of HMW-Ad to the total amount of adiponectin.

### Claim 16 (depends on 13)

The method as described in claim 13, wherein the evaluation is performed through correlation of an index with the amount of HMW-Ad or with the ratio of HMW-Ad to the total amount of adiponectin.

### Claim 17 (depends on 13)

The method as described in claim 13, wherein the disease or the pathological condition is type-II diabetes, arteriosclerotic disease, renal disease, hepatic disease, obesity, or metabolic syndrome.

### Claim 18 (depends on 13)

The method as described in claim 13, for evaluating onset, diagnosis, development, prognosis, or therapeutic effect of type-II diabetes, arteriosclerotic disease, renal disease, hepatic disease, obesity, or metabolic syndrome.

### Claim 19 (depends on 1)

The method of claim 1, wherein said target adiponectin multimer is middle molecular weight adiponectin (MMW-Ad), said method comprising:
(a) contacting a biological sample with a protease that digests ULMW-Ad and LMW-Ad,
(b) quantifying the total amount of adiponectin remaining in the sample treated in step (a),
(c) quantifying the amount of HMW-Ad in the biological sample, and
(d) subtracting the amount of HMW-Ad detected in (c) from the total amount of adiponectin quantified in (b),
wherein the amount of MMW-Ad corresponds to the amount quantified in (b) less the amount of HMW-Ad quantified in (c).

### Claim 20 (depends on 19)

The method of claim 19, wherein said protease is a protease derived from a microorganism.

### Claim 21 (depends on 19)

The method of claim 19, wherein said protease is a protease selected from the group consisting of protease S “Amano”, protease A “Amano”, sumizyme FP, sumizyme LP50D, and protease V8.

### Claim 22 (depends on 19)

The method of claim 19, wherein said protease is contacted with the biological sample at a concentration ranging from 0.01 to 100 U/mL or 0.01 to 100 mg/mL.

### Claim 23 (depends on 1)

The method of claim 1, wherein said target adiponectin multimer is low molecular weight adiponectin (LMW-Ad) and said method comprises:
contacting a biological sample with an anti-LMW-Ad antibody, and
determining the presence of LMW-Ad in said sample.

### Claim 24 (depends on 23)

The method of claim 23, wherein the anti-LMW-Ad antibody forms a complex with LMW-Ad, and the amount of the complex is used to determine the quantity of LMW-Ad in said biological sample.

### Claim 25 (depends on 23)

The method of claim 23, wherein said anti-LMW-adiponectin antibody is a monoclonal antibody.

### Claim 26 (depends on 23)

The method of claim 23, wherein said anti-LMW-adiponectin antibody is a polyclonal antibody.

### Claim 27 (depends on 23)

The method of claim 23 which is selected from the group consisting of an ELISA, CLEIA, RIA and LTIA.

### Claim 28 (depends on 1)

The method of claim 1, wherein said target adiponectin multimer is ultra low molecular weight adiponectin (ULMW-Ad) and said method comprises:
(a) quantifying the total amount of adiponectin in a biological sample,
(b) quantifying the amount of LMW-Ad, MMW-Ad and HMW-Ad in said sample, and
(c) subtracting the amount of LMW-Ad, MMW-Ad and HMW-Ad in said sample from the total amount of adiponectin in said sample,
wherein the amount of ULMW-Ad corresponds to the amount quantified in (a) less the amount quantified in (b).

### Claim 29 (depends on 1)

The method of claim 1, wherein said target adiponectin multimer is HMW-Ad (high molecular weight adiponectin) and said method comprises:
(a) digesting the adiponectin multimers other than HMW-Ad that are present in a sample suspected of containing HMW-Ad with at least one protease,
(b) immunologically quantifying the amount of HMW-Ad.