Method of selectively assaying adiponectin multimers
View Patent ↗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.
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.
2. 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.
3. 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.
4. 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.
5. The method according to claim 2 , wherein the protease is derived from a microorganism.
6. The method according to claim 2 , wherein the protease is selected from proteinase K, protease P “Amano,” protease N “Amano” and Umamizyme.
7. The method according to claim 2 , wherein the at least one protease includes proteinase K.
8. The method according to claim 2 , wherein the digesting step is performed at 4° C. to 60° C.
9. The method according to claim 2 , wherein the digesting step is performed from 5 minutes to 24 hours.
10. The method according to claim 2 , wherein HMW-Ad is immunologically quantified by sandwich ELISA (enzyme linked immunosorbent assay).
11. 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.
12. The method according to claim 1 , wherein the biological sample is obtained from a human.
13. 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.
14. The method as described in claim 13 , wherein the evaluation is performed on the basis of change in the amount of HMW-Ad.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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).
20. The method of claim 19 , wherein said protease is a protease derived from a microorganism.
21. 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.
22. 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.
23. 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.
24. 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.
25. The method of claim 23 , wherein said anti-LMW-adiponectin antibody is a monoclonal antibody.
26. The method of claim 23 , wherein said anti-LMW-adiponectin antibody is a polyclonal antibody.
27. The method of claim 23 which is selected from the group consisting of an ELISA, CLEIA, RIA and LTIA.
28. 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).
29. 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.