Analytical validation and biological evaluation of a high molecular-weight adiponectin ELISA.
BACKGROUND
Of the 3 circulating multimeric forms of adiponectin, the high-molecular-weight (HMW) form, as measured by size-exclusion and/or immunoblotting techniques, is a better index of insulin sensitivity for monitoring health and disease than is total adiponectin. We aimed to develop a simple ELISA to measure HMW adiponectin.


METHODS
We pretreated serum or plasma samples with digestion solution containing proteinase K (Millipore, ESDS). HMW (Millipore, EZHMWA-64K) and total adiponectin (Millipore, EZHADP-61K) concentrations were measured in treated and untreated samples, respectively, from 108 individuals and from 20 morbidly obese patients before and at 1, 3, 6, and 12 months after gastric-bypass surgery.


RESULTS
The ELISA has a dynamic range of 3-200 microg/L and a detection limit of 0.8 microg/L. Intraassay and interassay CVs were <4% and <10%, respectively. Sample-dilution curves paralleled the calibration curves. Fast protein liquid chromatography profiles of the proteinase K-treated samples revealed predominantly HMW adiponectin. Values for HMW adiponectin produced with this method are comparable with those obtained with Western blot analysis (y = 0.77x - 0.15; r = 0.96; n = 56). Body mass index (BMI)- and sex-related changes were more pronounced for HMW adiponectin and percentage of HMW adiponectin than for total adiponectin. HMW and total adiponectin increased after bypass surgery, but changes in HMW adiponectin were more pronounced and preceded changes in total adiponectin.


CONCLUSIONS
This simple, rapid ELISA for HMW adiponectin recognizes the HMW isoform, produces results closely correlated with those obtained with Western blotting, and appears to better distinguish BMI-, sex-, and weight loss-associated differences than assays for total adiponectin.
Sinha
Madhur K
MK
Millipore Bioscience Division, Millipore Corporation, St. Charles, MO 63304, USA. Madhur_Sinha@Millipore.com
Songer
Traci
T
Xiao
Qiang
Q
Sloan
John H
JH
Wang
Jin
J
Ji
Shaoquen
S
Alborn
William E
WE
Davis
Randy A
RA
Swarbrick
Michael M
MM
Stanhope
Kimber L
KL
Wolfe
Bruce M
BM
Havel
Peter J
PJ
Schraw
Todd
T
Konrad
Robert J
RJ
Scherer
Philipp E
PE
Mistry
Jehangir S
JS
D052242:Adiponectin / Q000097:blood / Q000737:chemistry; D001835:Body Weight; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015390:Gastric Bypass; D006801:Humans; D008297:Male; D008970:Molecular Weight; D009767:Obesity, Morbid / Q000378:metabolism / Q000601:surgery; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry; D012189:Retrospective Studies; D012737:Sex Factors
C499918:ADIPOQ protein, human; D052242:Adiponectin; D020033:Protein Isoforms