Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA

Madhur K. Sinha, Traci Songer, Qiang Xiao, John H. Sloan, Jin Wang, Shaoquen Ji, William E. Alborn, Randy A. Davis, Michael M. Swarbrick, Kimber L. Stanhope, Bruce M. Wolfe, Peter J. Havel, Todd Schraw, Robert J. Konrad, Philipp E. Scherer, Jehangir S. Mistry

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Abstract

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 μg/L and a detection limit of 0.8 μg/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. Conclusion: 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.

Original languageEnglish (US)
Pages (from-to)2144-2151
Number of pages8
JournalClinical Chemistry
Volume53
Issue number12
DOIs
StatePublished - Dec 2007

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Adiponectin
Molecular Weight
Enzyme-Linked Immunosorbent Assay
Molecular weight
Endopeptidase K
Surgery
Body Mass Index
Western Blotting
Gastric Bypass
Liquid chromatography
Immunoblotting
Liquid Chromatography
Calibration
Dilution
Insulin Resistance
Limit of Detection
Weight Loss
Digestion
Assays
Protein Isoforms

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Sinha, M. K., Songer, T., Xiao, Q., Sloan, J. H., Wang, J., Ji, S., ... Mistry, J. S. (2007). Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA. Clinical Chemistry, 53(12), 2144-2151. https://doi.org/10.1373/clinchem.2007.090670

Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA. / Sinha, Madhur K.; Songer, Traci; Xiao, Qiang; Sloan, John H.; Wang, Jin; Ji, Shaoquen; Alborn, William E.; Davis, Randy A.; Swarbrick, Michael M.; Stanhope, Kimber L.; Wolfe, Bruce M.; Havel, Peter J.; Schraw, Todd; Konrad, Robert J.; Scherer, Philipp E.; Mistry, Jehangir S.

In: Clinical Chemistry, Vol. 53, No. 12, 12.2007, p. 2144-2151.

Research output: Contribution to journalArticle

Sinha, MK, Songer, T, Xiao, Q, Sloan, JH, Wang, J, Ji, S, Alborn, WE, Davis, RA, Swarbrick, MM, Stanhope, KL, Wolfe, BM, Havel, PJ, Schraw, T, Konrad, RJ, Scherer, PE & Mistry, JS 2007, 'Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA', Clinical Chemistry, vol. 53, no. 12, pp. 2144-2151. https://doi.org/10.1373/clinchem.2007.090670
Sinha, Madhur K. ; Songer, Traci ; Xiao, Qiang ; Sloan, John H. ; Wang, Jin ; Ji, Shaoquen ; Alborn, William E. ; Davis, Randy A. ; Swarbrick, Michael M. ; Stanhope, Kimber L. ; Wolfe, Bruce M. ; Havel, Peter J. ; Schraw, Todd ; Konrad, Robert J. ; Scherer, Philipp E. ; Mistry, Jehangir S. / Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA. In: Clinical Chemistry. 2007 ; Vol. 53, No. 12. pp. 2144-2151.
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T1 - Analytical validation and biological evaluation of a high-molecular-weight adiponectin ELISA

AU - Sinha, Madhur K.

AU - Songer, Traci

AU - Xiao, Qiang

AU - Sloan, John H.

AU - Wang, Jin

AU - Ji, Shaoquen

AU - Alborn, William E.

AU - Davis, Randy A.

AU - Swarbrick, Michael M.

AU - Stanhope, Kimber L.

AU - Wolfe, Bruce M.

AU - Havel, Peter J.

AU - Schraw, Todd

AU - Konrad, Robert J.

AU - Scherer, Philipp E.

AU - Mistry, Jehangir S.

PY - 2007/12

Y1 - 2007/12

N2 - 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 μg/L and a detection limit of 0.8 μg/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. Conclusion: 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.

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