A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin.
Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).
Nakano
Yasuko
Y
Department of Medicinal Information, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Tokyo 142-8555, Japan. yanakano@pharm.showa-u.ac.jp
Tajima
Sachiko
S
Yoshimi
Ai
A
Akiyama
Haruyo
H
Tsushima
Motoo
M
Tanioka
Toshihiro
T
Negoro
Takaharu
T
Tomita
Motowo
M
Tobe
Takashi
T
D052242:Adiponectin / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000276:immunology; D000293:Adolescent; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D015394:Molecular Structure; D008970:Molecular Weight; D046912:Multiprotein Complexes; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
C499918:ADIPOQ protein, human; D052242:Adiponectin; D000911:Antibodies, Monoclonal; D046912:Multiprotein Complexes; D011994:Recombinant Proteins