Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

Abstract

Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10−19 moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy

“…Serum adiponectin is believed to enhance insulin sensitivity, and individuals with obesity, type 2 DM, and other metabolic disorders often have low serum adiponectin levels 9 10. In contrast to the serum levels, our preliminary results of the measurement of urinary adiponectin with an ultrasensitive ELISA that we developed suggest that the urinary adiponectin levels are higher in patients with DM than in normal (non-DM) subjects 11. We thus hypothesized that urinary adiponectin levels tend to increase with an increase in the risk of diabetic nephropathy.…”

“…The sensitivity of commercially available ELISA kits,16 17 however, is insufficient for the measurement of the low level of urinary adiponectin in normal subjects,20 and radioimmunoassay15 and more complicated ELISAs19 are not practical for application in most clinics and hospitals. Using our ultrasensitive ELISA, however, which only requires an ordinary microplate reader and no special apparatus, we could sufficiently detect the low urinary adiponectin levels in normal subjects 11. More recently, some research groups have also noticed the relationship between adiponectin levels and diabetic nephropathy 21.…”

“…An ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling was originally developed by Watabe and Ito 11 23–26. Shortly, a sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA.…”

“…The measurement methods for adiponectin were slightly modified from the previous studies 11. The antigen used was a recombinant human adiponectin derived from a human adiponectin oligomers/Acrp30 quantitative ELISA kit (Cat No DHWAD0; R&D Systems, Minneapolis, MN, USA).…”

Immunochromatographic Detection of MPB64 Secreted from Active BCG by Heating: Toward Same-Day Diagnosis of Tuberculosis

“…The present method detected approximately 10 4 CFU/ml, whereas the conventional culture method detected approximately <10 3 CFU/ml [11]. Combining the present method for eliciting MPB64 secretion by heating with a new ultrasensitive ELISA coupled with thionicotinamide–adenine dinucleotide (thio-NAD) cycling [16,17] might enable TB diagnosis as a same-day test with a higher LOD. This assay will be further developed in our future work.…”