Assay Principle

Schematic of Assay Principle

In the Adiponectin (Multimeric) ELISA, both Total and HMW adiponectin can be measured independently on the same plate. Samples are pretreated with or without proteases(1), diluted and then assayed for the different multimers of adiponectin as described below.

  1. To measure Total Adiponectin: Samples are pretreated with Sample Pretreatment Buffer (Citrate buffer + SDS) which reduces multimeric adiponectin to dimers1. Subsequent measurement in the ELISA quantifies the amount of all multimers of adiponectin in the sample.
  2. To measure HMW Adiponectin: Samples are pretreated with Protease II which selectively digests LMW and MMW. The remaining HMW fraction is treated with Sample Pretreatment Buffer which reduces these multimers to dimers while also stopping the protease digestion. Subsequent measurement in the ELISA quantifies the amount of reduced HMW adiponectin but does not detect the digested LMW and MMW adiponectin.
  3. To measure combined MMW Adiponectin + HMW Adiponectin: Samples are pretreated with Protease I which selectively digests only LMW adiponectin. The remaining MMW and HMW are then digested to dimers using Sample Pretreatment Buffer which also stops the protease digestion. Subsequent measurement in the ELISA quantifies the amount of reduced MMW + HMW adiponectin but does not detect the digested LMW.
  4. To measure MMW Adiponectin: MMW (hexamer) concentration is calculated by subtracting the concentration of HMW from the combined concentration of MMW + HMW adiponectin.
  5. To measure LMW Adiponectin: LMW (trimers + albumin-bound trimers) concentration is calculated by subtracting the combined concentration of MMW + HMW from Total adiponectin.

Fig 1: Selectivity of digestion by proteases. MMW=hexameric; LMW=trimeric. Excerpt from reference 1.

This ELISA utilizes an antibody "sandwich" for detection of adiponectin. The microplate wells have been coated with an anti-human adiponectin monoclonal antibody, and adiponectin in the standards and pretreated samples is captured during the first incubation. Washing removes all unbound material, and a biotin-conjugated anti-human adiponectin monoclonal antibody binds to the immobilized adiponectin in the wells. After the second incubation and subsequent wash step, HRP-labeled streptavidin is added. Following a third incubation and subsequent wash step, O-phenylenediamine (OPD) is added as substrate, and the colorimetric reaction is terminated with the addition of diluted H2SO4. The intensity of the color development, i.e., absorbance value, is proportional to the adiponectin concentration in the sample.

The ELISA is calibrated using purified native human adiponectin(2). Another benefit is the flexibility of the sample pre-treatment procedure. This has paved the way for the introduction of an assay utilizing the same antibodies that quantifies "total" adiponectin exclusively (see product here).

The ELISA itself is completed in less than three hours. Serum, EDTA and Heparinized plasma are all validated sample types for this assay. It has been our experience that samples which have endured up to three freeze/thaw cycles demonstrate greater than 90% recovery. Additionally, many researchers inquire about the long-term stability of adiponectin in archived samples. ALPCO has stability data at -70 °C for up to 1 year, although many researchers have published data collected from sample sets much older than that (3,4).

  1. 1. Ebinuma H, Clin Chem Acta 2006; 372(1-2).
  2. 2. Hada Y, Biochem Biophys Res Comm; 2007. 356: 487-493.
  3. 3. Heidemann, et al. 2008. Annals of Internal Medicine 149 (5): 307-316.
  4. 4. von Eynatten, et al. 2008. European Heart Journal 29: 1307-1315.

Preparing the Calibrator Stock:

Dilute 1:101 PRIOR to beginning the serial dilution series!