alpco

Assay Principle

Principle of the Assay:

In the Mouse HMW and Total Adipopnectin ELISA, both Total and HMW Adiponectin can be measured independently on the same plate. Samples are pretreated with or without protease, diluted and then assayed for adiponectin as described below.

To measure Total Adiponectin: Samples are not subjected to protease pretreatment but are diluted with sample pretreatment and dilution buffers; measurement in the ELISA quantifies the amount of all multimers of adiponectin without any modification to their respective structures. Total adiponectin includes HMW (12-mer and 18-mer), Mid Molecular Weight (MMW) (hexamer) and Low Molecular Weight (LMW) (trimer and albumin bound trimer).

To measure HMW Adiponectin: Samples are pretreated with the protease that specifically digests MMW, LMW and albumin-LMW.

Fig 1: Evaluation of the selective digestion of mouse adiponectin oligomers by protease. Mouse serum with or without protease pretreatment, were seperated by native PAGE and analyzed by Western blotting (detection antibody used for blot was the same as that used in our ELISA). Adapted from Ebinuma H, Clin Chim Acta 2009 page 182 Fig. 1A

Samples are then diluted with sample pretreatment and dilution buffers which stop the protease reaction. The remaining HMW adiponectin is then assayed in the ELISA:

Fig 2: Mouse sera pretreated with protease were fractionated by gel filtration chromatography on a Superdex 200 column and detected by our ELISA. Adapted from Ebinuma H, et al Clin Chim Acta 2009 page 182 Fig. 1B

The ELISA utilizes an antibody "sandwich" comprised of an anti-mouse adiponectin monoclonal (MoAb) and an anti-mouse polyclonal adiponectin antibody (PoAb). The microplate wells have been coated with anti-mouse adiponectin MoAb, and adiponectin in the standards and pretreated samples is captured by the antibody during the first incubation. Washing removes all unbound material, and a biotin- labeled PoAb is added, which binds to immobilized adiponectin in the wells. After the second incubation and subsequent washes, HRP-labeled streptavidin is added. Following a third incubation and subsequent wash step, O-phenylenediamine (OPD) is added as substrate. 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.