R&D Systems’ Quantikine Kit for measurement of the cytokine adiponectin (also known as Acrp30) is a quantitative sandwich ELISA. This ELISA is designed to measure adiponectin in cell culture supernatants, serum and plasma. The assay takes 4.5 hours to perform. The ELISA uses a monoclonal antibody specific for adiponectin; the antibody is pre-coated onto a 96-well individual strip microplate. A more detailed description of the procedure follows, but in general: Standards (recombinant human adiponectin) and samples are added to the wells and any adiponectin present is bound by the immobilized antibody. After washing the microplate to remove any unbound substances, an enzyme-linked (HRP) monoclonal antibody specific for adiponectin is added to the microplate wells. The microplate is washed once again, this time to remove unbound antibody-enzyme conjugate. At this point, the substrate solution is added to the wells and color development is allowed to proceed. Color development occurs in proportion to the amount of adiponectin bound in the initial step. After an incubation period, the color development is stopped and the intensity of the color is measured.
We have used this assay primarily to measure levels of adiponectin produced by primary differentiated adipocyte cell lines. All reagents are brought to room temperature before beginning the assay. The first step in the ELISA is to prepare the standards in the supplied calibrator diluent (RD5-5). Recombinant adiponectin standard is reconstituted in 2 ml of calibrator diluent to make a 250ng/ml solution, and allowed to incubate at room temperature, with gentle agitation, for at least 15 minutes. Once the adiponectin standard is prepared, dilutions are prepared in RD5-5 to generate adiponectin standards from 250 ng/ml to 3.9 ng/ml in doubling dilutions. The ELISA plate is removed from its foil pouch and the assay diluent (RD1F) is added to each well (100 ul). At this point 50 ul of each standard (in duplicate) and each sample are added to the ELISA plate. The ELISA plate is then incubated for 2hrs at room temperature. After incubation, the plate is aspirated and washed four times with the supplied wash buffer. Then 200 ul of adiponectin conjugate (monoclonal anti-adiponectin-HRP) is added to the plate and a further incubation of 2 hr at room temperature is performed. Once again, after the incubation, the plate is aspirated and washed 4 times. The next step is to add 200 ul of the substrate solution to the plate. The substrate solution is prepared by adding equal volumes of color reagent A (hydrogen peroxide) and color reagent B (TMB). The plate is then incubated for a further 20 minutes while the color develops (blue). Color development is stopped with the addition of the stop solution (2 N sulfuric acid) which turns the blue color to yellow. We have found that it is very important to mix the wells after addition of the stop solution to ensure correct conversion to yellow. Mixing is performed by pipetting the contents of the well up and down. After mixing, the plate can then be read in a microplate reader at 450 nm.
A standard curve is created from the standards on the plate and this is used to calculate the concentration of adiponectin in the samples. There are many types of software which will perform this step; we have used Molecular Devices SOFTmax® Pro. It is important to keep the level of adiponectin within the dynamic range of the assay. Samples may need to be diluted to bring them within that range if, on first measurement, they have an elevated adiponectin level. We have found the intra- and interassay precision to be very robust. In conclusion, R&D Systems’ ELISA for adiponectin is a simple, fast and reliable kit.
Boyd Scott
Senior Research Scientist
Vitae Pharmaceuticals
Discovery Biology