Basement membranes (BM) are growth factor enriched specialized extracellular matrix (ECM) protein complexes providing structural and functional support to cell monolayers separating from connective tissue. The ECM is a complex mixture of matrix molecules including glycosaminoglycans, fibronectin, collagens, laminins and non-matrix proteins such as growth factors. The composition of the ECM, rather than simply the presence of extracellular scaffold, is critical for regulating cell phenotype. ECMs determine body shape and stability, compartmentalization of organs and several cellular activities. These matrices include ubiquitously occurring basement membranes which are 20-200 nm broad deposits of specific proteins in close proximity to epithelial, muscle, fat and nerve cells.
Hyaluronic acid (HA) is a biologically derived non-sulfated glycosaminoglycan distributed in the basement membranes of epithelial, neuronal and cartilage tissues. The molecule (3000-4000 kDa) is also present in human blood samples and human synovial fluids. The major functions of HA are cell adhesion, migration, proliferation and differentiation.
The HA ELISA/EIA Kit from Echelon Biosciences is a non-radioactive 96-well assay kit for the rapid (3 hour) detection of HA (also called hyaluronan or hyaluronate) in human cell culture supernatants, serum or animal derived biological fluids. The detection range of the kit is 50-1600 ng/ml. The measurement is based on a competitive ELISA assay in which the absorbance is inversely proportional to HA concentration in the samples. The kit consists of the following components: a 96-well microplate coated with human HA, human HA stock solution at a concentration of 3200 ng/ml, HA detector, standard and sample diluent buffer, wash buffer concentrate, substrate buffer, stop solution, enzyme, yellow 96-well polystyrene bottom plate, p-nitrophenyl phosphate buffer and plate adhesive.
The samples can be diluted in diluent buffer if very concentrated. Since we utilized highly concentrated, conditioned-medium samples, dilution up to 100 fold was performed with the diluent buffers. (is any sample prep involved or can you mix straight supernatant, serum or other fluid?). Standards are generated using 1:2 serial dilutions of the stock solution to generate concentrations of 1600, 800, 400, 200, 100 and 50 ng/ml. Add 100 ul of samples and standards to the yellow 96-well polystyrene plate, including diluent buffer as blank controls. Add 50 ul of detector buffer to all samples and standards except the blank. Mix the plate gently, cover with plate seal and incubate for 30 min at 37°C. Following the incubation, add 100 ul of controls and samples to the 96-well microplate coated with anti-human HA. Mix contents, cover plate with seal and incubate for 30 min at 37°C. Remove solution from the plates, wash four times with 1X wash buffer solution and add 100 ul of working enzyme to each well. Mix contents, seal plate and incubate 30 min at 37°C. Wash plate 4 times with 1 X wash buffer and add 100 ul of working substrate to each well followed by incubation in the dark at room temperature. Stop reaction by adding 50 ul of stop solution to each well and measure absorbance at 405 nm; we use a Bio-Rad 680 plate reader.
We have used the HA ELISA Kit extensively in our laboratory for identifying the molecule in media supernatants and growth-factor enriched extracellular matrices. The ease of detection, quantification and comparison of positive experimental samples to negative controls (i.e. samples not containing HA) has been excellent in our experience. We truly recommend this assay kit for measurement of HA in human and animal biological fluids.