Measuring Hsp72 (HSPA1A) by Indirect Sandwich ELISA

The enzyme-linked immunosorbent assay (ELISA) is an immunological technique which is used to determine the presence or quantity of an antigen within a sample. ELISAs rely on the use of at least one antibody (Ab) specific for the antigen being measured. This antibody is covalently linked to an enzyme which is detected through the use of an enzymatic substrate, which can be colorimetric, fluorogenic, or chemiluminescent. The ELISA for Hsp72 described here is a typical indirect sandwich ELISA, which can be used for measuring Hsp72 from cellular/tissue extracts, tissue culture supernatant, and serum. Typically, a 96-well ELISA plate is coated with a specific antibody which captures Hsp72 from the sample, and another antibody specific for a different Hsp72 epitope is used to detect Hsp72. An enzyme-labelled species-specific antibody conjugate is then applied which is consequently detected using a colorimetric enzyme substrate. The quantity of Hsp72 present in the samples is interpolated using a standard curve of known amounts of pure Hsp72.