PBL's enzyme-linked immunosorbent assay (ELISA) for Mimetic Blue® SA/ Mimetic Blue® 1 is designed for use in monitoring ligand leakage from affinity adsorbents containing Mimetic Blue® SA, Mimetic Blue® 1, or equivalent ligands such as C.I. Reactive Blue 2. This assay utilises a purified rabbit polyclonal anti-Mimetic Blue® antibody in a competitive sandwich ELISA which has detection limits in the low nanomolar concentration range (Stuart et al). This is several orders of magnitude more sensitive than the limits of spectrophotometric detection.

The competitive sandwich ELISA is performed by incubating dilutions of sample and anti-Mimetic Blue® antibody in 96 -well microtitre plates coated with purified Mimetic Blue® haemoglobin conjugate. Bound antibody is detected with a goat anti-rabbit IgG peroxidase conjugate in conjunction with a chromogenic assay. Serial dilution of a Mimetic Blue® standard solution yields a standard curve from which the sensitivity of the assay may be determined and the concentration of Mimetic Blue® SA, Mimetic Blue® 1 or equivalent ligands in the samples may be estimated. The effect of interfering protein (e.g. eluted product) on the apparent free ligand concentration can be determined by constructing a standard curve in the presence of protein. Any difference between spiked and un-spiked standard curves is due to ligand sequestration by the protein(s).

Because certain similarities exist between the structures of Mimetic Blue® SA, Mimetic Blue® 1 and the reactive textile dye CI Reactive Blue 2, this assay may also be used to detect leakage from affinity supports containing C.I. Reactive Blue 2 (isomers- or isomeric mixtures of which are known variously as Procion® Blue H-B, Cibacron® Blue F-3GA and Cibacron® Blue 3GA). It is recommended that, where possible, separate standard curves are prepared for each dye ligand tested.

PBL's ELISA kit for Mimetic Blue® SA/Mimetic® Blue 1 contains sufficient materials to conduct ten plate assays (20 sample assays).

For more information on this assay please do not hesitate to contact us on sales@prometicbiosciences.com.