DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR CI REACTIVE BLUE-2 AND ITS APPLICATION TO A COMPARISON OF THE STABILITY AND PERFORMANCE OF A PERFLUOROCARBON SUPPORT WITH OTHER IMMOBILIZED CI REACTIVE BLUE-2 AFFINITY ADSORBENTS

An indirect competitive enzyme-linked immunosorbent assay capable of detecting the reactive triazine dye C.I. Reactive Blue 2 at concentrations down to 300 pM was developed, and representing a 3000-fold higher sensitivity over direct spectrophotometric analysis. An investigation of the cross-reactivity of the polyclonal antibody against various compounds structurally related to C.I. Reactive Blue 2 revealed that the immunoassay appeared to be specific for anthraquinone-containing dyes and was largely unaffected by substitutions at the triazine ring. These characteristics suggest that the immunoassay may be exploited to analyse the leaching of the ligand C.I. Reactive Blue 2 from dye-affinity adsorbents. The performance of a novel perfluoropolymer affinity support containing C.I. Reactive Blue 2 was compared with eight other commercially available dyed affinity adsorbents by three separate criteria: pressure-flow-rate characteristics, protein binding capacities and dye leakage under selected conditions. All the affinity adsorbents were subjected to six purification cycles of albumin from human plasma prior to comparison. In the pressure-flow-rate comparison, the perfluoropolymer support, in contrast to the other adsorbents, showed a non-linear relationship between pressure and flow-rate. Human serum albumin dynamic load capacities were determined by frontal analysis and were found to be in the range 10.1-48.5 mg/ml. The perfluoropolymer support displayed the lowest capacity, probably because of its lack of porosity and, consequently, low surface area. In the dye leakage analysis, the perfluoropolymer support exhibited the lowest leakage of immobilised dye under all conditions tested including exposure to sodium isothiocyanate (5 M), hydrochloric acid (1 M) and sodium hydroxide (1 M). This study suggests that although the novel C.I. Reactive Blue 2-perfluoropolymer support displays low protein binding capacity, it otherwise compares very favourably with a range of commercially available dye-affinity adsorbents.