Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column.
Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.
D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods; D008081:Liposomes / Q000737:chemistry*; D010539:Permeability; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry; D011078:Polychlorinated Biphenyls / Q000032:analysis* / Q000276:immunology; D013997:Time Factors
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010743:Phospholipids; D011078:Polychlorinated Biphenyls; D010741:Phospholipases A; D054467:Phospholipases A2; C007740:fluorexon