ABSTRACT
A homogeneous complement-mediated liposome immune lysis assay (LILA) was developed for determination of the herbicide atrazine. To dispose the antigen on the surface of lipid bilayer the atrazine was conjugated to a dimirystoylphosphatidylethanolamine (DMPE) carrier. Calcein was compared with sulforhodamine 101 as a fluorophore label for entrapping into the antigen-sensitized liposomes. The liposomes were incubated with rabbit anti-atrazine antibodies in the presence of guinea pig complement. Formation of the antigen-antibody complexes on the liposomal surface initiated the lytic action of the complement. As free competing atrazine inhibited the lytic reaction, the amount of calcein released was inversely proportional to the atrazine content in the probe. Concentration and kinetic dependences of the immunoassay were characterized to reach its maximal sensitivity. The developed assay allows detecting atrazine in concentrations up to 0.13 ng mL(-1) in the sample (0.04 ng mL(-1) in the final reaction mixture). The named sensitivity is two orders higher than those for the microplate enzyme-linked immunosorbent assay (ELISA) with the same antibodies which allows us to recommend LILA for environmental monitoring.
Subject(s)
Antibodies/chemistry , Atrazine/analysis , Complement System Proteins/chemistry , Herbicides/analysis , Immunoassay/methods , Liposomes/chemistry , Animals , Antibodies/immunology , Atrazine/immunology , Complement System Proteins/immunology , Environmental Monitoring/methods , Fluoresceins/chemistry , Guinea Pigs , Herbicides/immunology , Liposomes/immunology , Phosphatidylethanolamines/chemistry , Rabbits , Sensitivity and SpecificityABSTRACT
We have developed liposome sensitization by a protein, latrotoxin (LT), using immobilization of biotinylated LT via streptavidin with biotinylated phosphatidylethanolamine contained in liposomes. The use of such liposomes in the complement-dependent homogeneous liposome immune lysis assay (LILA) has allowed us to detect in the test sample as little as 2 micrograms/ml of polyclonal and 50-100 ng/ml of monoclonal IgG and IgM antibodies to LT. LT concentration in solution was determined by inhibition of immune lysis by free LT. The sensitivity of the LT assay varied from 1 x 10(-9) to 5-50 x 10(-9) M when antiserum (polyclonal antibodies) and monoclonal antibodies to LT were correspondingly used. The results show that a streptavidin-biotin spacer can be used to immobilize protein antigens on liposomes for a subsequent application in LILA. The suggested technique greatly simplifies the sensitization procedure and extends the applicability of the LILA.
