title: Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase: application to the peripheral proteins of photosystem 1.
abstract: An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.
authors.affiliation: Département de Biologie, CEN SACLAY, Gif-s-Yvette, France.
authors.forename: J
authors.initials: J
authors.lastname: Grassi
authors.forename: Y
authors.initials: Y
authors.lastname: Frobert
authors.forename: P
authors.initials: P
authors.lastname: Lamourette
authors.forename: B
authors.initials: B
authors.lastname: Lagoutte
mesh_terms: D000110:Acetylcholinesterase; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis; D001360:Avidin; D001710:Biotin; D002734:Chlorophyll / Q000032:analysis*; D006825:Hybridomas; D007124:Immunoenzyme Techniques; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins / Q000032:analysis*; D011863:Radioimmunoassay
chemical_list: D000911:Antibodies, Monoclonal; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D001360:Avidin; D002734:Chlorophyll; D001710:Biotin; D000110:Acetylcholinesterase