A simple, rapid and inexpensive technique to bind small peptides to polystyrene surfaces for immunoenzymatic assays.
Synthetic peptides are widely used in indirect ELISA to detect and characterize specific antibodies in biological samples. Small peptides are not efficiently immobilized on plastic surfaces by simple adsorption, and the conjugation to carrier proteins with different binding techniques is the method of choice. Common techniques to conjugate peptide antigens to carrier proteins and to subsequently purify such complexes are time consuming, expensive, and occasionally abrogate immunogenicity of peptides. In this report we describe a simple, fast and inexpensive alternative protocol to immobilize synthetic peptides to plastic surfaces for standard ELISA. The technique is based on use of maleimide-activated bovine serum albumin or keyhole limpet hemocyanin as a protein anchor adsorbed on the polystyrene surface of the microtiter plate. Following adsorption of the carrier protein, sulfhydryl-containing peptides are cross-linked with an in-well reaction, allowing their correct orientation and availability to antibody binding, avoiding the time consuming steps needed to purify the hapten-carrier complexes. The immunoreactivity of peptides was tested by using both monoclonal and polyclonal antibodies in standard ELISA assays, and compared with established coating methods.
Dipartimento di Scienze Biomediche, Università degli Studi di Sassari, Sassari, Italy. mantc@tiscali.it
Maria Antonietta
MA
Cuccuru
Daniele
D
Dessì
Paola
P
Rappelli
Pier Luigi
PL
Fiori
D000906:Antibodies / Q000276:immunology; D002352:Carrier Proteins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics* / Q000379:methods*; D008301:Maleimides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D011137:Polystyrenes / Q000737:chemistry*; D013499:Surface Properties
D000906:Antibodies; D002352:Carrier Proteins; D008301:Maleimides; D010455:Peptides; D011137:Polystyrenes; C043592:maleimide; D003545:Cysteine