Solid-phase immunoassay of serum antibodies to peptides. Covalent antigen binding to adsorbed phenylalanine-lysine copolymers.
Two small peptide antigens, glucagon and enkephalin (5-L-leucine), were covalently immobilised using either glutaraldehyde or bis-(sulphosuccinimidyl) suberate to an adsorbed layer of phenylalanine-lysine copolymer (PL) or partially acetylated PL (APL) on polystyrene. Both copolymers formed stable layers, particularly APL after adsorption from solution in distilled water. Adsorption of the copolymers under these conditions and subsequent coupling of the antigens yielded solid phases with low non-specific immunoglobulin binding characteristics in an enzyme-linked immunosorbent assay (ELISA) to detect peptide-specific antibodies in rabbit serum. The signal-to-noise ratio in this ELISA was dependent on the combination of copolymer, antigen and coupling reagent employed. Removal from the solid-phase of weakly bound antigen by washing with solutions containing Tween 20 or sodium dodecyl sulphate (SDS) increased assay sensitivity, which was 2-4-fold greater than when simple antigen adsorption was utilised. In the ELISA, the coefficient of variation was lower when covalent antigen coupling was employed.
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Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, U.K.
D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D004745:Enkephalins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005934:Glucagon / Q000276:immunology; D007118:Immunoassay; D008239:Lysine; D010455:Peptides / Q000276:immunology*; D010649:Phenylalanine; D011817:Rabbits
D000906:Antibodies; D004745:Enkephalins; D010455:Peptides; D010649:Phenylalanine; D005934:Glucagon; D008239:Lysine