Rapid Screening of Antigenically Reactive Fragments of αs1-Casein Using HPLC and ELISA

Abstract

Screening of antigenically reactive fragments of αs1-casein (αs1-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with αs1-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted with αs1-CN and incomplete Freund's adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti αs1-CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. αs1-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti αs1-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of αs1-CN by the direct and simple detection of specific antigen-antibody interaction.