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Production and application of monoclonal antibodies to ovine interleukin-1α and interleukin-1β

1994; Elsevier BV; Volume: 41; Issue: 3-4 Linguagem: Inglês

10.1016/0165-2427(94)90100-7

1873-2534

Paul J. Egan, Arna E. Andrews, Garry Barcham, M R Brandon, Andrew D. Nash,

Biochemical and Structural Characterization

Monoclonal antibodies (mAbs) were raised against recombinant ovine interleukin-1α and β (ovIL-1α and ovIL-1β). Five ovIL-1α specific mAbs and three ovIL-1β specific mAbs, all of the IgG1 isotype, were characterized. Four of the five ovIL-1α specific mAbs, designated 10.36, 10.49, 10.82 and 5.16, fell into two distinct groups based on several criteria. MAbs 10.36, 10.49 and 10.82 reacted with recombinant ovIL-1α in Western blot analysis, were potent in neutralizing ovIL-1α biological activity in vitro and bound to the same or a closely related epitope. MAb 5.16 also bound ovIL-1α in Western blot analysis, but was less potent in neutralizing ovIL-1α biological activity and bound to a different epitope. A fifth ovIL-1α specific mAb, 5.01, had some characteristics of antibodies from both groups. While the combination of mAb 5.16 with any of 10.36, 10.49 and 10.82 was suitable for detection of ovIL-1α in a sandwich immunoassay, the most sensitive detection of ovIL-1α utilized mAb 10.82 for capture and a rabbit polyclonal anti-ovIL-1α antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent. This combination of reagents had a detection limit for ovIL-1α of 5 pg ml−1 and could detect both recombinant and native ovIL-1α. Of the three ovIL-1β specific mAbs, (designated 2.93, 3.41 and 5.60) 3.41 and 5.60 recognized the same or a closely related epitope while 2.93 recognized an epitope more accessible on denatured ovIL-1β and proved most useful in Western blot analysis. Only mAb 3.41 was potent in neutralizing ovIL-1β biological activity in vitro. A sandwich immunoassay using mAb 3.41 to capture ovIL-1β and a rabbit polyclonal anti-ovIL-1β antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent had a sensitivity of 5 ng ml−1. The immunoassays were used to assess the relative proportions of IL-1α and IL-1β in the supernatant of lipopolysaccharide stimulated ovine alveolar macrophages with IL-1β found to be the predominant secreted species of ovIL-1.