Production and application of monoclonal antibodies to ovine interleukin-1 alpha and interleukin-1 beta.
Monoclonal antibodies (mAbs) were raised against recombinant ovine interleukin-1 alpha and beta (ovIL-1 alpha and ovIL-1 beta). Five ovIL-1 alpha specific mAbs and three ovIL-1 beta specific mAbs, all of the IgG1 isotype, were characterized. Four of the five ovIL-1 alpha 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-1 alpha in Western blot analysis, were potent in neutralizing ovIL-1 alpha biological activity in vitro and bound to the same or a closely related epitope. MAb 5.16 also bound ovIL-1 alpha in Western blot analysis, but was less potent in neutralizing ovIL-1 alpha biological activity and bound to a different epitope. A fifth ovIL-1 alpha 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-1 alpha in a sandwich immunoassay, the most sensitive detection of ovIL-1 alpha utilized mAb 10.82 for capture and a rabbit polyclonal anti-ovIL-1 alpha 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-1 alpha of 5 pg ml-1 and could detect both recombinant and native ovIL-1 alpha. Of the three ovIL-1 beta 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-1 beta and proved most useful in Western blot analysis. Only mAb 3.41 was potent in neutralizing ovIL-1 beta biological activity in vitro. A sandwich immunoassay using mAb 3.41 to capture ovIL-1 beta and a rabbit polyclonal anti-ovIL-1 beta 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-1 alpha and IL-1 beta in the supernatant of lipopolysaccharide stimulated ovine alveolar macrophages with IL-1 beta found to be the predominant secreted species of ovIL-1.
Egan
P J
PJ
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Vic., Australia.
Andrews
A E
AE
Barcham
G J
GJ
Brandon
M R
MR
Nash
A D
AD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western / Q000662:veterinary; D000939:Epitopes / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007375:Interleukin-1 / Q000276:immunology* / Q000302:isolation & purification; D008262:Macrophage Activation / Q000276:immunology; D016676:Macrophages, Alveolar / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000276:immunology; D012756:Sheep / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007375:Interleukin-1; D011994:Recombinant Proteins