Biochemical SocietyTransactions ( 1 992) 20
A specfic antibody to apolipoprotein B-48: a novel approach. Andrew S. Peel, Padma Komanduri, Christine M. Williams, Linda M. Morgan and Barry J. Gould. Nutritional Metabolism Group, School of Biological Sciences, University of Surrey, Guildford. GU2 5XH. U.K. Currently, there are no specfic, rapid assays that can distinguish the dietary and hepatic contribution to postprandial lipaemia. Apolipoprotein B-48 is uniquely associated with chylomicrons and their remnants of dietary origin [ 11, whereas apolipoprotein B-100 is associated with very low density lipoproteins and other lipoproteins of hepatic origin. Apo B-48 has an identical amino acid sequence to the N-terminal48% of apo B-100. By exploiting the hydrophilic nature of the charged apo B-48 C-terminal, in a largely hydrophobic protein, an antibody has been raised which specifically recognises apo B-48. The corresponding sequence of amino acids in apo B-100 are in the middle of a peptide chain and less likely to be surface orientated. Two heptapeptides were custom synthesised by the National Institute of Medical Research, Mill Hill, London. These corresponded to the two forms of apo B48 (ie. 2152 amino acid residues with an isoleucine Cterminus and 2151 residues with a methionine C-terminus [2,3] and included an N-terminal cysteine residue for conjugation. Peptides were coupled to ovalbumin using N-succinimidyl-6-maleimidocaproate; a heterobifunctional conjugating agent which cross-linked SH-groups on the peptides to NH2-groups on ovalbumin [4]. An estimation of the number of peptides coupled to each ovalbumin molecule was made by measuring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412nm. A ratio of eleven peptide molecules to each ovalbumin carrier molecule was produced. Antisera were raised to each peptide by emulsifying the conjugates (containing 200 pg of peptide) in non-ulcerative Freunds incomplete adjuvant (Moms) and injecting into two New Zealand white rabbits. The rabbits were boosted six weeks later with 100 pg of peptide and blood collected. Chylomicrons were prepared from human lymph by overlayering with NaCl (d=l.O06g/ml) and ultracentrifuging for 1 . 6 ~ 1 0 8g.min. [5]. Undiluted lymph and chylomicrons were run under denaturing conditions on an SDS-polyacrylamide linear gradient (520%) gel. The lymph sample gel was blotted onto a nitrocellulose membrane and incubated with anti-peptide antisera collected ten days after the boost. The Western blot was visualized using a streptavidin-biotin system, with a horseradish peroxidase label and 4-chloronapthol substrate. A high molecular weight band, slightly greater than 205 kDa and three lower molecular weight bands were observed in the chylomicron sample stained with Coomassie Blue (Fig f). A single, sh& band was seen Abbreviations used: Apo, apolipoprotein; SDS, sodium dodecylsulphate.
165s
669, 205116, 97.4. 66,
45\ 29
\
1
2
3
Fig. 1. SDS-PAGE of chvlomicrons stained for protein with Coomassie Blue R-250 and Western blot us'-1 2152 antiseq 1. Molecular weight markers (Sigma) 2. Chylomicrons 3. Westem blot of lymph detected with Ile-2152 antisera. . serum raised on the Western blot at about 205 kDa with against the Ile-2152 peptide. This is assumed to be apo B48 since the apparent molecular weight of apo B-48 is 264 kDa [6]. No band was observed with the Met-2151 antisera. The potential of using apo B-48 as a marker for particles of intestinal origin has long been recognised [7]. However, the similarity in structure between apo B-48 and apo B-100 has made it difficult to produce specfic apo B-48 antibodies. This is the first report of an antibody specfic for apo B-48. We intend to use the antibody to develop an immunological-based assay that can be used for the routine detection of lipoproteins of dietary origin. We thank Mr. Brian Morris for helpful discussions and acknowledge financial support from the Agricultural and Food Research Council and the Science and Engineering Research Council. 1. Patsch, J.R. (1987) in Bailliere's Clinical Endocrinology and Metabolism-Lipoprotein Metabolism, pp. 551-580. Bailliere Tindall, London 2. Hardman, D.A., Protter, A.A., Schilling, J.W. & Kane, J.P. (1987) Biochem. Biophys. Res. C o r n . 149, 1214-1219 3. Chen, S.H., Habib, G., Yang, C.Y., Gu, Z.W., Lee, B.R., Weng, S.A., Silberman, S.R., Cai, S.J., Deslypere, J.P., Rosseneu, M., Gotto JR, A.M., Li, W.H. & Chan, L. (1987) Science 23,363-366 4. Peeters, J.M., Hazendonk, T.G., Beuvery, E.C. & Tesser, G.I. (1989) J. Immun. Met. 120, 133-143 5. Bochenek, W.J., Kapuscinska, B., Slowinska, R. & Rodgers, J.B. (1987) Atherosclerosis 64, 167-172 6. Kane, J.P., Hardman, D.A. & Paulus, H.E. (1980) Proc. Natl. Acad. Sci. 77,2465-2469 7. Kane, J.P., Chen, G.C., Hamilton, R.L., Hardman, D.A., Malloy, M.J & Havel, R.J. (1983) Arteriosclerosis, 3,47-56
A specfic antibody to apolipoprotein B-48: a novel approach. Andrew S. Peel, Padma Komanduri, Christine M. Williams, Linda M. Morgan and Barry J. Gould. Nutritional Metabolism Group, School of Biological Sciences, University of Surrey, Guildford. GU2 5XH. U.K. Currently, there are no specfic, rapid assays that can distinguish the dietary and hepatic contribution to postprandial lipaemia. Apolipoprotein B-48 is uniquely associated with chylomicrons and their remnants of dietary origin [ 11, whereas apolipoprotein B-100 is associated with very low density lipoproteins and other lipoproteins of hepatic origin. Apo B-48 has an identical amino acid sequence to the N-terminal48% of apo B-100. By exploiting the hydrophilic nature of the charged apo B-48 C-terminal, in a largely hydrophobic protein, an antibody has been raised which specifically recognises apo B-48. The corresponding sequence of amino acids in apo B-100 are in the middle of a peptide chain and less likely to be surface orientated. Two heptapeptides were custom synthesised by the National Institute of Medical Research, Mill Hill, London. These corresponded to the two forms of apo B48 (ie. 2152 amino acid residues with an isoleucine Cterminus and 2151 residues with a methionine C-terminus [2,3] and included an N-terminal cysteine residue for conjugation. Peptides were coupled to ovalbumin using N-succinimidyl-6-maleimidocaproate; a heterobifunctional conjugating agent which cross-linked SH-groups on the peptides to NH2-groups on ovalbumin [4]. An estimation of the number of peptides coupled to each ovalbumin molecule was made by measuring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412nm. A ratio of eleven peptide molecules to each ovalbumin carrier molecule was produced. Antisera were raised to each peptide by emulsifying the conjugates (containing 200 pg of peptide) in non-ulcerative Freunds incomplete adjuvant (Moms) and injecting into two New Zealand white rabbits. The rabbits were boosted six weeks later with 100 pg of peptide and blood collected. Chylomicrons were prepared from human lymph by overlayering with NaCl (d=l.O06g/ml) and ultracentrifuging for 1 . 6 ~ 1 0 8g.min. [5]. Undiluted lymph and chylomicrons were run under denaturing conditions on an SDS-polyacrylamide linear gradient (520%) gel. The lymph sample gel was blotted onto a nitrocellulose membrane and incubated with anti-peptide antisera collected ten days after the boost. The Western blot was visualized using a streptavidin-biotin system, with a horseradish peroxidase label and 4-chloronapthol substrate. A high molecular weight band, slightly greater than 205 kDa and three lower molecular weight bands were observed in the chylomicron sample stained with Coomassie Blue (Fig f). A single, sh& band was seen Abbreviations used: Apo, apolipoprotein; SDS, sodium dodecylsulphate.
165s
669, 205116, 97.4. 66,
45\ 29
\
1
2
3
Fig. 1. SDS-PAGE of chvlomicrons stained for protein with Coomassie Blue R-250 and Western blot us'-1 2152 antiseq 1. Molecular weight markers (Sigma) 2. Chylomicrons 3. Westem blot of lymph detected with Ile-2152 antisera. . serum raised on the Western blot at about 205 kDa with against the Ile-2152 peptide. This is assumed to be apo B48 since the apparent molecular weight of apo B-48 is 264 kDa [6]. No band was observed with the Met-2151 antisera. The potential of using apo B-48 as a marker for particles of intestinal origin has long been recognised [7]. However, the similarity in structure between apo B-48 and apo B-100 has made it difficult to produce specfic apo B-48 antibodies. This is the first report of an antibody specfic for apo B-48. We intend to use the antibody to develop an immunological-based assay that can be used for the routine detection of lipoproteins of dietary origin. We thank Mr. Brian Morris for helpful discussions and acknowledge financial support from the Agricultural and Food Research Council and the Science and Engineering Research Council. 1. Patsch, J.R. (1987) in Bailliere's Clinical Endocrinology and Metabolism-Lipoprotein Metabolism, pp. 551-580. Bailliere Tindall, London 2. Hardman, D.A., Protter, A.A., Schilling, J.W. & Kane, J.P. (1987) Biochem. Biophys. Res. C o r n . 149, 1214-1219 3. Chen, S.H., Habib, G., Yang, C.Y., Gu, Z.W., Lee, B.R., Weng, S.A., Silberman, S.R., Cai, S.J., Deslypere, J.P., Rosseneu, M., Gotto JR, A.M., Li, W.H. & Chan, L. (1987) Science 23,363-366 4. Peeters, J.M., Hazendonk, T.G., Beuvery, E.C. & Tesser, G.I. (1989) J. Immun. Met. 120, 133-143 5. Bochenek, W.J., Kapuscinska, B., Slowinska, R. & Rodgers, J.B. (1987) Atherosclerosis 64, 167-172 6. Kane, J.P., Hardman, D.A. & Paulus, H.E. (1980) Proc. Natl. Acad. Sci. 77,2465-2469 7. Kane, J.P., Chen, G.C., Hamilton, R.L., Hardman, D.A., Malloy, M.J & Havel, R.J. (1983) Arteriosclerosis, 3,47-56
