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. 1988 May 15;252(1):237–245. doi: 10.1042/bj2520237

Rat monoclonal antibodies to rabbit and human serum low-density lipoprotein.

E Gherardi 1, A Hutchings 1, G Galfre 1, D E Bowyer 1
PMCID: PMC1149129  PMID: 2458711

Abstract

A total of 16 hybrid myeloma clones secreting monoclonal antibodies (McAb) to rabbit or human serum low-density lipoprotein (LDL) were derived from the fusion of spleen cells from LOU or DA rats immunized with rabbit or human LDL and the rat myeloma lines Y3 Ag1.2.3 or YB2/0. Anti-(rabbit LDL) McAb showed limited reactivity with LDL from human, rhesus-monkey, rat and mouse serum. Six out of seven anti-(human LDL) McAb reacted with rhesus-monkey LDL, and only one showed partial cross-reaction with rabbit LDL. Binding-competition experiments indicated that the epitopes recognized by the anti-(rabbit LDL) IgG could be grouped into two major clusters: McAb in the first cluster reacted either with apo-(lipoprotein B-100) (apoB-100) and apo-(lipoprotein B-74) (apoB-74) or with apoB-100 but not with apo-(lipoprotein B-48) (apoB-48), the lower-Mr form of apoB of intestinal origin; the McAb in the second cluster all reacted with apoB-48 in addition to apoB-100 or apoB-100 and apoB-74. The six anti-(human LDL) IgG bound to separate epitopes on LDL. Further data on the epitope specificity of these McAb were obtained by antibody blotting after partial proteolysis of apoB-100 with trypsin or staphylococcal V8 proteinase, and the data confirmed the results obtained with the binding-competition experiments. One McAb to rabbit LDL inhibited the binding of LDL to the fibroblast LDL receptor (50% inhibition at a McAb/LDL molar ratio of 10). A similar result was produced by two other McAb at higher concentrations of antibody.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Angelin B., Raviola C. A., Innerarity T. L., Mahley R. W. Regulation of hepatic lipoprotein receptors in the dog. Rapid regulation of apolipoprotein B,E receptors, but not of apolipoprotein E receptors, by intestinal lipoproteins and bile acids. J Clin Invest. 1983 Apr;71(4):816–831. doi: 10.1172/JCI110835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bilheimer D. W., Watanabe Y., Kita T. Impaired receptor-mediated catabolism of low density lipoprotein in the WHHL rabbit, an animal model of familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1982 May;79(10):3305–3309. doi: 10.1073/pnas.79.10.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown M. S., Kovanen P. T., Goldstein J. L. Regulation of plasma cholesterol by lipoprotein receptors. Science. 1981 May 8;212(4495):628–635. doi: 10.1126/science.6261329. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Chapman M. J. Animal lipoproteins: chemistry, structure, and comparative aspects. J Lipid Res. 1980 Sep;21(7):789–853. [PubMed] [Google Scholar]
  6. Cleveland W. L., Wassermann N. H., Sarangarajan R., Penn A. S., Erlanger B. F. Monoclonal antibodies to the acetylcholine receptor by a normally functioning auto-anti-idiotypic mechanism. Nature. 1983 Sep 1;305(5929):56–57. doi: 10.1038/305056a0. [DOI] [PubMed] [Google Scholar]
  7. Curtiss L. K., Edgington T. S. Immunochemical heterogeneity of human plasma apolipoprotein B. I. Apolipoprotein B binding of mouse hybridoma antibodies. J Biol Chem. 1982 Dec 25;257(24):15213–15221. [PubMed] [Google Scholar]
  8. Drapeau G. R. Cleavage at glutamic acid with staphylococcal protease. Methods Enzymol. 1977;47:189–191. doi: 10.1016/0076-6879(77)47023-x. [DOI] [PubMed] [Google Scholar]
  9. Drevon C. A., Attie A. D., Pangburn S. H., Steinberg D. Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts. J Lipid Res. 1981 Jan;22(1):37–46. [PubMed] [Google Scholar]
  10. Fisher A. G., Brown G. A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface. J Immunol Methods. 1980;39(4):377–385. doi: 10.1016/0022-1759(80)90238-0. [DOI] [PubMed] [Google Scholar]
  11. Galfrè G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
  12. Galfrè G., Milstein C., Wright B. Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG. Nature. 1979 Jan 11;277(5692):131–133. doi: 10.1038/277131a0. [DOI] [PubMed] [Google Scholar]
  13. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  14. Goldstein J. L., Kita T., Brown M. S. Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. N Engl J Med. 1983 Aug 4;309(5):288–296. doi: 10.1056/NEJM198308043090507. [DOI] [PubMed] [Google Scholar]
  15. Goldstein S., Chapman M. J. Comparative immunochemical studies of the serum low-density lipoprotein in several animal species. Biochem Genet. 1976 Dec;14(11-12):883–896. doi: 10.1007/BF00485121. [DOI] [PubMed] [Google Scholar]
  16. Goldstein S., Chapman M. J., Mills G. L. Biochemical and immunological evidence for the presence of an apolipoprotein B-like component in the serum low-density lipoproteins of several animal species. Atherosclerosis. 1977 Oct;28(2):93–100. doi: 10.1016/0021-9150(77)90147-2. [DOI] [PubMed] [Google Scholar]
  17. Huang L. S., Bock S. C., Feinstein S. I., Breslow J. L. Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6825–6829. doi: 10.1073/pnas.82.20.6825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hui D. Y., Innerarity T. L., Mahley R. W. Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Importance of apolipoprotein E. J Biol Chem. 1984 Jan 25;259(2):860–869. [PubMed] [Google Scholar]
  19. Hui D. Y., Innerarity T. L., Milne R. W., Marcel Y. L., Mahley R. W. Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48. J Biol Chem. 1984 Dec 25;259(24):15060–15068. [PubMed] [Google Scholar]
  20. Innerarity T. L., Pitas R. E., Mahley R. W. Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity. J Biol Chem. 1980 Dec 10;255(23):11163–11172. [PubMed] [Google Scholar]
  21. Kane J. P. Apolipoprotein B: structural and metabolic heterogeneity. Annu Rev Physiol. 1983;45:637–650. doi: 10.1146/annurev.ph.45.030183.003225. [DOI] [PubMed] [Google Scholar]
  22. Kane J. P., Hardman D. A., Paulus H. E. Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci U S A. 1980 May;77(5):2465–2469. doi: 10.1073/pnas.77.5.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kita T., Goldstein J. L., Brown M. S., Watanabe Y., Hornick C. A., Havel R. J. Hepatic uptake of chylomicron remnants in WHHL rabbits: a mechanism genetically distinct from the low density lipoprotein receptor. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3623–3627. doi: 10.1073/pnas.79.11.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Knott T. J., Rall S. C., Jr, Innerarity T. L., Jacobson S. F., Urdea M. S., Levy-Wilson B., Powell L. M., Pease R. J., Eddy R., Nakai H. Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization. Science. 1985 Oct 4;230(4721):37–43. doi: 10.1126/science.2994225. [DOI] [PubMed] [Google Scholar]
  26. Kroon P. A., Thompson G. M., Chao Y. S. Beta-very low density lipoproteins in cholesterol-fed rabbits are of hepatic origin. Atherosclerosis. 1985 Sep;56(3):323–329. doi: 10.1016/0021-9150(85)90007-3. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Law S. W., Lackner K. J., Hospattankar A. V., Anchors J. M., Sakaguchi A. Y., Naylor S. L., Brewer H. B., Jr Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8340–8344. doi: 10.1073/pnas.82.24.8340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mao S. J., Kazmar R. E., Silverfield J. C., Alley M. C., Kluge K., Fathman C. G. Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies. Biochim Biophys Acta. 1982 Nov 12;713(2):365–374. doi: 10.1016/0005-2760(82)90255-7. [DOI] [PubMed] [Google Scholar]
  30. Mao S. J., Patton J. G., Badimon J. J., Kottke B. A., Alley M. C., Cardin A. D. Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies. Clin Chem. 1983 Nov;29(11):1890–1897. [PubMed] [Google Scholar]
  31. Marcel Y. L., Hogue M., Theolis R., Jr, Milne R. W. Mapping of antigenic determinants of human apolipoprotein B using monoclonal antibodies against low density lipoproteins. J Biol Chem. 1982 Nov 25;257(22):13165–13168. [PubMed] [Google Scholar]
  32. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marcovina S., Kottke B. A., Mao S. J. Monoclonal antibodies can precipitate low-density lipoprotein. II. Radioimmunoassays with single and combined monoclonal antibodies for determining apolipoprotein B in serum of patients with coronary artery disease. Clin Chem. 1985 Oct;31(10):1659–1663. [PubMed] [Google Scholar]
  34. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  35. Milne R. W., Theolis R., Jr, Verdery R. B., Marcel Y. L. Characterization of monoclonal antibodies against human low density lipoprotein. Arteriosclerosis. 1983 Jan-Feb;3(1):23–30. doi: 10.1161/01.atv.3.1.23. [DOI] [PubMed] [Google Scholar]
  36. Nelson C. A., Tasch M. A., Tikkanen M., Dargar R., Schonfeld G. Evolution of low density lipoprotein structure probed with monoclonal antibodies. J Lipid Res. 1984 Aug;25(8):821–830. [PubMed] [Google Scholar]
  37. Patton J. G., Alley M. C., Mao S. J. Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure. J Immunol Methods. 1982 Dec 17;55(2):193–203. doi: 10.1016/0022-1759(82)90031-x. [DOI] [PubMed] [Google Scholar]
  38. Schumaker V. N., Robinson M. T., Curtiss L. K., Butler R., Sparkes R. S. Anti-apoprotein B monoclonal antibodies detect human low density lipoprotein polymorphism. J Biol Chem. 1984 May 25;259(10):6423–6430. [PubMed] [Google Scholar]
  39. Shechter Y., Maron R., Elias D., Cohen I. R. Autoantibodies to insulin receptor spontaneously develop as anti-idiotypes in mice immunized with insulin. Science. 1982 Apr 30;216(4545):542–545. doi: 10.1126/science.7041258. [DOI] [PubMed] [Google Scholar]
  40. Shoulders C. C., Myant N. B., Sidoli A., Rodriguez J. C., Cortese C., Baralle F. E., Cortese R. Molecular cloning of human LDL apolipoprotein B cDNA. Evidence for more than one gene per haploid genome. Atherosclerosis. 1985 Dec;58(1-3):277–289. doi: 10.1016/0021-9150(85)90073-5. [DOI] [PubMed] [Google Scholar]
  41. Sparks J. D., Bolognino M., Trax P. A., Sparks C. E. The production and utility of monoclonal antibodies to rat apolipoprotein B lipoproteins. Atherosclerosis. 1986 Sep;61(3):205–211. doi: 10.1016/0021-9150(86)90139-5. [DOI] [PubMed] [Google Scholar]
  42. Tikkanen M. J., Cole T. G., Hahm K. S., Krul E. S., Schonfeld G. Expression of apolipoprotein B epitopes in very low density lipoprotein subfractions. Studies with monoclonal antibodies. Arteriosclerosis. 1984 Mar-Apr;4(2):138–146. doi: 10.1161/01.atv.4.2.138. [DOI] [PubMed] [Google Scholar]
  43. Tikkanen N. J., Dargar R., Pfleger B., Gonen B., Davie J. M., Schonfeld G. Antigenic mapping of human low density lipoprotein with monoclonal antibodies. J Lipid Res. 1982 Sep;23(7):1032–1038. [PubMed] [Google Scholar]
  44. Tsao B. P., Curtiss L. K., Edgington T. S. Immunochemical heterogeneity of human plasma apolipoprotein B. II. Expression of apolipoprotein B epitopes on native lipoproteins. J Biol Chem. 1982 Dec 25;257(24):15222–15228. [PubMed] [Google Scholar]
  45. Watt T. S., Watt R. M. Detection of unique antigenic determinants on human plasma low density lipoprotein and on delipidated apolipoprotein B. Proc Natl Acad Sci U S A. 1983 Jan;80(1):124–128. doi: 10.1073/pnas.80.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wei C. F., Chen S. H., Yang C. Y., Marcel Y. L., Milne R. W., Li W. H., Sparrow J. T., Gotto A. M., Jr, Chan L. Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxyl-terminal fragment of human apolipoprotein B-100. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7265–7269. doi: 10.1073/pnas.82.21.7265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Young S. G., Witztum J. L., Casal D. C., Curtiss L. K., Bernstein S. Conservation of the low density lipoprotein receptor-binding domain of apoprotein B. Demonstration by a new monoclonal antibody, MB47. Arteriosclerosis. 1986 Mar-Apr;6(2):178–188. doi: 10.1161/01.atv.6.2.178. [DOI] [PubMed] [Google Scholar]
  48. Zupko K., Waltenbaugh C., Diamond B. Use of anti-idiotypic antibodies to identify a receptor for the T-cell I-J determinant. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7399–7403. doi: 10.1073/pnas.82.21.7399. [DOI] [PMC free article] [PubMed] [Google Scholar]

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