Abstract
Two monoclonal antibodies of types IgG2b and IgG2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(γ-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N 1-acetylspermidine (N1-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.
Similar content being viewed by others
References
Bartos D, Campbell RA, Bartos F, Grettie DP (1975) Direct determination of polyamines in human serum by radioimmunoassay. Cancer Res 35:2056–2060
Bartos F, Bartos D, Dolney AM, Grettie DP, Campbell RA (1978) Radioimmunoassay of spermidine in human serum. Res Commun Chem Pathol Pharmacol 19:295–309
Fawcett DW (1986) A textbook of histology, 11th edn. Saunders, Igaku-Shoin, pp 716–730
Fujiwara K (1994) An evaluation of polyamine immunocytochemistry using immunocytochemical model systems. Histochemistry 101:287–294
Fujiwara K, Asada A, Kitagawa T, Yamamoto K, Ito T, Tsuchiya R, Sohda M, Nakamura N, Hara K, Tomonaga Y, Ichimaru M, Takahashi S (1983) Preparation of polyamine antibody and its use in enzyme immunoassay of spermine and spermidine with β-d-galactosidase as a label. J Immunol Methods 61:217–226
Fujiwara K, Araki M, Kitagawa T, Inoue Y (1993) A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model. Histochemistry 99:477–483
Garthwaite I, Stead AD, Rider CC (1989) A monoclonal antibody-based immunoassay for the polyamines spermine and spermidine. Biochem Soc Trans 17:1056–1057
Garthwaite I, Stead AD, Rider CC (1993) Assay of the polyamine spermine by a monoclonal antibody-based ELISA. J Immunol Methods 162:175–178
Graham RC, Karnowsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302
Hodgson AJ, Penke B, Erdei A, Chubb IW, Somogyi P (1985) Antisera to γ-aminobutyric acid. 1. Production and characterization using a new model system. J Histochem Cytochem 33:229–239
Hougaard DM, Fujiwara K, Larsson L-I (1986) Polyamine cytochemistry: comparisons between cytochemical, autoradiographic, immunocytochemical and chemical results in the prostate. Histochem J 18:321–328
Hougaard DM, Fujiwara K, Larsson L-I (1987) Immunocytochemical localization of polyamines in normal and neoplastic cells. Comparisons to the formaldehyde-fluorescamine and o-phthalaldehyde method. Histochem J 19:643–650
Igarashi K, Watanabe Y, Nakamura K, Kojima M, Fujiki Y, Hirose S (1978) Changes of substrate specificity by polyamines of ribonucleases which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides. Biochem Biophys Res Commun 67:1070–1077
Kato K, Naoe T, Hiraiwa A, Namikawa R, Suzuki S, Yamada K, Shiku H (1985) Production and analysis of HH 10 monoclonal antibodies reactive to immature hematopoietic cells and their use for monitoring acute leukemia cells. Jpn J Cancer Res (Gann) 76:524–531
Larsson L-I (1988) Immunocytochemistry, theory and practice. CRC Press, Boca Raton, Florida
Nakane PK, Pierce GB (1966) Enzyme-labeled antibodies: preparation and application for localization of antigens. J Histochem Cytochem 14:929–931
Pegg AG (1986) Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 234:249–262
Pegg AE, McCann PP (1988) Polyamine metabolism and function in mammalian cells and protozoans. ISI Atlas of science: biochemistry, pp 11–18
Rich DH, Gesellchen PD, Tong A, Cheung A, Buckner CK (1975) Alkylating derivatives of amino acids and peptides. Synthesis of N-maleoylamino acid [1-(N-maleoylglysyl) exytosin, and [1-(N-maleoyl-11-aminoundecanoyl)cysteinyl]oxytosin. Effect on vasopressin-stimulated water loss from isolated toad bladder. J Med Chem 18:1004–1010
Rudkin BB, Mamont PS, Seiler N (1984) Decreased protein-synthetic activity is an early consequence of spermidine depletion in rat hepatoma tissue-culture cells. Biochem J 217, 731–741
Schipper RG, Jonis JA, Rutten RGJ, Tesser GI, Verhofstad AAJ (1991) Preparation and characterization of polyclonal and monoclonal antibodies to polyamines. J Immunol Methods 136:23–30
Sternberger LH (1979) Immunocytochemistry, 2nd edn. John Wiley & Sons, New York
Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790
Takemoto T, Nagamatsu Y, Oka T (1983) The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland. Biochem Biophys Acta 740:73–79
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Fujiwara, K., Furukawa, K., Shiku, H. et al. Production and characterization of monoclonal antibodies against the polyamine, spermine: immunocytochemical localization in rat tissues. Histochemistry 102, 397–404 (1994). https://doi.org/10.1007/BF00268911
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00268911