Summary
Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.
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Abbreviations
- AEC:
-
3-amino-9-ethylcarbazole
- AP:
-
Alkaline phosphatase
- BSA:
-
Bovine serum albumin
- CGG:
-
Chicken gamma globulin
- c-IF:
-
Cytoplasmic immunofluorescence
- DAB:
-
3,3-Diamino benzidine HCl
- ELISA:
-
Enzyme linked immunosorbent assay
- Fl:
-
Fluorescein
- F/P ratio:
-
Number of fluorochrome molecules per molecule of protein
- Ig:
-
Immunoglobulin
- Mab:
-
Monoclonal antibody
- OPD:
-
Ortho-phenylenediamine-dihydrochloride
- PBS:
-
Phosphate buffered saline
- PBS-T:
-
PBS with 0.2% Tween-20
- PO:
-
Peroxidase
- RAM/PO:
-
Peroxidase labeled rabbit antiserum directed against mouse immunoglobulins
- Rh:
-
Tetramethyl rhodamine
- RT:
-
Room temperature
References
Amzel LM, Poljak RJ (1979) Three-dimensional structure of immunoglobulins. Annu Rev Biochem 48:961–977
Bergquist NR, Nilsson P (1974) The conjugation of immunoglobulins with tetramethyl rhodamine isothiocyanate by utilisation of dimethyl sulphoxide (DMSO) as a solvent. J Immunol Methods 5:189–198
Boorsma DM (1984) Direct immunoenzyme double staining applicable for monoclonal antibodies. Histochemistry 80:103–106
Bosman FT, Lindeman J, Kuiper G, Van der Wal A, Kreunig J (1977) The influence of fixation on immunoperoxidase staining of plasma cells in paraffin sections of intestinal biopsy specimens. Histochemistry 53:57–62
Feller AC, Parwaresch MR, Wacker H-W, Rad Zun H-J, Lennert K (1983) Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils. Histochem J 15:557–562
Haaijman JJ (1977) Quantitative immunofluorescence microscopy; methods and applications. Thesis, Leiden, The Netherlands
Haaijman JJ, Deen C, Kröse CJM, Zijlstra JJ, Coolen J, Radl J (1984) Monoclonal antibodies in immunocytology: a jungle full of pitfalls. Immunol Today 5:56–58
Haaijman JJ, Van Dalen JPR (1974) Quantitation in immunofluorescence microscopy. A new standard for fluorescein and rhodamine emission measurements. J Immunol Methods 5:359–374
Hijmans W, Schuit HRE, Hulsing-Hesselink E (1971) An immunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann NY Acad Sci 177:290–305
Hijmans W, Schuit HRE, Klein F (1969) An immunofluorescence procedure for the detection of intracellular immunoglobulins. Clin Exp Immunol 4:457–472
Hsu SM, Soban E (1982) Color modification of diamino benzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem 30:1079–1082
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497
Milstein C, Wright B, Cuello AC (1983) The discrepancy between the crossreactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. Mol Immunol 20:113–123
Nakane PK, Kawaoi A (1974) Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 22:1084–1091
Radl J (1981) Immunoglobulin levels and abnormalitics in aging humans and mice. In: Adler WH, Nordin AA (eds) Immunological techniques applied to aging research. CRC Press, Boca Raton, FL, USA, pp 121–139
Schauenstein K, Böck G, Wick G (1982) The use of lasers to determine the fluorescence characteristics of fluorescein derivatives in immunofluorescence assays. In: Wick G, Traill KN, Schauenstein K (eds) Immunofluorescence technology. Elsevier Biomedical Press, Amsterdam, pp 27–35
Streefkerk JG (1972) Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J Histochem Cytochem 20:829–830
Vandesande F (1983) Immunohistochemical double staining techniques. In: Cuello AC (ed) Immunohistochemistry. IBRO Handbook series, Methods in the neurosciences, vol III. Wiley and Sons, Chichester England, pp 257–272
Van Dalen JPR, Knapp W, Ploem JS (1973) Microfluorometry on antigen-antibody interactions in immunofluorescence using antigens covalently bound to agarose beads. J Immunol Methods 2:383–392
Voss EW (ed) (1984) Fluorescein hapten: an immunological probe. CRC Press, Boca Raton, FL, USA
Voss EW, Eschenfeldt W, Root RT (1976) Fluorescein: a complete antigenic group. Immunochemistry 13:447–456
Watt RM, Voss EW (1977) Mechanism of quenching of fluorescein by anti-fluorescein IgG antibodies. Immunochemistry 14:533–541
Wilchek M, Bayer EA (1984) The avidin-biotin complex in immunology. Immunol Today 5:39–43
Wofsy L, Baker PC, Thompson K, Goodman J, Kimura J, Henry C (1974) Hapten-sandwich labeling, I. A general procedure for simultaneous labeling of multiple cell surface antigens for fluorescence and electronmicroscopy. J Exp Med 140:523–537
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In honour of Prof. P. van Duijn
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Haaijman, J.J., Coolen, J., Kröse, C.J.M. et al. Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry. Histochemistry 84, 363–370 (1986). https://doi.org/10.1007/BF00482964
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DOI: https://doi.org/10.1007/BF00482964