Summary
125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1∶1.4, contained over 90% ferritin-IgG “monomers”; 70–90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aoki T, Hammerling U, DeHarven E, Boyse EA, Old LJ (1969) Antigenic structure of cell surfaces. J Exp Med 130:979–1002
Baumeister W, Hahn M (1978) Specimen supports. In: Hayat MA (ed) Principles and techniques of electron microscopy, vol 8. Van Nostrand Reinhold, New York, pp 1–112
Knutson DW, Kijlstra A, Lentz H, van Es LA (1978) Isolation of stable aggregates of IgG by zonal ultracentrifugation in sucrose gradients containing protein. Immunol Commun 8:337–345
McConhahey PJ, Dixon FJ (1966) A method of trace iodination of protein for in vivo studies. Int Arch Allergy Appl Immunol 29:185–189
Otto H, Tahamiya H, Vogt A (1973) A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde. Comparison between the one-stage and the two-stage method. J Immunol Methods 3:137–146
Rudick RA, Herndon RM, Knutson DW (1982) Isolation and ultrastructural characteristics of model soluble immune complexes (ICx) using ferritin conjugates of monoclonal anti-human IgG (A-IgG-F). Clin Res 30 (2):356A
Schultz JS, Frelinger JA, Kim SK, Shreffler DC (1975) The distribution of Ia antigens on the H-2 complex on lymph node cells by immunoferritin labelling. Cell Immunol 16:125–134
Siess E, Wieland O, Miller F (1971) A simple method for the preparation of pure and active γ-globulin-ferritin conjugates using glutaraldehyde. Immunology 20:659–665
Singer SJ, Schick AF (1961) The properties of specific stains for electron microscopy prepared by conjugation of antibody molecules with ferritin. J Biophys Biochem Cytol 9:519–537
Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley & Sons, New York, pp 59–82
Striker GE, Donati EJ, Petrali JP, Sternberger LA (1966) Postembedding staining for electron microscopy with ferritin-antibody conjugates. Exp Mol (Suppl) 3:52–58
Author information
Authors and Affiliations
Additional information
Dr. Rudick is the recipient of Teacher-Investigator Development Award PHS 1 KO7 NS 00791-01
Rights and permissions
About this article
Cite this article
Rudick, R.A., Bloechl, E.K. & Knutson, D.W. Preparation of monoclonal antibody-ferritin conjugates of high specific activity. Histochemistry 80, 269–272 (1984). https://doi.org/10.1007/BF00495776
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00495776