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. 1972 Jul;69(7):1771–1775. doi: 10.1073/pnas.69.7.1771

Solid-Phase Conjugation of Ferritin to Fab-Fragments of Immunoglobulin G for Use in Antigen Localization on Thin Sections

J P Kraehenbuhl 1, J D Jamieson 1
PMCID: PMC426799  PMID: 4505654

Abstract

A technique for the solid-phase conjugation of ferritin to monovalent antibodies (Fab-fragments of immunoglobulin G) is described in which Fab-fragments from sheep antiserum to rabbit Fab-fragments are first adsorbed to Sepharose containing insolubilized rabbit immunoglobulin G, and subsequently allowed to couple with ferritin previously activated with toluene-2,4-diisocyanate. The conjugate, after elution from the adsorbent, is separated from free ferritin and unconjugated Fab by gel chromatography. We have used this conjugate as a common second-step reagent for the surface localization of intracellular antigens on thin sections of tissue embedded in albumin. The first-step reagent consists of specific rabbit antibodies (IgG) directed against the tissue component under study. An example of the use of this technique for the localization of trypsinogen in bovine exocrine pancreatic cells is given.

Keywords: immuno-electron microscopy, protein embedment, affinity chromatography, trypsinogen, bovine pancreas

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Selected References

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

  1. BOREK F., SILVERSTEIN A. M. Characterization and purification of ferritin-antibody globulin conjugates. J Immunol. 1961 Nov;87:555–561. [PubMed] [Google Scholar]
  2. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  3. GREENE L. J., HIRS C. H., PALADE G. E. On the protein composition of bovine pancreatic zymogen granules. J Biol Chem. 1963 Jun;238:2054–2070. [PubMed] [Google Scholar]
  4. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol. 1967 Aug;34(2):577–596. doi: 10.1083/jcb.34.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. KELLER P. J., COHEN E. Enzymic composition of some cell fractions of bovine pancrease. J Biol Chem. 1961 May;236:1407–1413. [PubMed] [Google Scholar]
  6. Kawarai Y., Nakane P. K. Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method. J Histochem Cytochem. 1970 Mar;18(3):161–166. doi: 10.1177/18.3.161. [DOI] [PubMed] [Google Scholar]
  7. Kraehenbuhl J. P., De Grandi P. B., Campiche M. A. Ultrastructural localization of intracellular antigen using enzyme-labeled antibody fragments. J Cell Biol. 1971 Aug;50(2):432–445. doi: 10.1083/jcb.50.2.432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Leduc E. H., Scott G. B., Avrameas S. Ultrastructural localization of intracellular immune globulins in plasma cells and lymphoblasts by enzyme-labeled antibodies. J Histochem Cytochem. 1969 Apr;17(4):211–224. doi: 10.1177/17.4.211. [DOI] [PubMed] [Google Scholar]
  9. Mancini G., Carbonara A. O., Heremans J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 1965 Sep;2(3):235–254. doi: 10.1016/0019-2791(65)90004-2. [DOI] [PubMed] [Google Scholar]
  10. McLean J. D., Singer S. J. A general method for the specific staining of intracellular antigens with ferritin-antibody conjugates. Proc Natl Acad Sci U S A. 1970 Jan;65(1):122–128. doi: 10.1073/pnas.65.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nakane P. K., Pierce G. B., Jr Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol. 1967 May;33(2):307–318. doi: 10.1083/jcb.33.2.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nicolson G. L., Marchesi V. T., Singer S. J. The localization of spectrin on the inner surface of human red blood cell membranes by ferritin-conjugated antibodies. J Cell Biol. 1971 Oct;51(1):265–272. doi: 10.1083/jcb.51.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. PORTER R. R. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J. 1959 Sep;73:119–126. doi: 10.1042/bj0730119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. SCHICK A. F., SINGER S. J. On the formation of covalent linkages between two protein molecules. J Biol Chem. 1961 Sep;236:2477–2485. [PubMed] [Google Scholar]

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