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

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.