HACH: A Polymer Designed to Optimize Protein Antigen Localization

Abstract

Abstract: The purpose of this study was to determine whether a polymer could be formed from relatively innocuous monomeric ingredients and, if so, if it might serve as a suitable embedding medium for maximizing antigen retention. Such a polymer, HACH, was made up from a mixture of 2-hydroxyhexanedial and carbohydrazide. It polymerized spontaneously at room temperature within 24 hr. Preservation of protein antigenicity and subsequent immunocytochemical localization were demonstrated by three methods. To determine whether protein antigens were retained up to the polymerization stage, we studied hemoagglutination of red blood cells using antibodies directed against their protein antigens. In these trials, HACH-treated cells exhibited the same agglutination responses as control, untreated cells. Second, a guinea pig antibody was used to immunodecorate insulin in β cells of the islets of Langerhans. The number of gold particles, indicating sites where the antibody was bound, was several-fold greater in HACH- than in Lowicryl K4M-embedded pancreatic β cells. To assess the limit of detection of protein antigens in thin sections, an example of a protein present in mitochondria, lipoamide dehydrogenase, was also studied. An indirect procedure for immunodecoration, employing rabbit immunoglobulin G followed by gold-tagged secondary antibody, indicated that the enzyme was present at several sites within cross-sectioned mitochondria. The results suggest that the HACH polymer will be useful for the localization of antigens that are present in relatively few copies.