HACH - A POLYMER DESIGNED TO OPTIMIZE PROTEIN ANTIGEN LOCALIZATION

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 r etention. Such a polymer, HACH, was made up from a mixture of 2-hydrox yhexanedial and carbohydrazide. It polymerized spontaneously at room t emperature within 24 hr. Preservation of protein antigenicity and subs equent immunocytochemical localization were demonstrated by three meth ods. To determine whether protein antigens were retained up to the pol ymerization stage, we studied hemoagglutination of red blood cells usi ng antibodies directed against their protein antigens. In these trials , HACH-treated cells exhibited the same agglutination responses as con trol, untreated cells. Second, a guinea pig antibody was used to immun odecorate insulin in beta cells of the islets of Langerhans. The numbe r of gold particles, indicating sires where the antibody was bound, wa s several-fold greater in HACH- than in Lowicryl K4M-embedded pancreat ic beta cells. To assess the limit of detection of protein antigens in thin sections, an example of a protein present in mitochondria, lipoa mide dehydrogenase, was also studied. An indirect procedure for immuno decoration, employing rabbit immunoglobulin G followed by gold-tagged secondary antibody, indicated that the enzyme was present at several s ites 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.