Identification of Rectal Genglion Cells using Monoclonal Antibodies

Abstract

Many histopathologists are relunctant to make a diagnosis of Hirschsprung's disease on the basis of section rectal biopsies, possibly due both to doubt as to the amount of submucosa that must be scanned before absence of ganglion cells is indicative of aganglionosis and the relative difficulty of accurate identification of submucosal ganglia. The aim of this study was to produce a ganglion cell specific monoclonal antibody that could be used as a reliable marker for the detection of ganglion cells in suction rectal biopsy specimens. Balb/c mice were immunised with human lumbar sympathetic ganglia. Following fusion of mouse spleen cells with Sp2 myeloma cells, 704 hybrodomas were produced with 67 producing nonspecific antibodies and 4 hybridomas producing ganglion cell specific monoclonal antibody. All 4 hybridomes were re-cloned twice by limiting dilution and cells were stored in liquid nitrogen. One clone (F7) which gave the highest titre was subsequently passaged in mice to produce high titre ascites fluid monoclonal antibody. This antibody reacted with rectal ganglion cells from dog, rabbit and pig as well as from humans but was completely negative with a wide range of other human and animal tissues. Using indirect immunofluorescence this antibody stained brightly all ganglion cells in specimens of ganglionic bowel but was negative when reacted with rectal tissue from three patients with Hirschsprung's disease tested so far. This method provides a new and easy approach for the identification of ganglion cells in rectal biopsies in suspected cases of Hirschsprung's disease.