Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-24
2004-12-28
Huff, Sheela J. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007900, C435S007920, C436S064000, C436S503000
Reexamination Certificate
active
06835549
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to an in vitro immunoassay method for diagnosing human gastric intestinal metaplasia which comprises the steps of (a) contacting a gastric tissue sample of a subject suspected of having human gastric intestinal metaplasia cells with the monoclonal antibody DAS-1, or a fragment thereof, which monoclonal antibody is produced by the hybridoma deposited under ATCC accession number HB 9397 and which reacts with human gastric intestinal metaplasia antigen; and (b) detecting immunoreactivity between the gastric tissue and the monoclonal antibody, such immunoreactivity indicating a positive diagnosis of human gastric intestinal metaplasia. This invention also pertains to an in vivo immunoassay method for diagnosing human gastric intestinal metaplasia which comprises the steps of (a) administering to a human, suspected of having human gastric intestinal metaplasia, the monoclonal antibody DAS-1, or a fragment thereof, which monoclonal antibody is produced by the hybridoma deposited under ATCC accession number HB 9397 and which reacts with human gastric intestinal metaplasia antigen and is tagged with an isotope; and (b) detecting immunoreactivity between the human gastric intestinal metaplasia cells and the monoclonal antibody by external scanning, such immunoreactivity indicating a positive diagnosis of human gastric intestinal metaplasia.
2. Description of the Background
Gastric carcinoma remains the second most common cause of cancer deaths in the world. In some of the developing countries, gastric carcinoma is the leading cause of human cancer. The cause of this increase, particularly in the developing countries, is not clear, however,
Helicobacter pylori
(
H. Pylori
) infection has been considered to be a risk factor for gastric carcinoma by the World Health Organization on the basis of several studies all over the world (Parsonnet et al., New Engl J Med 325:1127-1131 (1991)). It is well established that gastric intestinal metaplasia is a pre-cancerous condition, however, it is not known whether there is a sub-group of patients with gastric intestinal metaplasia who are more prone to cancer. Some literature suggests that gastric intestinal metaplasia with colonic phenotype of metaplasia changes is more prone to cancer and other literature suggests that small intestinal metaplastic changes are more prone to cancer.
In addition to the fully developed intestinal metaplasia, cellular variants suggesting partial or incomplete forms of metaplasia were observed in both the benign and the malignant cells, Goldman et al., Laboratory Investigation, 18, 203-210 (1968). The tumor cells exhibited alterations in the surface microvilli, terminal webs, mucin granules, plasma membranes, and nuclei, as well as the presence of large lysosomal structures. A correlative study between the morphology and the staining reactions of the mucin granules in 1 u sections was undertaken. In the normal foveolar and surface mucous cells, these granules were dark and small and stained strongly with periodic acid, Schiff only, whereas the pale granules in the mature goblet cells reacted with Alcian Blue as well. No such correlation could be made in the benign cells with partial metaplasia and in the tumor cells. The metaplastic cells however resembled those seen in the small intestine and paneth cells were present as well. Earlier studies using various enzyme histochemistry also suggested that metaplasia associated with carcinoma are identical to small intestine. Planteydt et al. J. Path. Bact. 80:317-323 (1960)).
Gastric atrophy and intestinal metaplasia are considered the earliest phenotypic changes in the cascade of events leading from normal mucosa to intestinal-type gastric cancer, and epidemiological evidence links
Helicobacter pylori
to gastric epithelial malignancies. Rugge et al., Digestive Diseases and Sciences, 41, 950-955 (1996). To evaluate any causal relationship between bacterial infection and atrophic metaplastic lesions, gastric pathology was histologically and histochemically evaluated in 267 consecutive, non-ulcerous, untreated subjects, with attention given to the phenotypes of intestinal metaplasia. The prevalence of
Helicobacter pylori
infection was 61%. Intestinal metaplasia (particularly types II and III), suggestive of colonic phenotype, was significantly associated with both
Helicobacter pylori
detection and increasing age. The development of intestinal metaplasia proved more significantly linked with
Helicobacter pylori
infection than with age with no interaction.
Helicobacter pylori
can be considered among the major causal agents of mucosal lesions involved in the multistep process of gastric carcinogenesis justifying an attempt to eradicate this bacterial infection.
Using hybridoma technology, a monoclonal antibody (7E
12
H
12
, IgM isotype) was developed against a colon epithelial antigen associated with ulcerative colitis (Das et al.,
J. Immunol.
139:77, 1987). The monoclonal antibody 7E
12
H
12
binds specifically to colonocytes along baso-lateral and brush border areas and it does not react with 13 other epithelial organs or any other parts of the gastrointestinal tract including the esophagus, stomach, and small intestine.
The colon epithial specific protein (CEP) recognized by 7E
12
H
12
mAb was predominantly localized at the plasma membrane in the apical (brush border area) and basolaceral domains of colonocytes (Das, K. M. et al.,
J. Immunol.
1987;139:77-84). Using two and three color immunofluorescence assay (Halstensen, T. S. et al.,
Gut
1993;34:650-657), the 7E
12
H
12
reactive epitope was also localized exclusively in colonic enterocytes, but not in small intestinal enterocytes, with increasing intensity caudally, expanding to intense cytoplasmic expression in the rectum. However, the mAb DAS-1 antibody has been shown to react with several precancerous conditions such as Barrett's Epithelium and chronic cystitis profunda, U.S. Pat. No. 5,888,743, Pantuck et al., J. Urol., 158, 1722 (1977). However, the mAb DAS-1 antibody does not react with normal gastroesophageal mucosa and normal urinary bladder but does react with adenocarcinoma from Barrett's Epithelium and urinary bladder carcinoma.
SUMMARY OF THE INVENTION
The present invention pertains to an in vitro immunoassay method for diagnosing human gastric intestinal metaplasia which comprises the steps of:
(a) contacting a gastric tissue sample of a subject suspected of having human gastric intestinal metaplasia cells with the monoclonal antibody DAS-1, or a fragment thereof, which monoclonal antibody is produced by the hybridoma deposited under ATCC accession number HB 9397 and which reacts with human gastric intestinal metaplasia antigen; and
(b) detecting immunoreactivity between the gastric tissue and the monoclonal antibody, such immunoreactivity indicating a positive diagnosis of human gastric intestinal metaplasia.
Preferably, the human gastric intestinal metaplasia antigen is colon epithial specific protein.
Preferably, the antibody or fragment is directly or indirectly attached to a detectable label. Preferably detecting immunoreactivity is performed by immunoperoxidase staining, immunofluorescence, immunoelectronmicroscopy, or ELISA, and more preferably the immunoassay method is immunoperoxidase staining. Most preferably, the immunoperoxidase staining comprises (a) deparaffinizing the intestinal tissue by heating; (b) immersing the deparaffinized tissue in xylene; (c) rehydrating the tissue in decreasing concentrations of alcohol; (d) washing the rehydrated tissue in neutral PBS; (e) reducing the aldehydes of the washed tissue of step (d); (f) reacting the tissue with normal goat serum, the monoclonal antibody, biotinylated goat anti-mouse antibody and avidin-biotin-peroxidase complex; (g) treating the reacted tissue with diaminobenzidine; (h) washing the diaminobenzidine-treated tissue; (i) staining the washed tissue of step (h) with hematoxylin, eosin or both; and (j) examining the stained tissue under a mi
Huff Sheela J.
Licata & Tyrrell P.C.
University of Medicine & Dentistry of New Jersey
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