Generation of monoclonal antibodies to involved ileum of Crohn's disease: Characterization of a panel of antibodies with Goblet cell membrane and brush border-specific reactivity

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Abstract

There are no known intestinal cell phenotypic markers characteristic of the transmural idiopathic inflammatory diseases of the small bowel. To address this issue, we developed monoclonal antibodies specific for Crohn's disease tissue by generating a library of hybridomas specific for intestine following murine immunization with intestinal epithelial cells from a patient with Crohn's disease. The epithelial cells were initially harvested from a fresh operative specimen by gentle scraping and digestion with 1% collagenase on ice. Cells were then washed, evaluated for viability, and polytron homogenized. After immunization and subsequent fusion, hybridoma cell lines producing distinct antibody-binding patterns were identified by indirect immunoepifluorescence (IIEF). Wells representing distinct patterns were subcloned and carried to limiting dilution. Of the multiple hybridomas studied, the predominant target antibodies were goblet cell and glycoprotein-like molecules. Patterns of antibody binding identified by IIEF included: brush border, goblet cell, surface glycoprotein, enterocyte membranes, basilar crypt inclusions, circumferential goblet cell, and a heterogeneous goblet cell glycoprotein pattern. Molecular weight determination and cross-reactivity with various human tissues were studied by immunoblotting following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Several hybridomas were specific for the intestine but were not specific for Crohn's disease.

Original languageEnglish (US)
Pages (from-to)935-942
Number of pages8
JournalAmerican Journal of Gastroenterology
Volume83
Issue number9
StatePublished - 1988

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Goblet Cells
Hybridomas
Microvilli
Ileum
Crohn Disease
Monoclonal Antibodies
Cell Membrane
Antibodies
Intestines
Immunization
Glycoproteins
Epithelial Cells
Basilar Membrane
Enterocytes
Membrane Glycoproteins
Ice
Inflammatory Bowel Diseases
Immunoblotting
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Gastroenterology

Cite this

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title = "Generation of monoclonal antibodies to involved ileum of Crohn's disease: Characterization of a panel of antibodies with Goblet cell membrane and brush border-specific reactivity",
abstract = "There are no known intestinal cell phenotypic markers characteristic of the transmural idiopathic inflammatory diseases of the small bowel. To address this issue, we developed monoclonal antibodies specific for Crohn's disease tissue by generating a library of hybridomas specific for intestine following murine immunization with intestinal epithelial cells from a patient with Crohn's disease. The epithelial cells were initially harvested from a fresh operative specimen by gentle scraping and digestion with 1{\%} collagenase on ice. Cells were then washed, evaluated for viability, and polytron homogenized. After immunization and subsequent fusion, hybridoma cell lines producing distinct antibody-binding patterns were identified by indirect immunoepifluorescence (IIEF). Wells representing distinct patterns were subcloned and carried to limiting dilution. Of the multiple hybridomas studied, the predominant target antibodies were goblet cell and glycoprotein-like molecules. Patterns of antibody binding identified by IIEF included: brush border, goblet cell, surface glycoprotein, enterocyte membranes, basilar crypt inclusions, circumferential goblet cell, and a heterogeneous goblet cell glycoprotein pattern. Molecular weight determination and cross-reactivity with various human tissues were studied by immunoblotting following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Several hybridomas were specific for the intestine but were not specific for Crohn's disease.",
author = "Prindiville, {Thomas P} and Cantrell, {M. C.} and Gershwin, {M. Eric}",
year = "1988",
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AU - Gershwin, M. Eric

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N2 - There are no known intestinal cell phenotypic markers characteristic of the transmural idiopathic inflammatory diseases of the small bowel. To address this issue, we developed monoclonal antibodies specific for Crohn's disease tissue by generating a library of hybridomas specific for intestine following murine immunization with intestinal epithelial cells from a patient with Crohn's disease. The epithelial cells were initially harvested from a fresh operative specimen by gentle scraping and digestion with 1% collagenase on ice. Cells were then washed, evaluated for viability, and polytron homogenized. After immunization and subsequent fusion, hybridoma cell lines producing distinct antibody-binding patterns were identified by indirect immunoepifluorescence (IIEF). Wells representing distinct patterns were subcloned and carried to limiting dilution. Of the multiple hybridomas studied, the predominant target antibodies were goblet cell and glycoprotein-like molecules. Patterns of antibody binding identified by IIEF included: brush border, goblet cell, surface glycoprotein, enterocyte membranes, basilar crypt inclusions, circumferential goblet cell, and a heterogeneous goblet cell glycoprotein pattern. Molecular weight determination and cross-reactivity with various human tissues were studied by immunoblotting following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Several hybridomas were specific for the intestine but were not specific for Crohn's disease.

AB - There are no known intestinal cell phenotypic markers characteristic of the transmural idiopathic inflammatory diseases of the small bowel. To address this issue, we developed monoclonal antibodies specific for Crohn's disease tissue by generating a library of hybridomas specific for intestine following murine immunization with intestinal epithelial cells from a patient with Crohn's disease. The epithelial cells were initially harvested from a fresh operative specimen by gentle scraping and digestion with 1% collagenase on ice. Cells were then washed, evaluated for viability, and polytron homogenized. After immunization and subsequent fusion, hybridoma cell lines producing distinct antibody-binding patterns were identified by indirect immunoepifluorescence (IIEF). Wells representing distinct patterns were subcloned and carried to limiting dilution. Of the multiple hybridomas studied, the predominant target antibodies were goblet cell and glycoprotein-like molecules. Patterns of antibody binding identified by IIEF included: brush border, goblet cell, surface glycoprotein, enterocyte membranes, basilar crypt inclusions, circumferential goblet cell, and a heterogeneous goblet cell glycoprotein pattern. Molecular weight determination and cross-reactivity with various human tissues were studied by immunoblotting following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Several hybridomas were specific for the intestine but were not specific for Crohn's disease.

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