Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in bruch's membrane of the aging eye

Behnom Farboud, Amy Aotaki-Keen, Toshio Miyata, Leonard M Hjelmeland, James T. Handa

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Purpose: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. Methods: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. Results: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. Conclusions: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

Original languageEnglish (US)
JournalMolecular Vision
Volume5
Issue number11
StatePublished - 1999

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Bruch Membrane
Epitopes
Antibodies
Bovine Serum Albumin
High Pressure Liquid Chromatography
Staining and Labeling
Extracellular Matrix
pentosidine
Rabbits
glycolaldehyde

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in bruch's membrane of the aging eye. / Farboud, Behnom; Aotaki-Keen, Amy; Miyata, Toshio; Hjelmeland, Leonard M; Handa, James T.

In: Molecular Vision, Vol. 5, No. 11, 1999.

Research output: Contribution to journalArticle

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abstract = "Purpose: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. Methods: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. Results: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. Conclusions: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.",
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T1 - Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in bruch's membrane of the aging eye

AU - Farboud, Behnom

AU - Aotaki-Keen, Amy

AU - Miyata, Toshio

AU - Hjelmeland, Leonard M

AU - Handa, James T.

PY - 1999

Y1 - 1999

N2 - Purpose: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. Methods: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. Results: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. Conclusions: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

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