# PURIFICATION OF EPIDERMOLYSIS BULLOSA ACQUISITA ANTIGEN

> **NIH NIH R01** · STANFORD UNIVERSITY · 1990 · $196,833

## Abstract

Epidermolysis bullosa acquisita (EBA) is a severe blistering skin
disease in which the epidermis separates from the dermis through
the basement membrane zone (BMZ).  Blister formation within the
BMZ begins beneath the lamina densa where autoantibodies are
deposited.  Using autoantibodies from EBA patients' sera, we
identified the EBA antigen as a large matrix molecule of 290,000
daltons that is distinct from bullous pemphigoid antigen, laminin,
elastin, fibronectin and collagens I, IV and V.  Our current goal is
to purify and further characterize the EBA antigen using both
conventional protein chemistry and molecular biology.  0.4 mm
keratotomed slices of human skin will be de-epidermized in 1M
salt at 4 degrees C for 96 hours, the epidermis discarded and the
dermal BMZ extracted in 6M GuHC1, 0.05 M Tris-HC1, pH 9.2,
0.002 EDTA, with reducing agents.  Proteins below 100 kd will be
removed by dialysis and ultrafiltration.  Collagens will be
separated from EBA antigen by chromatography on a phenyl
boronate affinity column.  EBA antigen will be further purified by
DEAE ion exchange and molecular sieve chromatography.  If
further purification is needed, hydroxyapatite chromatography,
affinity chromatography on a fibronectin column and an anti-EBA
antigen monoclonal antibody column will be attempted.  An ELISA
assay will be developed for rapid antigen and antibody detection.
Purified EBA antigen will be characterized by isoelectric
focusing, protease mapping, glyconse mapping, hexosamine
determination, amino acid analysis, molecular sieve
chromatography for native size, and rotart shadowing for
molecular shape.  In parallel, EBA antigen purification and
characterization will be attempted by recombinant DNA
technology:  antigen blotted to activated glass filters will be
placed in a gas-phase microsequanator.  With an amino acid
sequence, a corresponding balanced oligonucleotide probe will be
synthesized and labeled and a human skin fibroblast cDNA library
will be screened.  Probe positive clones will be cultured and DNA
purified.  Positive identification of the clones will include
transcribing RNA from the positive cDNA clones or isolating it by
positive selection.  This newly synthesized mRNA will be used in a
rabbit reticulocyte cell-free translation system to make product
(EBA antigen) which will be verified by SDSPAGE, blotting,
immunoprecipitation and amino acid sequencing.

## Key facts

- **NIH application ID:** 3156599
- **Project number:** 5R01AR033625-08
- **Recipient organization:** STANFORD UNIVERSITY
- **SAM.gov UEI:** HJD6G4D6TJY5
- **Principal Investigator:** DAVID T. WOODLEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1990
- **Award amount:** $196,833
- **Award type:** 5
- **Project period:** 1990-03-01 → 1992-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3156599

## Citation

> US National Institutes of Health, RePORTER application 3156599, PURIFICATION OF EPIDERMOLYSIS BULLOSA ACQUISITA ANTIGEN (5R01AR033625-08). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3156599. Licensed CC0.

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