Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA.
A partial cDNA clone (called BP cDNA) with coding sequences for the carboxy-terminal region of bullous pemphigoid (BP) antigen has been recently isolated and sequenced. In order to determine whether specific peptides encoded by the cDNA could be used to raise antibodies against BP antigen, fusion proteins derived from fragments of the BP cDNA and 17-mer or 19-mer synthetic peptides, corresponding to its deduced amino acid sequence, were used to generate rabbit antibodies. Three restriction enzyme fragments, 1179 bp (5' end), 264 bp (middle), and 546 bp (3' end), of the 1992 open reading frame (ORF) of BP cDNA were subcloned in frame into pEX plasmids to make beta-galactosidase fusion proteins FP1, FP2, and FP3, respectively. Fusion proteins of the predicted molecular weight, and which bound anti-beta-galactosidase antibodies, were produced, confirming the length of the predicted ORF. Rabbits immunized with FP1, but not FP3, produced antibodies, similar to authentic antibodies from BP patients, which: 1) bound the epidermal basement membrane at titers over 10,000, as determined by indirect immunofluorescence; 2) bound the basement membrane on the roof of 1 M NaCl-split skin; 3) immunoprecipitated the 230-kD BP antigen; and 4) bound the hemidesmosome, as determined by immunoelectron microscopy. Rabbits immunized with FP2 also produced lower titer BP-like antibodies. We further showed that short hydrophilic synthetic peptides, contained in FP1, could induce similar BP-like antibodies in rabbits at immunofluorescence titers up to 2560. These rabbit antibodies should prove useful for further studies on the function and structure of particular epitopes of BP antigen as well as on the pathophysiology of disease.
Tanaka
T
T
Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Korman
N J
NJ
Shimizu
H
H
Eady
R A
RA
Klaus-Kovtun
V
V
Cehrs
K
K
Stanley
J R
JR
D000818:Animals; D000906:Antibodies / Q000235:genetics*; D000917:Antibody Formation; D001426:Bacterial Proteins / Q000235:genetics; D004247:DNA / Q000276:immunology; D000939:Epitopes / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D008854:Microscopy, Electron; D010391:Pemphigoid, Bullous / Q000235:genetics / Q000276:immunology*; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012872:Skin Diseases, Vesiculobullous / Q000276:immunology*; D001616:beta-Galactosidase / Q000235:genetics
D000906:Antibodies; D001426:Bacterial Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D004247:DNA; D001616:beta-Galactosidase