Spontaneously beating neonatal rat heart myocyte culture-a model to characterize angiotensin II at(1) receptor autoantibodies in patients with preeclampsia.
This report focuses on angiotensin II AT(1) receptor autoantibodies (anti-AT(1)-AABs) in preeclamptic women. An enzyme-linked immunosorbent assay was described. Biotinylated peptide was incubated with anti-AT(1)-AABs. Streptavidin-coated magnetic particles bind the protein-autoantibody complex. Detection of anti-AT(1)-AABs was performed using anti-human IgG3 peroxidase-coupled antibody. The color reaction of tetramethylbenzidine solution was stopped by adding 0.5 M H(2)SO(4). Optical density was measured at 450 nm (620 nm reference filter). Seventy-nine percent of anti-AT(1)-AAB-positive patients (measured by bioassay) showed an increase in optical density (>145%). The same biotinylated peptide was successfully used for purification of 6/6 anti-AT(1)-AABs. Chronotropic effects of purified antibodies were registered on primary cultured neonatal rat cardiomyocytes with the computer imaging system IMAGOQUANT. Western blot of coimmunoprecipitation of angiotensin II AT(1) receptor shows one band (molecular weight >40.0 kDa) in potassium thiocyanate eluate.
Wallukat
Gerd
G
Max Delbrück Center, Immunology of Cardiovascular Diseases, Robert-Rössle-Str. 10, D-13092 Berlin, Germany.
Nissen
Eberhard
E
Neichel
Dajana
D
Harris
Jason
J
D000818:Animals; D000831:Animals, Newborn; D001323:Autoantibodies / Q000032:analysis* / Q000276:immunology*; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000276:immunology*; D011225:Pre-Eclampsia / Q000276:immunology*; D011247:Pregnancy; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000276:immunology*
D001323:Autoantibodies; D044140:Receptor, Angiotensin, Type 1