Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods.
Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.
Kohno
T
T
Department of Biochemistry, Medical College of Miyazaki.
Yamaguchi
K
K
Ishikawa
E
E
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000276:immunology*; D000818:Animals; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D004136:Dinitrobenzenes / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis*; D008969:Molecular Sequence Data; D011137:Polystyrenes; D011817:Rabbits; D012710:Serum Albumin, Bovine; D001616:beta-Galactosidase / Q000378:metabolism
D000936:Antigen-Antibody Complex; D004136:Dinitrobenzenes; D007074:Immunoglobulin G; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; D000803:Angiotensin I; D001616:beta-Galactosidase