Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein A and neomycin phosphotransferase II in two-chamber-well microtiter plates.
A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [gamma-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10 microliter-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen.
Hunger
H D
HD
Academy of Sciences of the GDR, Department of Human Molecular Genetics, Berlin, German Democratic Republic.
Flachmeier
C
C
Schmidt
G
G
Behrendt
G
G
Coutelle
C
C
D000818:Animals; D000941:Antigens / Q000032:analysis; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019868:Kanamycin Kinase; D009355:Neomycin / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011137:Polystyrenes; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012709:Serum Albumin / Q000032:analysis; D013205:Staphylococcal Protein A / Q000235:genetics
D000941:Antigens; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D013205:Staphylococcal Protein A; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D009355:Neomycin