New, sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization.
A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 x 10(-21) mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.
Hauber
R
R
Abteilung für Klinische Chemie, Universität München, FRG.
Miska
W
W
Schleinkofer
L
L
Geiger
R
R
D000469:Alkaline Phosphatase; D015153:Blotting, Western / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D015342:DNA Probes; D049454:Firefly Luciferin; D008156:Luciferases; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D011506:Proteins / Q000302:isolation & purification*
D015342:DNA Probes; D011506:Proteins; D049454:Firefly Luciferin; D004247:DNA; D008156:Luciferases; D000469:Alkaline Phosphatase