Enzyme-linked coagulation assay: V. Amplified blotting assays using snake venom conjugates.
We have previously reported an ultrasensitive microtiter plate assay, enzyme-linked coagulation assay (ELCA), which can measure a factor X activator isolated from Russell's viper venom (RVV-XA) at concentrations less than 0.1 amol/sample. The high sensitivity of this assay is derived from enzyme amplification via the clotting cascade in combination with the utilization of enzyme-labeled solution-phase and unlabeled solid-phase fibrinogen. Modification of the ELCA assay to detect RVV-XA directly bound to nitrocellulose, ELCA blot, as described in this report, allowed the detection of blotted RVV-XA at amounts as low as 2 fg. The high sensitivity of the ELCA blot was utilized to develop an immunodetection system for Western blots, the ELCA immunoblot, and a biotin/avidin protein stain for blotted membranes, biotin/avidin ELCA blot. For the ELCA immunoblot, using RVV-XA-labeled antibodies we were able to detect blotted placental alkaline phosphatase at amounts two orders of magnitude lower than those when using peroxidase-labeled antibodies. Using an avidin-RVV-XA conjugate in the biotin/avidin ELCA blot, 1 ng of biotinylated fibrinogen and 100 pg of biotinylated placental alkaline phosphatase, which had been subjected to electrophoresis and transferred to a nitrocellulose membrane, were visualized. These data support the general utility of the ELCA system for assay amplification on solid-phase matrices and demonstrate considerable potential of this methodology in "blotting" applications.
Durkee
K H
KH
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
Cheng
T M
TM
Doellgast
G J
GJ
D000469:Alkaline Phosphatase / Q000032:analysis; D001360:Avidin; D001710:Biotin; D015153:Blotting, Western / Q000379:methods; D010450:Endopeptidases / Q000032:analysis* / Q000276:immunology; D005340:Fibrinogen / Q000032:analysis; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques; D008666:Metalloendopeptidases; D008832:Microchemistry
D001360:Avidin; D001710:Biotin; D005340:Fibrinogen; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D008666:Metalloendopeptidases; C033858:russellysin