ABSTRACT
RVV-X, the factor X activator from Russell's viper venom, has been isolated using affinity chromatography on agarose columns of a monoclonal antibody specific for this enzyme. Upon testing acid, alkaline, and high concentrations of MgCl2 for elution, it was found that use of high concentrations of MgCl2 was most effective in elution of RVV-X. It was nondenaturing and yielded 90% recovery of homogeneous enzyme without measurable contamination by other proteins of the venom.
Subject(s)
Chromatography, Affinity/methods , Endopeptidases/isolation & purification , Magnesium Chloride , Metalloendopeptidases , Viper Venoms/enzymology , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Electrophoresis, Polyacrylamide Gel , Endopeptidases/immunology , Hydrogen-Ion Concentration , Immunosorbent Techniques , Mice , Mice, Inbred BALB C/immunology , Molecular WeightABSTRACT
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.
Subject(s)
Endopeptidases/analysis , Immunoblotting/methods , Metalloendopeptidases , Alkaline Phosphatase/analysis , Avidin , Biotin , Blotting, Western/methods , Endopeptidases/immunology , Fibrinogen/analysis , Humans , Immunoenzyme Techniques , MicrochemistryABSTRACT
To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently. The substance P level in lumbar spinal ganglia remained fairly constant, while the level of SP from cervical and thoracic regions declined significantly. At the maximal depletion dosage (173 micrograms of capsaicin/kg), SP concentration decreased 72.3% in cervical spinal ganglia, 45.5% in thoracic ganglia and 56.1% in the atrium. The lack of SP depletion in lumbar ganglia indicates that capsaicin acted locally on cardiac afferents rather than systemically. Data from this study suggest that capsaicin-sensitive neurons of the heart have cell bodies in the lower cervical spinal ganglia as well as in the upper thoracic spinal ganglia.
