Development of an avidin-biotin competitive inhibition assay and validation of its use for the quantitation of human intervertebral disc serine proteinase inhibitory proteins.
A simple convenient method has been developed for the quantitation of serine proteinase inhibitors (SPIs) in tissue extracts. The method is based on the competitive binding to trypsin and chymotrypsin immobilized using glutaraldehyde on 96-well microtiter plate wells of native SPIs and a biotinylated secretory proteinase inhibitor (SLPI) standard. The bound SLPI standard was visualized using an avidin-alkaline phosphatase conjugate and inhibition curves were determined using absorbancy measurements at 405 nm. The standard assay had a range between 0.02 and 1 microgram SLPI/well and a lower detection limit of 20 ng SLPI/well; an improved microassay had a detection limit of 2 ng SLPI/well. Only active free inhibitor was detected in the assay since denatured and/or enzyme-inhibitor complexes did not bind to the plates. A range of SPI species was demonstrable in human bronchial mucus and intervertebral disc SPI samples using this technique. Quantitation of SPI levels in a number of intervertebral disc samples indicated that the SPIs were depleted in degenerate discs compared to nondegenerate discs (P less than 0.05, n = 12). Since the immobilized trypsin and chymotrypsin microplates used in this assay may be prepared in advance (and are stable at 4 degrees C for at least 1 month) the remaining two steps of the assay (the inhibition step and visualization) may be completed in 2-3 h; thus the assay is simple, convenient, and fast. All reagents (other than the biotinylated SLPI standard) are readily available commercially, and in principle the assay could be adapted to other systems provided defined biotinylated standards were available.
Melrose
J
J
Raymond Purves Research Laboratories, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, New South Wales, Australia.
Ghosh
P
P
D001360:Avidin; D001667:Binding, Competitive; D001710:Biotin; D002918:Chymotrypsin / Q000737:chemistry; D003238:Connective Tissue / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007403:Intervertebral Disc / Q000201:enzymology*; D009093:Mucus / Q000201:enzymology; D015842:Serine Proteinase Inhibitors / Q000032:analysis*; D014357:Trypsin / Q000737:chemistry
D004800:Enzymes, Immobilized; D015842:Serine Proteinase Inhibitors; D001360:Avidin; D001710:Biotin; D002918:Chymotrypsin; D014357:Trypsin; D005976:Glutaral