Development of a novel substrate capture immunoassay for the detection of a neutral metalloproteinase capable of degrading basement membrane (type IV) collagen.
This paper describes the development of a modified substrate capture immunoassay useful in the detection of a neutral metalloproteinase enzyme, type IV collagenase. Capture and immobilization of this enzyme was achieved by coating the microtiter plate with gelatin, an alternative substrate for this enzyme. The captured enzyme could then be detected by affinity-purified rabbit anti-type IV collagenase antibodies prepared against synthetic peptides from the amino terminus of the enzyme. Soluble type IV collagenase was readily detected in samples known to contain this enzyme. Using purified enzyme this assay method was shown to detect less than 50 ng of latent type IV collagenase. Using EDTA, an inhibitor of this metalloproteinase, gelatin binding was shown to be independent of catalytic activity.
Wacher
M P
MP
Tumor Invasion and Metastasis Section, National Cancer Institute, Bethesda, MD 20892.
Krutzsch
H C
HC
Liotta
L A
LA
Stetler-Stevenson
W G
WG
D000595:Amino Acid Sequence; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases; D004492:Edetic Acid; D004792:Enzyme Precursors / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D003012:Microbial Collagenase / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D011817:Rabbits; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D003094:Collagen; D004492:Edetic Acid; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase