Enzyme activity amplification method for increasing assay sensit

Details Enzyme activity amplification method for increasing assay sensit Enzyme activity amplification method for increasing assay sensit

1986-04-15

1990-06-26

Naff, David M.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

435 4, 435 7, 435174, 435177, 435181, 435288, C12P 100, G01N 33535, C12N 1100, C12N 1106

Patent

active

049371886

ABSTRACT:
Enzyme amplification is achieved by covalently bonding enzyme to a supporting material via a molecular chain which is a substrate for the enzyme, then introducing a small amount of enzyme in the free state to this system, causing release of a large amount of bound enzyme. In an alternative embodiment, complementary enzymatically inactive fragments of an active enzyme, which fragments can recombine to form active enzyme, are covalently attached to separate support materials by a molecular chain material which is a substrate for the active enzyme, and these two fragment-support conjugates are connected in series. Upon application of free enzyme or free complementary enzyme to one of these fragment-support conjugates, followed by application of the resulting product mixture to the second fragment-support conjugate, a large amount of free enzyme is ultimately produced. In a second alternative embodiment, two different active enzymes are each attached to separate supporting materials by different leashes, in which the leash for the first enzyme only is cleaved in the system by the second enzyme, and the leash for the second enzyme only is cleaved in the system by the first enzyme. These two materials are connected in series, and upon application of the second enzyme to the first enzyme-support conjugate, followed by application of the released first enzyme to the second enzyme-support conjgate material, ultimately a large amount of released active second enzyme is produced. The amplification of enzyme activity has uses in analytical chemistry such as to increase sensitivity of a standard immunoassay.

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