US4463090A — Cascade amplification enzyme immunoassay

Claim 1

Independent Claim 248 words

In an enzyme immunoassay wherein a fully active enzyme ligand is coupled to an immuno-reactive substance which will react with an assayed substance when said assayed substance is present, and a substrate for the fully active enzyme is provided, which substrate can be catalyzed by said enzyme only if such reaction has occurred, the improvement wherein a three-stage cascade assay is performed by: using either one of the complement pair of (i) a second proenzyme, or (ii) a second activator for the second proenzyme, as the coupled ligand instead of the fully active enzyme; inducing a third-stage catalysis by providing the remaining complementary second proenzyme or second activator so as to afford a catalytic activation of the second proenzyme to a second enzyme when the assayed substance is present; inducing a second-stage catalysis when the assayed substance is present by providing a first proenzyme capable of being catalytically activated to a first enzyme by the second enzyme; inducing a first-stage catalysis when the assayed substance is present by providing a substrate responsive to the first enzyme; and measuring the change in said substrate when the assayed substance is present; wherein each proenzyme is selected from one of the group consisting of inactive or less active forms of enzymes which are transferases or hydrolases, or active forms of any enzymes encapsulated in liposomes, whereby the sensitivity of the assay is geometrically increased by the multiplication of the third-stage turnover rate, the second-stage turnover rate, and the first-stage turnover rate.

Claim 2

Dependent Claim 248 words

In an enzyme immunoassay wherein a fully active enzyme ligand is coupled to an immuno-reactive substance which will react with an assayed substance when said assayed substance is present, and a substrate for the fully active enzyme is provided, which substrate can be catalyzed by said enzyme only if such reaction has occurred, the improvement wherein a three-stage cascade assay is performed by: using either one of the complement pair of (i) a second proenzyme, or (ii) a second activator for the second proenzyme, as the coupled ligand instead of the fully active enzyme; inducing a third-stage catalysis by providing the remaining complementary second proenzyme or second activator so as to afford a catalytic activation of the second proenzyme to a second enzyme when the assayed substance is present; inducing a second-stage catalysis when the assayed substance is present by providing a first proenzyme capable of being catalytically activated to a first enzyme by the second enzyme; inducing a first-stage catalysis when the assayed substance is present by providing a substrate responsive to the first enzyme; and measuring the change in said substrate when the assayed substance is present; wherein each proenzyme is selected from one of the group consisting of inactive or less active forms of enzymes which are transferases or hydrolases, or active forms of any enzymes encapsulated in liposomes, whereby the sensitivity of the assay is geometrically increased by the multiplication of the third-stage turnover rate, the second-stage turnover rate, and the first-stage turnover rate.

Claim 3

Dependent Claim 17 words

The improvement of claim 1 wherein the coupled ligand is the second activator for the second proenzyme.

Claim 4

Dependent Claim 133 words

The improvement of claim 1 wherein a four-stage cascade assay is performed by additionally: using either one of the complement pair of (i) a third proenzyme, or (ii) a third activator for the third proenzyme, as the coupled ligand instead of the fully active enzyme, inducing a fourth-stage catalysis by providing the remaining complementary third enzyme or third activator so as to afford a catalytic activation of the third proenzyme to a third enzyme when the assayed substance is present; and inducing the third-stage catalysis by providing a second proenzyme capable of being catalytically activated to the second enzyme by the third enzyme; whereby the sensitivity of the assay is geometrically increased by the multiplication of the fourth-stage turnover rate, the third-stage turnover rate, the second-stage turnover rate, and the first-stage turnover rate.

Claim 5

Dependent Claim 17 words

The improvement of claim 3 wherein the coupled ligand is the third activator for the third proenzyme.

Claim 6

Dependent Claim 23 words

The improvement of claim 1 or 3 wherein the hydrolases are peptide hydrolases and the enzyme encapsulated in a liposome is an oxidoreductase.

Claim 7

Dependent Claim 15 words

The improvement of claim 5 wherein the peptide hydrolases are serine proteases or carboxyl proteases.

Claim 8

Dependent Claim 93 words

The assay of claim 1 wherein: the second proenzyme is pepsinogen A, B, or C; the second activator is carbonic anhydrase or a substance which acidifies the assay; the second enzyme is pepsin A, B, or C which acts as the first activator; the first proenzyme is chymotrypsinogen A, B, or C; the first enzyme is chymotrypsin A, B, or C; and the substrate is an aminoacyl ester or an aminoacyl amide, or is N-tosyl-L-leucine chlormethyl ketone if the first enzyme is chymotrypsin C, or any of these substrates with a marking label.

Claim 9

Dependent Claim 29 words

The assay of claim 7 wherein the aminoacyl ester or aminoacyl amide substrate is: N-acetyl-L-tryptoplasmin amide; N-benzyloxy carboxyl-L-tyrosine-p-nitrophenyl ester; 7-glutarylphenylalanineamido-4-methylcoumarin; or any of these substrates with a marking label.

Claim 10

Dependent Claim 82 words

The assay of claim 1 wherein: the second proenzyme is trypsinogen; the second activator is enterokinase, trypsinogen kinase, or peptidase A; the second enzyme is trypsin which acts as the first activator; the first proenzyme is chymotrypsinogen A, B, or C; the first enzyme is chymotrypsin A, B, or C; and the subtrate is an aminoacyl ester or an aminoacyl amide, or is N-tosyl-L-leucine chlormethyl ketone if the first enzyme is chymotrypsin C, or any of these substrates with a marking label.

Claim 11

Dependent Claim 29 words

The assay of claim 9 wherein the aminoacyl ester or aminoacyl amide substrate is: N-acetyl-L-tryptoplasmin amide; N-benzyloxy carboxyl-L-tyrosine-p-nitrophenyl ester; 7-glutarylphenylalanineamido-4-methyleoumarin; or any of these substrates with a marking label.

Claim 12

Dependent Claim 68 words

The assay of claim 1 wherein: the second proenzyme is plasminogen; the second activator is streptokinase, urokinase, staphylokinase, cytofibrokinase, or plasmakinase; the second enzyme is plasmin which acts as the first activator; the substrate is fibrinogen with or without a marking label; the cleaved substrate product is fibrin with or without a marking label; and the amount of fibrin is measured without the use of a further substrate.

Claim 13

Dependent Claim 67 words

The assay of claim 1 wherein: the second proenzyme is prothrombin; the second activator is thrombokinase or prothrombin-activating proteinase; the second enzyme is thrombin which acts as the first activator; the substrate is fibrinogen with or without a marking label; the cleaved substrate product is fibrin with or without a marking label; and the amount of the fibrin is measured without the use of a further substrate.

Claim 14

Dependent Claim 63 words

The assay of claim 1 wherein: the second proenzyme is prokallikrein; the second activator is Factor XIIa; the second enzyme is kallikrein which acts as the first activator; the first proenzyme is kininogen with or without a marking label; the first enzyme is kinin with or without a marking label; and the amount of kinin is measured without the use of a substrate.

Claim 15

Dependent Claim 55 words

The assay of claim 1 wherein: the second proenzyme is inactive phosphorylase kinase; the second activator is protein kinase; the second enzyme is activated phosphorylase kinase which acts as the first activator; the first proenzyme is phosphorylase b; the first enzyme is phosphorylase a; and the substrate is glycogen with or without a marking label.

Claim 16

Dependent Claim 56 words

The assay of claim 1 wherein: the second proenzyme is inactive angiotensinogen; the second activator is renin; the second enzyme is partially active angiotensin I which acts as the first proenzyme; the first activator is dipeptidyl carboxypeptidase; the first enzyme is fully activated angiotensin II; and the substrate is kaolin with or without a marking label.

Claim 17

Dependent Claim 186 words

A cascade amplification enzyme immunoassay kit for performing the improved assay of claim 1 consisting essentially of: (a) an immuno-reactive substance which will react with an assayed substance when said substance is present, said immuno-reactive substance having a coupled ligand which is either one of the complement pair of (1) a second proenzyme capable of activation to a second enzyme, or (2) a second activator for the second proenzyme; (b) the uncoupled remaining member of the complement pair; (c) an uncoupled first proenzyme capable of being catalytically activated to a first enzyme by the second enzyme; and (d) a substrate capable of being catalytically acted upon by the first enzyme; each reagent being present in an amount sufficient to perform at least one assay, so that if the assayed substance is present the immuno-reactive substance with the coupled ligand will bind to it and also be present, the second proenzyme will become catalytically activated to a second enzyme, the second enzyme will catalytically activate the first proenzyme to a first enzyme, and the first enzyme will catalytically act upon the substrate, thereby effecting a three-fold cascade.

Claim 18

Dependent Claim 14 words

The improved kit of claim 16 wherein the coupled ligand is the second activator.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1987-07-31	1988-01-30	1988-07-31	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1991-07-31	1992-01-30	1992-07-31	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1995-07-31	1996-01-30	1996-07-31	$7700.00	Window Closed / Lapsed

Cascade amplification enzyme immunoassay

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