US5320944A — Immunoassay using magnetic particle

Claim 1

Independent Claim 174 words

An immunoassay method comprising the steps of: (a) reacting a sample containing an antigen with an antibody which specifically binds to said antigen, so as to bind said antigen to said antibody, wherein said antibody is immobilized on a 0.2 to 3.0 μm magnetic particle comprising a core and a coating layer on the surface of said core, wherein said core comprises an organic polymer and said coating layer comprises a metal oxide formed by adding ferrous ions or ferrous ions and an ion selected from the group consisting of zinc, cobalt, nickel, manganese, copper, vanadium, antimony, lithium, molybdenum, titanium, rubidium, aluminum, silicon, chromium, tin, calcium, cadmium, indium ions and mixtures thereof to deoxidized water in which cores of organic polymer are suspended to form a resulting solution, and subsequently adding an oxidizing agent solution to the resulting solution to provide a ferrite coating on said core; (b) measuring an amount of bound antigen; and (c) correlating the amount of bound antigen obtained in step (b) with an amount of antigen in the sample.

Claim 2

Dependent Claim 174 words

An immunoassay method comprising the steps of: (a) reacting a sample containing an antigen with an antibody which specifically binds to said antigen, so as to bind said antigen to said antibody, wherein said antibody is immobilized on a 0.2 to 3.0 μm magnetic particle comprising a core and a coating layer on the surface of said core, wherein said core comprises an organic polymer and said coating layer comprises a metal oxide formed by adding ferrous ions or ferrous ions and an ion selected from the group consisting of zinc, cobalt, nickel, manganese, copper, vanadium, antimony, lithium, molybdenum, titanium, rubidium, aluminum, silicon, chromium, tin, calcium, cadmium, indium ions and mixtures thereof to deoxidized water in which cores of organic polymer are suspended to form a resulting solution, and subsequently adding an oxidizing agent solution to the resulting solution to provide a ferrite coating on said core; (b) measuring an amount of bound antigen; and (c) correlating the amount of bound antigen obtained in step (b) with an amount of antigen in the sample.

Claim 3

Dependent Claim 174 words

An immunoassay method comprising the steps of: (a) reacting a sample containing an antibody with an antigen which specifically binds to said antibody, so as to bind said antibody to said antigen, wherein said antigen is immobilized on a 0.2 to 3.0 μm magnetic particle comprising a core and a coating layer on the surface of said core, wherein said core comprises an organic polymer and said coating layer comprises a metal oxide formed by adding ferrous ions or ferrous ions and an ion selected from the group consisting of zinc, cobalt, nickel, manganese, copper, vanadium, antimony, lithium, molybdenum, titanium, rubidium, aluminum, silicon, chromium, tin, calcium, cadmium, indium ions and mixtures thereof to deoxidized water in which cores of organic polymer are suspended to form a resulting solution, and subsequently adding an oxidizing agent solution to the resulting solution to provide a ferrite coating on said core; (b) measuring an amount of bound antibody; and (c) correlating the amount of bound antibody obtained in step (b) with an amount of antibody in the sample.

Claim 4

Dependent Claim 174 words

An immunoassay method comprising the steps of: (a) reacting a sample containing an antibody with an antigen which specifically binds to said antibody, so as to bind said antibody to said antigen, wherein said antigen is immobilized on a 0.2 to 3.0 μm magnetic particle comprising a core and a coating layer on the surface of said core, wherein said core comprises an organic polymer and said coating layer comprises a metal oxide formed by adding ferrous ions or ferrous ions and an ion selected from the group consisting of zinc, cobalt, nickel, manganese, copper, vanadium, antimony, lithium, molybdenum, titanium, rubidium, aluminum, silicon, chromium, tin, calcium, cadmium, indium ions and mixtures thereof to deoxidized water in which cores of organic polymer are suspended to form a resulting solution, and subsequently adding an oxidizing agent solution to the resulting solution to provide a ferrite coating on said core; (b) measuring an amount of bound antibody; and (c) correlating the amount of bound antibody obtained in step (b) with an amount of antibody in the sample.

Claim 5

Dependent Claim 15 words

An immunoassay method according to claim 1, wherein said method is an enzyme immunoassay method.

Claim 6

Dependent Claim 15 words

An immunoassay method according to claim 2, wherein said method is an enzyme immunoassay method.

Claim 7

Dependent Claim 32 words

An immunoassay method according to claim 1, wherein said particle is further coated with a polymer selected from the group consisting of silane, nylon and polystyrene prior to immobilization of the antibody.

Claim 8

Dependent Claim 32 words

An immunoassay method according to claim 2, wherein said particle is further coated with a polymer selected from the group consisting of silane, nylon and polystyrene prior to immobilization of the antigen.

Claim 9

Dependent Claim 30 words

The immunoassay method according to claim 1, wherein said organic polymer of the core comprises an polystyrene or a polymer comprising at least one of an acrylate and a methacrylate.

Claim 10

Dependent Claim 30 words

The immunoassay method according to claim 2, wherein said organic polymer of the core comprises an polystyrene or a polymer comprising at least one of an acrylate and a methacrylate.

Claim 11

Dependent Claim 21 words

The immunoassay method according to claim 1, wherein said coating layer is a magnetite layer or a mixed crystal ferrite layer.

Claim 12

Dependent Claim 21 words

The immunoassay method according to claim 2, wherein said coating layer is a magnetite layer or a mixed crystal ferrite layer.

Claim 13

Dependent Claim 73 words

The immunoassay method according to claim 5, wherein (a) said sample comprises said antigen; (b) said antibody is immobilized on said magnetic particle; and (c) said method further comprises adding an enzyme-labeled antibody, which specifically binds to said antigen to the sample; and separating unreacted enzyme labeled antibody from the sample by washing out said unreacted enzyme-labeled antibody, adding an enzyme substrate specific for the enzyme and measuring the activity of antibody-bound enzyme.

Claim 14

Dependent Claim 72 words

The immunoassay method according to claim 6, wherein (a) said sample comprises said antigen; (b) said antibody is immobilized on said magnetic particle; and (c) said method further comprises adding an enzyme-labeled antigen, which specifically binds to said antibody to the sample; and separating unreacted enzyme-labeled antigen from the sample by washing out said unreacted enzyme-labeled antigen, adding an enzyme substrate specific for the enzyme and measuring the activity of antigen-bound enzyme.

Claim 15

Dependent Claim 45 words

The immunoassay method according to claim 11, wherein said enzyme substrate is selected from the group consisting of (i) ABTS and luminol-H 2 O 2 for peroxidase; (ii) (2'-pyro-tri-cyclodecab)-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt, p-nitrophenylphosphate and methylumbelliferyl phosphate for alkaline phosphatase; and (iii) p-nitrophenyl-β-o-galactose and methylumbelliferyl-β-o-galactose for β-galactosidase.

Claim 16

Dependent Claim 45 words

The immunoassay method according to claim 12, wherein said enzyme substrate is selected from the group consisting of (i) ABTS and luminol-H 2 O 2 for peroxidase; (ii) (2'-pyro-tri-cyclodecab)-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt, p-nitrophenylphosphate and methylumbelliferyl phosphate for alkaline phosphatase; and (iii) p-nitrophenyl-β-o-galactose and methylumbelliferyl-β-o-galactose for β-galactosidase.

Claim 17

Dependent Claim 19 words

The immunoassay method according to claim 13, wherein said enzyme substrate is (2'-pyro-tricyclo[3.3.1.1 3 ,7 ]-decan)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane disodium salt.

Claim 18

Dependent Claim 19 words

The immunoassay method according to claim 14, wherein said enzyme substrate is (2'-pyro-tricyclo[3.3.1.1 3 ,7 ]-decan)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane disodium salt.

Claim 19

Dependent Claim 73 words

The immunoassay method according to claim 3, wherein (a) said sample comprises said antigen; (b) said antibody is immobilized on said magnetic particle; and (c) said method further comprises adding an enzyme-labeled antigen, which specifically binds to said antibody to the sample; and separating unreacted enzyme labeled antigen from the sample by washing out said unreacted enzyme-labeled antigen, adding an enzyme substrate specific for the enzyme and measuring the activity of antigen-bound enzyme.

Claim 20

Dependent Claim 73 words

The immunoassay method according to claim 4, wherein (a) said sample comprises said antibody; (b) said antigen is immobilized on said magnetic particle; and (c) said method further comprises adding an enzyme-labeled antibody, which specifically binds to said antibody to the sample; and separating unreacted enzymelabeled antibody from the sample by washing out said unreacted enzyme-labeled antibody, and adding an enzyme substrate specific for the enzyme and measuring the activity of antibody-bound enzyme.

Claim 21

Dependent Claim 48 words

The immunoassay method according to claim 17, wherein said enzyme substrate is selected from the group consisting of (i) ABTS and luminol-H 2 O 2 for peroxidase; (ii) (2'-pyro-tricyclo[3.3.1.1. 3 ,7 ]decan)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt, p-nitrophenylphosphate and methylumbelliferyl phosphate for alkaline phosphatase; and (iii) p-nitrophenyl-β-o-galactose and methylumbelliferyl-β-o-galactose for β-galactosidase.

Claim 22

Dependent Claim 48 words

The immunoassay method according to claim 18, wherein said enzyme substrate is selected from the group consisting of (i) ABTS and luminol-H 2 O 2 for peroxidase; (ii) (2'-pyro-tricyclo[3.3.1.1 3 ,7 ]-decan)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt, p-nitrophenylphosphate, and methylumbelliferyl phosphate for alkaline phosphatase; and (iii) p-nitrophenyl-β-o-galactose and methylumbelliferyl-β-o-galactose for β-galactosidase.

Claim 23

Dependent Claim 19 words

The immunoassay method according to claim 19, wherein said enzyme substrate is 2'-pyro-tricyclo[3.3.1.1 3 ,7 ]-decan)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane disodium salt.

Claim 24

Dependent Claim 19 words

The immunoassay method according to claim 20, wherein said enzyme substrate is 2'-pyro-tricyclo[3.3.1.1 3 ,7 ]-decan)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane disodium salt.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1997-06-13	1997-12-13	1998-06-14	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2001-06-13	2001-12-13	2002-06-14	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2005-06-13	2005-12-13	2006-06-14	$7700.00	Window Closed / Lapsed

Immunoassay using magnetic particle

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