Simultaneous immunoassay for the determination of antigens and antibodies

Claim 2

An immunochemical assay to determine the presence or concentration of an antigenic substance in a fluid, comprising: (a) forming a ternary complex of an antibody or antibody fragment conjugated to a first label, a second antibody or antibody fragment conjugated to a second label, and said antigenic substance; and (b) detecting a signal produced in the presence of at least one substrate, by an interaction between said first label and said second label enhanced by their proximity to each other bound to said antigenic substance.

Claim 3

An immunochemical assay to determine the presence or concentration of an antibody in a fluid, comprising: (a) forming a ternary complex of an antigenic substance conjugated to a first label, a second antigenetic substance conjugated to a second label, and said antibody; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antibody.

Claim 4

An immunochemical assay to determine the presence or concentration of an antibody in a fluid, comprising: (a) forming a ternary complex of an antigenic substance conjugated to a first label, a second antigenetic substance conjugated to a second label, and said antibody; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antibody.

Claim 5

The immunochemical assay according to claim 1, wherein the antibody conjugated to the first label is a monoclonal antibody specific for a first epitope on the antigenic substance, and the antibody conjugated to the second label is a monoclonal antibody specific for a second epitope on the antigenic substance.

Claim 6

The immunochemical assay according to claim 1, wherein the antibody fragment is a monovalent fragment (Fab) produced by digestion of the antibody with papain.

Claim 7

The immunochemical assay according to claim 1, wherein the antibody fragment is a bivalent fragment (Fab₂) produced by digestion of the antibody with pepsin.

Claim 8

An immunochemical assay to determine the presence or concentration of an antigenic substance in a fluid, comprising: (a) in the presence of the antigenic substance to be determined, forming a ternary complex of the antigenic substance conjugated to a first label, the antigenic substance conjugated to a second label, and an antibody to said antigenic substance; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antibody.

Claim 9

An immunochemical assay to determine the presence or concentration of an antigenic substance in a fluid, comprising: (a) in the presence of the antigenic substance to be determined, forming a ternary complex of the antigenic substance conjugated to a first label, the antigenic substance conjugated to a second label, and an antibody to said antigenic substance; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antibody.

Claim 10

An immunoassay to determine the presence or concentration of antibody in fluid, comprising: (a) in the presence of the antibody to be determined, forming a ternary complex of the antibody conjugated to a first label, the antibody conjugated to a second label, and an antigenic substance; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antigenic substance.

Claim 11

An immunoassay to determine the presence or concentration of antibody in fluid, comprising: (a) in the presence of the antibody to be determined, forming a ternary complex of the antibody conjugated to a first label, the antibody conjugated to a second label, and an antigenic substance; and (b) detecting a signal produced in the presence of at least one substrate, by the interaction between said first label and said second label enhanced by their proximity to each other bound to said antigenic substance.

Claim 12

The immunochemical assay according to claim 1, 2, 6 or 7, wherein said first label and said second label are each enzymes, and said signal is produced by the sequential action of said first enzyme label on or in the presence of a first substrate, and said second enzyme label on or in the presence of a second substrate, said second substrate being a product of the action of said first enzyme label on said first substrate.

Claim 13

The immunological assay according to claim 8, wherein said signal is an increased rate of appearance of said second substrate.

Claim 14

The immunological assay according to claim 8, wherein said signal is an increased rate of disappearance of said first substrate.

Claim 15

The immunological assay according to claim 8, wherein said signal is an increased rate of a set of cyclic reactions, in which said first substrate is regenerated by the action of said second enzyme label on or in the presence of said second substrate.

Claim 16

The immunochemical assay according to claim 9, wherein the first enzyme label is glucose oxidase, the second enzyme label is horseradish peroxidase, the first substrate is glucose, the second substrate is hydrogen peroxide, and the signal is a color change in the presence of orthophenylenediamine.

Claim 17

The immunochemical assay according to claim 9, wherein the first enzyme label is horseradish peroxidase, the second enzyme label is luminol, the first substrate is hydrogen peroxide, the second substrate is oxygen, and the signal is light.

Claim 18

The immunochemical assay according to claim 9, wherein the first enzyme label is phosphenol pyruvate kinase, the second enzyme label is luciferase, the first substrate is phophenol pyruvate, and the signal is light.

Claim 19

A process for determining the presence or concentration of antibody to an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution of the antigenic substance, said solution containing the antigenic substance conjugated to a first enzyme label and the antigenic substance conjugated to a second enzyme label, in order to form a ternary complex of an antibody in the sample, said first enzyme-labelled antigen and said second enzyme-­labelled antigen; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said substrate; (c) measuring a signal produced by the reaction of said second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of antibody to said antigenic substance, in order to determine the presence of antibodies in said fluid sample; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of the antibody prepared according to steps (a) - (c), in order to determine the concentration of said antibodies in said fluid.

Claim 20

A process for determining the presence or concentration of antibody to an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution of the antigenic substance, said solution containing the antigenic substance conjugated to a first enzyme label and the antigenic substance conjugated to a second enzyme label, in order to form a ternary complex of an antibody in the sample, said first enzyme-labelled antigen and said second enzyme-­labelled antigen; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said substrate; (c) measuring a signal produced by the reaction of said second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of antibody to said antigenic substance, in order to determine the presence of antibodies in said fluid sample; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of the antibody prepared according to steps (a) - (c), in order to determine the concentration of said antibodies in said fluid.

Claim 21

A process for determining the presence or concentration of an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution of antibodies to the antigenic substance, said solutions containing antibodies conjugated to a first enzyme label and antibodies conjugated to a second enzyme label, in order to form a ternary complex of an antigenic substance in the fluid, said first enzyme-labelled antibody and said second enzyme-labelled antibody; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said substrate; (c) measuring a signal produced by the reaction of said second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of said antigenic substance, in order to determine the presence of said antigenic substance in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said antigenic substance prepared according to steps (a) - (c), in order to determine the concentration of the antigenic substance in said fluid.

Claim 22

A process for determining the presence or concentration of an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution of antibodies to the antigenic substance, said solutions containing antibodies conjugated to a first enzyme label and antibodies conjugated to a second enzyme label, in order to form a ternary complex of an antigenic substance in the fluid, said first enzyme-labelled antibody and said second enzyme-labelled antibody; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said substrate; (c) measuring a signal produced by the reaction of said second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of said antigenic substance, in order to determine the presence of said antigenic substance in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said antigenic substance prepared according to steps (a) - (c), in order to determine the concentration of the antigenic substance in said fluid.

Claim 23

The process according to claim 15, wherein the antibodies in the fluid are antibodies to the humane immune deficiency virus.

Claim 24

The process according to claim 16, wherein the antigen in the fluid is human immune deficiency virus.

Claim 25

A process for determining the presence or concentration of an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution containing the antigenic substance, said solution containing the antigenic substance conjugated to a first enzyme label, the antigenic substance conjugated to a second enzyme label, and antibodies to said antigenic substance, in order to form a ternary complex in which the antigenic substance in the sample competes with enzyme labelled antigen for binding sites on the antibody; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said first substrate; (c) measuring a signal produced by the production of said first second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of said antigenic substance, in order to determine the presence of the antigenic substance in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said antigenic substance prepared according to steps (a) - (c), in order to determine the concentration of the antigenic substance in said fluid.

Claim 26

A process for determining the presence or concentration of an antigenic substance in a fluid, comprising the steps of: (a) contacting the fluid with a solution containing the antigenic substance, said solution containing the antigenic substance conjugated to a first enzyme label, the antigenic substance conjugated to a second enzyme label, and antibodies to said antigenic substance, in order to form a ternary complex in which the antigenic substance in the sample competes with enzyme labelled antigen for binding sites on the antibody; (b) contacting the ternary complex solution with a substrate for said first enzyme label, in order to form a reaction product of said first enzyme label and said first substrate; (c) measuring a signal produced by the production of said first second enzyme label in the presence of the reaction product formed in step (b); (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a) - (c), said control sample being free of said antigenic substance, in order to determine the presence of the antigenic substance in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said antigenic substance prepared according to steps (a) - (c), in order to determine the concentration of the antigenic substance in said fluid.

Claim 27

A process according to claim 19, wherein the antigenic substance in the fluid is thyroxine.