Anti-idiotype assay using fluorescent energy transfer

Claim 2

An immunoassay for detecting an analyte comprising: a first binding partner immunologically reactive with said analyte; a second binding partner immunologically reactive with said . first binding partner and whose spatial relationship to said - first binding partner is altered upon imnunological reaction of said analyte with said first. binding partner; and - means associated with said first and second binding partners for providing a detectable signal responsive to said altered spatial relationship.

Claim 3

The immuncassay as claimed in Claim 1 wherein said associated means comprises a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength distinguishably detectable from said first emission wavelength. -

Claim 4

The immunoassay as claimed in Claim 2 wherein one of said donor or acceptor molecules is associated with the first binding partner and the other of said molecules is associated with the second binding partner.

Claim 5

The immunoassay as claimed in any one of claims 1 to 3 wherein said first and second binding partners are selected from the group consisting of 'polyclonal antibodies, monoclonal antibodies, F(ab)' antibodies, F(ab) antibodies and antibody fragments.

Claim 6

The immunoassay as claimed in Claim 4 wherein said second binding partner is selected from the group consisting of anti-idiotype antibodies and anti-framework antibodies.

Claim 7

The immunoassay as claimed in any one of Claims 1 to 5 wherein said responsive detectable signal is selected from the group consisting of increased or decreased donor fluorescent emission, increased or decreased acceptor fluorescent emission, increased or decreased donor emission time constant, and any combination of the foregoing.

Claim 8

An immunoassay for detecting an immunologically reactive analyte or for determining the structure thereof wherein the binding of said analyte to an antibody having an idiotype changes the spatial relationship between said antibody and an anti-idiotype antibody capable of binding said idiotype, said change in spatial relationship or relative motion being detectable by fluorescent means.

Claim 9

An immunoassay for detecting an immunologically reactive analyte or for determining the structure thereof wherein the binding of said analyte to an antibody having an idiotype changes the spatial relationship between said antibody and an anti-idiotype antibody capable of binding said idiotype, said change in spatial relationship or relative motion being detectable by fluorescent means.

Claim 10

A homogeneous immunoassay for detecting an analyte, having an immunologically reactive antigenic determinant, in response to the change in spatial relationship between 1) an antibody specific for the antigenic determinant and characterized with an idiotype or 2) a framework and an anti-idiotype or anti-framework antibody.

Claim 11

A homogeneous immunoassay for detecting an analyte, having an immunologically reactive antigenic determinant, in response to the change in spatial relationship between 1) an antibody specific for the antigenic determinant and characterized with an idiotype or 2) a framework and an anti-idiotype or anti-framework antibody.

Claim 12

The immunoassay as claimed in Claim 7 or Claim 8 wherein the changes are detected by measuring the change in fluorescence emission or the change in fluorescence emission time constant.

Claim 13

The immunoassay as claimed in any one of claims 7 to 9 wherein the fluorescent means comprises a fluorescent energy transfer pair.

Claim 14

The immunoassay as claimed in Claim 10 wherein the fluorescent energy transfer pair comprises a donor molecule having a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second wavelength distinguishably detectable from said first emission wavelength.

Claim 15

The immunoassay as claimed in any one of claims 7 to 11 wherein at least one of the antibodies is an incomplete antibody.

Claim 16

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength, dtstinguishably detectable from said first emission wavelength, said assay method comprising the steps of: providing the sample containing the analyte to be analyzed; further providing a first binding partner specific for the analyte and labeled with either the acceptor or the donor molecule of the acceptor-donor pair; forming a first mixture by contacting said first binding partner with a second binding partner specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions which allow any immunological reaction to occur, said second binding partner having a spatial relationship with said first binding partner which can be altered upon immunological reaction of said analyte with said first binding partner; contacting said sample with said first mixture to form a second mixture; incubating said second mixture under conditions whereby immunological binding of said analyte with said first binding partner is permitted to occur; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescence emission or the increase in the second fluorescence emission and, determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 17

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength, dtstinguishably detectable from said first emission wavelength, said assay method comprising the steps of: providing the sample containing the analyte to be analyzed; further providing a first binding partner specific for the analyte and labeled with either the acceptor or the donor molecule of the acceptor-donor pair; forming a first mixture by contacting said first binding partner with a second binding partner specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions which allow any immunological reaction to occur, said second binding partner having a spatial relationship with said first binding partner which can be altered upon immunological reaction of said analyte with said first binding partner; contacting said sample with said first mixture to form a second mixture; incubating said second mixture under conditions whereby immunological binding of said analyte with said first binding partner is permitted to occur; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescence emission or the increase in the second fluorescence emission and, determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 18

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength, said assay method comprising the steps of: contacting the sample containing the analyte to be measured with a first binding partner specific for the analyte and labeled with either the acceptor or the donor molecule of the acceptor-donor pair under conditions permitting any immunological reaction to occur; further contacting said sample and said first binding partner with a second binding partner, specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions whereby said second binding partner competes with said analyte for binding sites on said first binding partner ; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescence emission or the increase in the second fluorescence emission and determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 19

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength, said assay method comprising the steps of: contacting the sample containing the analyte to be measured with a first binding partner specific for the analyte and labeled with either the acceptor or the donor molecule of the acceptor-donor pair under conditions permitting any immunological reaction to occur; further contacting said sample and said first binding partner with a second binding partner, specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions whereby said second binding partner competes with said analyte for binding sites on said first binding partner ; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescence emission or the increase in the second fluorescence emission and determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 20

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength distinguishably detectable from said first emission wavelength, said assay method comprising the steps of: mixing in a single step: the sample containing the analyte to be measured, a first binding partner specific for the analyte and labeled with either . the acceptor or the donor molecule of the acceptor-donor fluorescent energy transfer pair; and a second binding partner specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions which allow any imnunological reaction to occur whereby binding of the analyte to said first binding partner alters the spatial relationship between said first and second binding partners; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescent emission or the increase in the second fluorescent emission and determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 21

An immunoassay method for analyzing for the presence of an analyte in a sample for use with a detectable label system comprising a fluorescent energy transfer pair having a donor molecule with a first excitation wavelength and a first emission wavelength and an acceptor molecule capable of being excited by said first emission wavelength and having a second emission wavelength distinguishably detectable from said first emission wavelength, said assay method comprising the steps of: mixing in a single step: the sample containing the analyte to be measured, a first binding partner specific for the analyte and labeled with either . the acceptor or the donor molecule of the acceptor-donor fluorescent energy transfer pair; and a second binding partner specific for said first binding partner and labeled with the other of said donor or acceptor molecule under conditions which allow any imnunological reaction to occur whereby binding of the analyte to said first binding partner alters the spatial relationship between said first and second binding partners; irradiating said second mixture with light having a wavelength capable of exciting said donor molecule but not having a wavelength capable of substantially exciting said acceptor molecule; and detecting either the decrease in the first fluorescent emission or the increase in the second fluorescent emission and determining the presence of said analyte on the basis of said decrease or increase in fluorescence.

Claim 25

The method as claimed in claim 18 wherein one or both of said binding partners are monoclonal antibodies.

Claim 26

Reagent kit for use in a competitive homogeneous immunoassay system for an analyte using fluorescent energy transfer between a donor whose emission is capable of exciting an acceptor molecule having a fluoresce of emission distinguishable from the donor emission and where the excitation wavelength employed to excite the donor molecule results in no appreciable excitation of the acceptor molecule, said kit comprising: a first binding partner specific for the analyte to be assayed and labeled with either the donor molecule or the acceptor molecule of the donor-acceptor fluorescent energy transfer pair; and a second binding partner labeled with the other of said donor or acceptor molecule of said donor-acceptor fluorescent energy transfer pair, said second binding partner capable of reacting with said first binding partner and whose spatial relationship with said first binding partner is altered upon immunological reaction between said analyte to be determined and said first binding partner.

Claim 27

Reagent kit for use in a competitive homogeneous immunoassay system for an analyte using fluorescent energy transfer between a donor whose emission is capable of exciting an acceptor molecule having a fluoresce of emission distinguishable from the donor emission and where the excitation wavelength employed to excite the donor molecule results in no appreciable excitation of the acceptor molecule, said kit comprising: a first binding partner specific for the analyte to be assayed and labeled with either the donor molecule or the acceptor molecule of the donor-acceptor fluorescent energy transfer pair; and a second binding partner labeled with the other of said donor or acceptor molecule of said donor-acceptor fluorescent energy transfer pair, said second binding partner capable of reacting with said first binding partner and whose spatial relationship with said first binding partner is altered upon immunological reaction between said analyte to be determined and said first binding partner.