CA1297406C

CA1297406C - Method of detecting and quantifying ligands in liquids

Info

Publication number: CA1297406C
Application number: CA000556618A
Authority: CA
Inventors: Richard C. Hevey
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-01-15
Filing date: 1988-01-15
Publication date: 1992-03-17
Anticipated expiration: 2009-03-17
Also published as: JPH02502122A; AU1342488A; WO1988005538A1

Abstract

METHOD OF DETECTING AND QUANTIFYING
LIGANDS IN LIQUIDS

Abstract of the Disclosure A method of determining a ligand of interest in a liquid is described. The method, which makes it possible to detect and quantify a ligand in a liquid, makes use of a biotin-avidin system which can be used to carry out immunoassays in both heterogeneous and homogeneous format. The basic components used in the method are a biotin-labeled substance (which is biotin-lableled ligand or biotin-labeled specific binding partner), biotin-labeled fluorophore, a liquid to be analyzed for the ligand of interest, specific binding partner for the ligand of interest and avidin. The method can be used to determine the presence (or absence) and the quantity of avidin and biotin in a sample, using fluorescence polarization.

Description

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METHOD OF DETECTING AND QUANTIFYING
LIGANDS IN LIQUIDS

Background Nonisotopic immunoassays are widely used in 05 clinical and research contexts for the determination of both the presence and ~he quantity of analytes such-as proteins, nucleotide sequences, drugs, steroids, etc. Nonisotopic immunoassays can be divided into two types: heterogeneous assays and homogeneous assays.
In heterogeneous assays, a solid support (e.g., beads, a column) is used; some of the labeled reagent becomes bound to the support, while the remainder does not. A procedure is required to separate bound and free labeled reagent.
In homogeneous assays, no separation is re-quired, thus eliminating the need for an additional step. There are at least five major types of homogeneous immunoassays routinely used. One of these, fluorescence polarization immunoassays (FPIA~, can be used to measure small quantities of substances te.g., in the nanogram-per-milliliter range). They make use of the fact that molecules can exist in a ground or (lowest energy) state and, after exposure to incident radiation, an excited or higher energy state. Absorption of energy from this source results in promotion of one or more electrons in a molecule to highex energy levels. As this jump ; occurs t the electron may lose a small percentage of the absorbed energy (e.g., via collisons with other ~YP

~7~6 molecules, etc). As its electrons return from higher energy levels to ground state, the excited molecule can radiate energy. The energy generated in this way is, however, less than that originally 05 involved in exciting the molecule. As a result, the wavelength of the light emitted (here, fluorescent light) is longer than that of the light used to excite the molecule. Emitted light energy can be detected using standard equipment, such as a detec-tor positioned at a right angle to the incidentlight beam. S. Bakerman (ed.), Chem Cues: Fluores-cence Polarization Immunoassay, Laboratory Mana~e-ment, 16-18 (July 1983); D. Freifelder, Fluorescence Spectroscopy, In: Physical Biochemistry: Applica-tions to Biochemistry and Molecular Biology (2ded.), 537-572 (1982).
An understanding of FPIA also requires an understanding of polarized light. Ordinary light can be thought of as a number of electromagnetic waves, each in a single plane; each wave passes through the central axis or path of the light beam.
Polarized light, however, is light in which only one wave plane occurs (the others having been eliminated or screened out). When a fluorescent molecule is oriented such that its dipoles lie in the same plane as the light waves, it absorbs the polarized light.
As it returns to its ground state, the molecule emits light in the same plane.
Two additional factors of importance in FPIA
are time-related. First, the fluorescence lifetime of the molecule being used must be considered. The .

lifetime is the interval between excitation of the molecule by a polarized light burst and emission by the molecule of a similar burst. Second, the rotational relaxation time of the molecule - the 05 time necessary for an excited molecule to move out of alignment so that emitted polarized light is emitted in a direction different from its excita-tion must be taken into accountO Small molecules (e.g., haptens) rotate rapidly in solution; their rotational relaxation times are shorter than molecular fluorescence lifetime. As a result, after having absorbed polarized light, such small mole-cules become randomly oriented by the time a burst of polarized emitted light is obtained. Larger molecules ~e.g., immunoglobulins) rotate relatively slowly and have rotational times longer than the typical fluorescence lifetime.
Fluorescence polarization measurements rely on the fact that polarized excitation radiation gives rise to polarized emission radiation if no molecular rotation of the fluorophore occurs. A fluorophore is a fluoEescent molecule or a compound which has the property of absorbing light at one wavelength and emitting it at a longer wavelength. As des-cribed above, the fluorophore bound to a smallmolecular hapten experiences molecular rotation at a rate that is rapid compared to the lifetime of the excited state prior to emission. Thus, the light is depolarized when bound to a small molecule. When antibody binds the fluorophore-antigen, rotation decreases dramatically because of the large size (molecular weight - 150,000) of the antibody, causing the emitted light to remain polarized.
Immunoassays utilize this phenomenon as fol-lows: with antibody and fluorophore-labeled hapten 05 or fluorophore-labelled antigen present, binding occurs between antibody and fluorophore and hapten or between antibody and fluorophore-antigen and ; little fluorescence depolarization occurs. As antigen to be analyzed is added, it binds to anti-body competitively, fluorophore-antigen is not bound and depolarization is observed. The depolarization is a Punction of antigen concentration and con-stitutes a quantitative assay.
Although the principle of FPIA has been known since the 1970's and feasible instrumentation for the assay with flow-cell and digital read-out has been available since 1973, FPIA has had relatively limited use clinically because it is limited in the size of analytes for whose detection it can be used.
The FPIA method is simple, rapid and precise, but its sensitivity is limited. Because only a rela-tively small change in the polarization occurs, the method is not applicable to antigens whose molecular mass exceeds about 20,000 Daltons.

Disclosure of the Invention The present invention relates to a method for determining a ligand in a liquid using fluorescence polarization. The invention relates to a method of detecting and quantifying a ligand, which makes use of a biotin-avidin complex system. The invention also relates to a method or specifically determin-ing the amount of avidin and ~iotin in a liquid.
The biotin-avidin complex system can be used to carry out immunoassays in both heterogeneous format 05 and homogeneous format. It is particularly well suited to determining the presence and the quantity of large molecules (e.g., those having molecular weight of about 20,000 Daltons or more) in a liquid in a homogeneous format. It thus makes it possible to extend the use of fluorescence polarization immunoassay to the detection and quantification of substances (e.g., ligands) of molecular weiyht above 20,000 Dalton.
According to the method of the present inven-tion, a substance is covalently coupled to biotin toform a biotin-labeled substance; the substance is either a ligand or a specific binding partner for the ligand, resulting in the production of, respec-tively, a biotin-labeled ligand and a biotin-labeled specific binding partner for the ligand. Biotin is also covalently coupled to a fluorescent material (biotin-labeled fluorophore). Moreover, this linkage of biotin to a fluorophore also forms the basis for use of the method of the present inven-tion, discussed below, to measure fluorescencepolarization of a solution containing an unknown quantity of biotin or avidin.
The basic components used in the method of the present invention for quantifying ligands in liquids ara: 1) a biotin-labeled substance; 2) a biotin-labeled fluorophore; 3) a li~uid or sample ~ .

(containing the ligand whose presence and/or quantity are to be determined -- the ligand of interest); 4) specific binding partner Eor the ligand of interest; and 5) avidin.
05 In one embodiment of the method of the inven-tion, immunoassay is carried out in homogeneous format. That is, one method for using the biotin-avidin complex in determining the presence and quantity of a ligand in a liquid according to the present invention is as follows: A known volume of liquid to be analyzed for the ligand of interest is incubated (combined) with a known quantity of the ligand of interest which is labeled with biotin; a quantity o~ speci~ic binding partner for the ligand of interest; a biotin-labeled fluorophore; and avidin. The resulting combination is maintained under conditions appropriate for specific binding reactions (reaction between the ligand and its specific binding partner) to occur. Fluorescence polarization is determined using known techniques, befors and after the addition of avidin. The difference in fluorescence polarization (e.g., with and without avidin present~ can be done sequentially or simultaneously. That is, a known quantity of avidin is added, either along with the other compo nents or after the other components have been combined and specific binding reactions have oc-curred. In the first case, the change in fluores-cence polarization is measured simultaneously; in the latter, it is measured sequentially. The change serves as an indication of the presence and quantity ~2~

of ligand in the liquid. It is also possible to compare the fluorescence polarization oE th~ combin-ation with that of an avidin-free control. Back-ground fluorescence polarization (that is caused by 05 the components themselves) can be determined and subtracted, as described below~ to increase the sensitivity of the assay.
A method of this invention which can also be used in a homogeneous format makes use of a biotin-labeled fluorophore, a liquid to be analyzed for a ligand of interest, a specific binding partner for the ligand, ligand bound to biotin, and avidin.
In this method of determining the presence and quantity of a liyand in a liquid, the biotin-bound ligand is pre-reacted with its specific binding partner or with avidin. In the first case, the resulting complex (biotin-bound ligand/specific binding partner), a biotin-labeled fluorophore and avidin are added to a known volume of liquid to be analyzed for the ligand of interest. In the second case, the resulting complex (biotin-bound ligand/
avidin), a biotin-labeled fluorophore and a specific binding partner are added to a known volume of liquid to be analyzed for the ligand of interest.
The change in fluorescence polarization (before and after addition of avidin or ligand) is measured and serves as an indicator of the presence and quantity of the ligand of interest. Alternatively, the fluorescence polarization of the combination can he compared with that of an avidin-free control.

In another embodiment of this method, an in-soluble phase containing a specific binding partner for the ligand of interest is used. This embodi~ent is useful in the determination of ligands which have 05 one or more binding points or sites for their specific binding partners. The components of the system are incubated (combined) under conditions appropriate for specific binding reactions to occur (e.g., between ligand and its specific binding partner). Following the specific binding reactions, the fluorescence polarization of the soluble phase is determined.
Specifically, the presence and quantity of a ligand in a ].iquid are determined as follows: An insoluble phase containing a speciic binding partner for the ligand is incubated with a known volume of liquid to be analyzed for the ligand of interest and biotin-labeled ligand. Unreacted reagents (which are in the soluble phase) are separated from the insoluble phase after incubation and fluorescence polarization of the soluble phase is measured. A biotin-labeled fluorophore and a known quantity of avidin are then added to the soluble phase. Fluorescence polarization is again measured using known techniques. The change in fluorescence polarization is determined and serves as an indication of the presence and quantity of ligand in the liquid. Alternatively, fluorescence polarization of the incubated combination can be compared with that of an avidin-free control.

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In another embodiment, an insoluble phase containing a specific binding partner for the ligand of interest is reacted with a ligand which has at least two binding points or sites for its specific 05 binding partner Biotin-labeled binding substance and avidin are added. The components are combined under conditions appropriate for specific hinding reactions to occur (e.g., between ligand and its specific binding partner) and for biotin and avidin to bind. The soluble phase is separated from the insoluble phase and an aliquot of uncoupled avidin (e.g., of the soluble phase~ is removed and reacted with a biotin-labeled fluorophore. After specific bindiny reactions have occurred, the fluorescent polarization of the soluble phase is determined.
An alternative embodiment of this invention which can be used to determine the presence and quantity of a ligand which has more than one bindiny point or site for the specific binding partner involves use of an insoluble phase containing a specific binding partner for the ligand of interest.
In this embodiment, the insoluble phase is incubated with a known quantity of liquid to be analyzed for the ligand of interest and biotin-labeled specific binding partner for the ligand ~which is bound to the solid phase specific binding partner). Incu-bation is carried out under conditions appropriate for specific binding reactions to occur. Unreacted reagents are separated from the insoluble phase and a known quantity of avidin is added to the insoluble phase. Fluorescence polarization of the insoluble . .. ~ , ., ,,", ... .

phase is determined using known techniques. Unreact-ed reagents are again separa~ed from the insoluble phase and a biotin-labeled fluorophore is added either to the insoluble phase or to the separated liquid phase. Fluorescence polarization due to 05 binding of biotin-labeled fluorescent material, either to the avidin on the insoluble phase or to unreacted avidin in the liquid phase, is determined.
The change in fluorescence polarization is de-termined and serves as an indication of the presence and quantity of ligand in the liquid.
The method of the present invention can be used for the determination of nucleotide sequences (i.e., DN~, RNA) of interest. Its use in determining the presence and quantity of DNA in a liquid is as follows:
A known volume of liquid containing DNA of interest is placed upon a nitrocellulose solid phase (e.g., nitrocellulose filter). Biotin-labeled DNA
having a nucleotide sequence complementary to the DNA of interest is added to the nitrocellulose solid phase. Bound and free biotin-labeled DNA are separated and reacted with avidin and biotin-labeled fluorophore. The degree of fluorescence polariza-tion caused by binding o~ the biotin-fluorophore to avidin is determined using known techniques.
The ligand determination performed according to the method of the present invention can be ac-complished through use of either a noncompetitive binding reaction or a competitive binding reaction.
The method of this invention makes it possible to accuratelv determine the presence and quantity of ~`

.. ... ..

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a ligand of interest in a liquid quickly, without use of radioactive reagents.
In a similar manner, the method o~ this in-vention concerning the specific assay for biotin or 05 avidin makes it possible to carry out assays rapidly (i.e., in approximately ~ive minutes). The linkage of biotin to a fluorophore achieves a five to-ten fold increase in sensitivity over the best presently available methods for determining biotin or avidin.
Use of the present invention for quantifying avidin or biotin alone makes it possible to detect much smaller quantities of avidin and biotin in a sample than can be detected with presently available methods. The method makes use of the faat that complexed and uncomplexed fluorescent molecules (i.e., fluorescent molecules linked to a high molecular weight molecule and fluorescent molecules linked to a small molecule) can be distinguished by measurement of the degree of polarization of the light they emit. Thus, small molecules tumble rapidly in solution, with the result that excitation by polarized light is followed by depolarization of emitted light. Large molecules, however, tumble more slowly, with the result that some of the emitted radiation remains polarized. Complexed and uncomplexed molecules can be distinguished on the basis of their degree of polarization.
It is possible, using the method of the present invention, to detect smaller quantities of avidin (e.g., about 5 ng/ml) and biotin (e.g., as low as about 40 pg./ml.~ in a sample than can he detected with existing methods of avidin/biotin analysis.

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_ief Description of the Drawings Figure 1 is a schematic representation of the reactions occurring when biotin-labeled fluorophore, biotin-labeled ligand and avidin are combined and of the effect on fluorescence polarization.
Figure 2 is a standard curve for assay of avidin. The change in polarization of a biotin-fluorescein complex is shown as a function of avidin concentration. Biotin-fluorescein concen-tration is ~ x 10 9M in O.lM phosphate bufferedsaline (pH=7.4) Figure 3 is a schematic representation of an immunoassay carried out in homogeneous format according to the method of the present invention.
Figure 4 is a schematic representation oP an immunoassay carried out in heterogeneous format according to the method of the present invention.
Figure 5 is a schematic representation of the effect of biotin-antigen, in the presence of 80 ng/ml avidin, on the polarization of biotin-fluores-cein. Data points are single determinations.
Figure 6 is a schematic representation of the effect of anti-rabbit IgG, in the presence of 60 ng/ml biotin-rabbit IgG and 80 ng/ml avidin, added sequentially, on the polarization of biotin-fluorescein. Error bars are the high, low, and average of two different day runs normalized to the same initial polarization with zero antibody controls.

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Figure 7 xepresents ~he effect of rabbit Ig~ in the presence of avidin:biotin-anti-rabbit IgG on the polarization of biotin-fluoroscein.

Detailed Description of the Invention 05 The method of determining a ligand of interest in a liquid through the measurement of fluorescence polarization which a subject of the present inven-tion makes use of the high binding effect between biotin and avidin. This method can be used in both homogeneous binding processes and heterogeneous binding processes; however, competitive homogeneous or lloncompetitive homogeneous binding processes are preferred.
Determlnation of the presence and quantity of a ligand of interest in a liquid is carried out through use of a biotin fluorescent polarization detection system. Use of this system takes advan-tage of the high binding affinity of avidin for biotin as a means of detecting and quantitating biotin or biotin-bound ligand. It is also possible to use anti-biotin antibody in place of avidin; for example, a monoclonal anti-biotin antibody can be used.
The basic components used in the method o~ the present invention are: 1~ a biotin-labeled reagent, which is generally a biotin-labeled ligand, but can also be a biotin-labeled specific binding partner;
2) a biotin-labeled fluorophore; 3) a liquid or sample to be analyzed for a ligand of interest; 4) a specific binding partner for the ligand of interest;
and 5) avidin.

~14-Generally, these components are combined at one time or in a selected sequence; the determining consideration as to which procedure is used is the ability of the selected approach to result in binding of avidin to biotin in the desired manner 05 (e.g., one which allows measurement of fluorescence polarization and its use as an indicator of the presence and/or quantity of the ligand of interest in the liquid).
The method of the present invention can be used in both homogeneous binding processes or immuno-assays (i.e., those which do not require separation of bound and free fractions of the labeled reagent~
and heterogeneous binding processes or immunoassays (i.e., those which re~uire a distinct step to separate the two reagent fractions).
Figure 1 is a schematic representation of reactions which occur among biotin-labeled ligand or analyte, biotin-labeled fluorophore and avidin when they are combined. A fluorophore is a fluorescent material and has the property of absorbing light at one wavelength and emitting it at another (longer) wavelength. As shown, avidin binds to biotin-labeled ligand, blocking avidin binding sites otherwise available for binding to biotin-labeled fluorophore. Avidin also binds with biotin-labeled fluorophore, resulting in fluorescent complex.
Finally, some biotin labeled fluorophore remains unbound (free) and undergoes rapid rotation.
The fact that fluorescence radiation from small molecules remains polarized when the small molecule is complexed to a larger molecule is also the basis for determining avidin and biotin concentrations in a sample using the method of the present invention.
The assay of the present invention makes use of 05 this fact in that a small fluorescent biotin conjugate, formed by linking a fluorescent material (fluorophore) and biotin. A fluorophore is a fluorescent material and has the property of ab-sorbing light at one wavelength. The link between the two is preferably covalent. As described herein, the assay makes use of fluorescein, but other fluorophores which exhibit polarization (e.g., rhodamines, pyrine derivatives, umbellipherones, porphyrins) can bç used.
The assay ~or avidin carried out by the method of the present invention is based on measurement of the fluorescence polarization of a solution in which a fluorescein-biotin conjugate and avidin bind. The fluorescence of a solution containing an unknown quantity of avidin is compared with a standard curve, such as that shown in Figure 2, and the concentration of avidin in the sample thus de-termined. An avidin concentration as low as approx-imately 5 ng/ml is detectable using this method; the assay range is about 20~fold.
The assay for biotin carried out by the method of the present invention is based on measurement of fluorescence polarization of a solution containing an unknown quantity of biotin. According to this ~ 30 method, avidin is added to a solution having an `~; unknown biotin concentration; the solution is ~2~0~

incubated briefly ~e.g., about 2-3 minutes) at room temperature. A fluorescent biotin conjugate (e,g., biotin-fluorescein) is then added in sufficient guantity to result in a final concentration of 1 x 05 10 9M; the resulting combination is incubated again briefly (e.g., about 2-3 minutes). Fluorescence polarization is then measured and compared with a standard curve. Using the method of the present invention, it is possible to conduct assays of samples in which the biotin concentration is ap-proximately 40-400 pg/ml.
The sensitivities of presently available avidin-biotin assays are compared with that of the present method in Table 1.

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Table 1. Sensitivities of available avidin:biotin assays Method -Sensitivity Source [avidin] [biotin]
05 ng/ml ng/ml Fluorometric 170,000 250 Mock et al.
Colormetric50,000 100 Green Colormetric3 - 100 Niedbala et al.
Radioisotopic 5,000 50 Green 10 Radioisotopic 100 - O'Malley Potentiometric 5,000 20 Gebause ~
Spectrophoto- Rechnite metric 3,000 5 Carrico Radioisotopic10,000 0.6 Rettenmeier 15 Yluorometric 40 0.5 Al-Hakiem Fluorometric 5 0.1 present invention Assay conditions are room temperature with sequential additions o~ biotin, avidin, and biotin-fluorescein and short incubation periods (e.g., approximately three minutes) after addition of avidin and after addition of biotin-fluorescein.
Polarization values were stable for at least several hours and serum levels up to 1% did not interfere.
The biotin-avidin complex method of the present invention can be employed in homogeneous immuno-assays, as represented in Figure 3~ As shown, the following components are used: 1) a biotin-labeled ~7~ 6 ligand (here, a biotin-labeled antigen~; 2) a biotin-labeled fluorophore; 3) an unknown quantity of a ligand of interest (here, an antigen) present in a liquid sample of known volume; 4) a specific 05 binding partner for the ligand of interest (here, antibody specific for antigen); and 5) avidin.
In this case, the following are incubated (combined) under conditions appropriate for specific binding reactions ~i.e., reactions between ligand (antigen) and its specific binding partner (anti-body)) to occur: 1) a known quantity of liquid to be analyzed for the ligand (antigen) of interest; 2) a known quantity of biotin-labeled ligand (antigen);

3) a known quantity of specific binding partner (antibody); 4) biotin-labeled fluorophore; and 5) a quantity of avidin. Components 1-4 can be combined simultaneously or sequentially. For greater sensi-tivity, it is also possible to combine the compon-ents sequentially and to vary the order in which the ; 20 components are combined. For example, it is poss-ible to combine biotin-labeled ligand, an unknown quantity of a ligand of interest (in a known volume of liquid) and a specific binding partner and to incubate them. Avidin is added, followed by a biotin-labeled fluorophore. The components can also be combined sequentially in the following order:
biotin-labeled ligand; an unknown quantity of ligand (in a known volume of liquid); specific binding partner for the ligand of interest; biotin-labeled fluorophore; and avidin.

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Fluorescence polarization can be measured, using known techniques, before and afte:r the addi-tion of avidin. Alternatively, fluorescence polari-zation can be measured after all five components OS have been combined and the resulting measurement compared with that for an avidin-free control (i.e., which includes components 1-4 listed above). An increase in polarization when avidin is present is indicative of its binding to the biotin-labeled fluorophore. The extent of increase in polarization is inversely related to the quantity of the ligand of interest present in the liquid analyzed. For example, as the quantity of antigen present in the liquid to be analyzed decreases, biotin-antigen binding with antibody increases. The biotin in the biotin-antigen-antibody complex is not available to react with added avidin. Thus, more binding of the biotin-fluorophore to avidin occurs than is the case when a large quantity of antigen is present in the liquid. As a result, much of the biotin-labeled fluorophore binds to avidin; the avidin-biotin fluorophore is fluorescent and polarization is greater than is the case when a large quantity of antigen is present. The amount of ligand of inter-est in a liquid can be determined by comparing thefluorescence polarization of the liquid with that of a standard of known concentration. For example, it can be compared with a standard curve prepared by measuring the fluorescence polarization of liquids of varying known avidin concentrations. ~lterna-tively; it can be determined by comparing the ~z~

fluorescence polarization of the liquid with that of a standard liquid whose fluorescence polarization is also read.
It is also possible to substract background 05 fluorescence, and thus increase the assay1s sensi-tivity. ~o subtract background fluorescence due to the presence of the components themselves, the following steps are carried out: 1) a known quantity of liquid to be analyzed for the ligand of interest, a known quantity of biotin-labeled ligand, a known quantity of specific binding partner and avidin are combined; 2) fluorescence polarization of the combination is measured. This results in a measure-ment of the fluorescence polarization of the sample itself. Comparison with an avidin-free control, AS
described above, results in determination of the quantity of ligand of interest present in the sample. The bac~ground fluorescence of the avidin-free control can also be subtracted or removed by combining components 1, 2 and 3 listed above, measuring fluorescence, adding component 4, again measuring fluorescence and determining the differ-ence between the two measurements. Then fluores-cence polarization of the avidin-free combination is determined; this provides measurement of fluores cence polarization of the control.
If a homogeneous immunoassay is to be carried out for a ligand of interest which is an antibody, the procedure is similar to that described above for determination of an antigen. Such an assay is useful for determining antibodies of interest such ~' as anti-rubella antibodies, anti-DNA antibodies and anti-insulin antibodies. Here, however, a known quantity of liquid to be analyzed for an antibody of interest is incubated with a known quantity of 05 biotin-labeled specific binding partner (which in this case is biotin-labeled antigen, such as biotin-labeled virus or insulin), biotin-labeled fluorophore and avidin. The components can be combined simultaneously or sequentially and, as described above, the order in which they are combined can be varied (e.g., to increase sensitivity).
As also described above in the description of antigen assay, fluoreæcence polarization can be measured be~ore and after the addition of avidin, to produce a measurement of the change in fluorescence polarization. Alternatively, it can be determined after avidin is added and compared with the fluores-cence polarization of an avidin-free control. Here, to~, background fluorescence of the sample can be subtracted, thus increasing the assay's sensitivity.
Use of the method of the present invention in homogeneous binding processes may be varied in that biotin-labeled ligand (e.g., biotin-labeled antigen~
may be pre-reacted with its specific binding partner (antibody) to form a biotin-labeled ligand/specific binding partner (biotin-labeled antigen/antibody) complex. The resulting complex is incubated with a biotin-labeled fluorophore, a liquid sample of known volume containing an unknown quantity of a ligand of interest and avidin. As described above, fluores-cence polarization is measured before and after the addition of avidin and the change determin~d. The extent of fluorescence polarization varies inversely with the quantity of antigen present in the sample;
05 that is, as the quantity of antigen increases, polarization decreases.
In another embodiment, biotin-labeled specific binding partner is pre-reacted with avidin to produce a biotin-labeled specific binding partner/
avidin. The resulting complex is incubated with a known volume of liquid sample having an unknown quantity of ligand. Biotin-labeled fluorophore is added and fluorescence polarization is determined.
The amount of polarization is compared with that of a control which does not contain avidin.
An example oP the use of the method of the present invention in heterogeneous immunoassays is represented in Figure 4. As represented, the components used are: 1) a biotin-labeled reagent (here, a biotin-labeled antigen); 2) a biotin-labeled fluorophore; 3) an unknown quantity of a ligand of interest (here, an antigen) in a known volume of ligand ; 4) a spesific binding partner bound to a solid phase (here, antibody specific for antigen); and 5) avidin. In this case, a known quantity of liquid to be analyzed for the ligand (antigen) of interest, biotin-labeled ligand and an insoluble phase containing a specific binding partner (antibody) for the ligand are incubated (combined) under conditions appropriate for specific binding reactions to occur. After incubation, the :,:
insoluble phase and the soluble phase of the re-action mixture are separated. A known volume of the soluble phase is incubated with a biotin-labeled fluorophore and a known quantity of avidin, as 05 illustrated in the lower portion of ~igure ~.
Fluorescence polarization is measured, using known techniques, before and after the addition of avidin.
Alternatively, fluorescence polarization of the final combination can be measured (after incubationj and compared with fluorescence polarization of an avidin-free control. An increase in polarization when avidin is present is indicative of its binding to the biotin-labeled fluorophore. The extent of increase in polarization is indirectly related to the quantity of the li~and of interest present in the liquid analyzed. Fluorescence polarization measurements are compared with a standard (e.g., representing fluorescence polarization of solutions of ~nown concentrations) to determine the quantity of ligand of interest present.
Here, too, it is possible to subtract back-ground fluorescence and thus increase the assay's sensitivity. This is done in the following manner:
A known quantity of liquid to be analyzed for the ligand of interest, biotin-labeled ligand and an insoluble phase containing a specific binding partner for the ligand are incubated (combined) under conditions appropriate for specific binding reactions to occur. The insoluble phase and the soluble phase are then separated. A known volume of the soluble phase is combined with avidin and 7~

fluorescence is measured. Biotin-labeled fluoro-phore is subsequently added and fluorescence is again measured. The determination of fluorescence polarization can thus be obtained with background 05 subtracted. An alternative approach to using the method of the present invention in heterogeneous binding processes is possible if the ligand to be detected in liquid has more than one point or site at which it can bind to a specific binding partner.
In this case, an insoluble phase containing a specific binding partner (e.g., antibody) for the ligand (e.g., antigen) to be detected is incubated either sequentially or simultaneously, with a known volume of the ]iquid to be analyzed and biotin-labeled specific binding partner for the liyandbound to the solid phase specific binding partner.
The soluble (liquid) phase (unreacted reagents) and the insoluble phase are separated and a known quantity of avidin is incubated with the insoluble phase. The soluble phase (unreacted avidin) and the insoluble phase (bound avidin) are separated and a biotin-labeled fluorophore is incubated with either the unreacted avidin in the soluble phase or the avidin on the insoluble phase.
Fluorescence polarization is measured, using known techniques, before and after the last incubation (with biotin labeled fluorophore). An increase in polarization is due to binding of the biotin-labeled fluorophore to either the avidin bound to the insoluble phase or the unreacted avidin in the soluble phase. In the first case, in which avidin ~2~7~

; bound to the solid phase is being measured, fluores-cence polarization is directly related to the amount of ligand of interest (e.g., antigen) present in the sample; fluorescence polarization increases as 05 ligand increases. In the second case, in which free avidin is being measured, fluorescence polarization is inversely related to the quantity of ligand present; fluorescence polarization decreases as ligand concentration increases. In addition, the soluble phase of the first incubation can be reacted with avidin and biotin-fluorophore sequentially.
In the case of competitive binding reactions, biotin bound ligand and a biotin-labeled ~luorescent compound ~fluorophore) compete for binding to avidin. As the concentration of biotin-bound ligand increases, binding o~ the biotin-labeled fluorescent compound decreases; that is, if the concentration of biotin-bound ligand is high, binding of biotin-fluorophore to avidin is less than if the concentra-tion of biotin-bound ligand is low. According to the theory of fluorescence polarization, binding of biotin-fluorophore to avidin results in an increase in polarization; the intensity of polarization is related to the concentration of biotin-fluorophore ; ~ 25 bound to avidin.
In the case o~ non-competitive binding re-actions, sequential addition of materials (e.g., biotin, ligand, avidin) can be employed to yield as sensitive assay as with the competitive approach.
Ligands which may be determined according to the present invention are compounds for which one or .

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more specific binding partners can be provided.
Such a specific binding partner is any substance or group of substances which has a specific binding affinity for the ligand, to the exclusion of other 05 substances. Ligands which may be determined through the method of the present invention include, for example, antigens, antibodies, haptens (drugs or other small organic molecules), nucleic acid se-quences (e.g., DNA, RNA) and other substances which 1~ have naturally occurring receptors.
When the ligand is an antigen, the specific binding partner used to detect the antigen is normally the corresponding antibody produced when the antigen is introduced into the blood stream of a vertebrate. Examples of antigens which may be determined according to the instant invention include, for example, polypeptide and protein hormones, human IgE and alpha fetoprotein, a fetal antigen that also occurs in serum of patients with hepatoma and embryonal adenocarcinoma. When the ligand is an antibody, the eliciting antigen may be employed as a specific binding partner. Assay of antibody titers is particularly useful in the diagnosis of, for example, infectious diseases such a syphillus, rubella and infection caused by haemo-lytic streptococci.
When the ligand is a hapten (i.e., a protein-free substance which does not itself elicit antibody formation), specific binding partner utilized to detect the hapten is an antibody produced when the hapten, bound to an antigenic carrier, is introduced 37L~i into the blood stream of a vertebrate. Examples of haptens which may be determined according to the instant invention include steroids such as estrone, estradiol, testosterone, pregnanediol and 05 progesterone; vitamins such as B12 and folic acid;
triodothyronine, thyroxine, histamine, serotonine, digoxin, prostaglandins, adrenalin, noradrenalin, morphine, hormones, and antibiotics, such as peni-cillin.
When the ligand is a substance having a natur ally occurring receptor, the receptor can be utilized as the specific binding partner for detect-ing the ligand, if the receptor can be isolated in a form specific or the ligand. Ligands which have lS naturally occurring receptors include, for example, thyroxine, many steroids, polypeptides, such as insulin and angiotensin and many others. Receptors for this class of ligands are usually proteins or nucleic acids.
Ligands which are determined àccording to the instant invention with the aid of a non-competitive binding process (i.e., the "Sandwichl' technique) must have at least two points or sites for binding to their specific binding partner in order to bind with both the insoluble phase containing specific binding partner and biotin labeled specific binding partner. This is not necessary when a competitive binding process is employed.
Preparation of the biotin labeled substance (i.e., biotin-labeled specific binding partner or biotin-labeled ligand) may be accomplished by simply mixing the entity to be labeled with biotin N-~2~t7~

hydroxysuccinimide ester (BNHS) in a suitable solvent such as dimethylformamide. Although the use of BNHS is preferred, other suitable reagents and/or methods may be employed.
05 Biotin-labeled fluorescent compound (fluoro-phore~ can be any fluorescent compound, labeled with biotin, which exhibits polarization. For example, fluorescein, rhodamines, pyrine-derivatives, umbellipherones and porphyrins can be used. Bio-tin-fluorescein is particularly useful in the method of the present invention and can be prepared in the following manner: Fluorescein isothiocyanate, 10 mg (25 umoles), is dissolved in 2 mls of dimethyl-formamide; 8 ul of ethylene diamine (120 umoles) are added at room temperature. The precipitate formed is recrystallized in dimethylformamide, filter and dried. (The precipitate may form immediately;
however, the reaction is allowed to proceed for 40 hours.) The fluorescein-ethylene diamine adduct, 6.5 mg (14 umoles) is dissolved in 2 mls of dimethylsufoxide; 9 mg (26 umoles) of N-hydroxysuccinimidobiotin are added and the resulting mixture allowed to stand at room temperature for several hours. The conjugate is purified by chromatographing 100 ul of the above reaction mixture on a silica gel column ~2X1O5 cm, 230-400 mesh, 60A pore size), preequilibrated and eluted with methanol.
Preparation of the insoluble phase containing specific binding partner for the ligand to be determined can be accomplished by known methods.

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For example, the specific binding partner can be attached to a solid carrier by cross-linking, by covalent binding or by physical coupling. Examples of solid carriers used in the instant invention 05 include polypropylene tubes, polystyrene microtiter plates and nylon beads. When the ligand to b~
detected is an antigen, preparation of the insoluble phase can be accomplished by simply coating the tubes or plates with the appropriate antibody. When nylon beads are used, the appropriate antibody may be coalently coupled to the beads by the method of Faulstich et al. described in FEBS Letters, 48:226 (1974).
Liquids in which the presence and quantity of a ligand of interest can be determined may be naturally-occurring or synthetic. In many cases, the liquid will be a biological fluid, such as serum, plasma, whole blood, urine, amniotic fluid and cerebrospinal fluid. Ligands may also be determined by dissolving or dispersing the ligand in an appropriate non-aqueous liquid.
Determination of fluorescence polarization is carried out using known techniques and standard equipment. See, for example, Freifelder, D., Fluorescence Spectroscopy, In: Physical Biochemistry (2d ed.), 537 572, W.H. Freemant & Co. (1982).
The present invention will now be illustrated by the following examples, which are not to be considered limiting in any way.

EXAMPLE 1: Synthesis of Fluorescent Biotin Conjugate Avidin, biotin, N-hydroxysuccinimidobiotin, and fluorescein isothiocyanate were obtained from Sigma Chemical Co. ~11 solvents and buffers were of 05 reagent grade quality.
Polarization values were determined using an SLM fluorometer with xenon arc lamp, a monochrometer in the excitation beam set to 491 nm and a 10 nm handwidth 520 nm filter in the emission beam.
Polarizing filters were placed in the excitation and emission beams so the emission filter could be aligned parallel with or perpendicular to the excitation filter. An SLM SPC-822 data processing module was used in the photon counting mode to determine polarization values.
Synthesis of biotin-fluorescein. Fluorescein isothiocyanate, 10 mg (25 umoles), was dissolved in 2 ml. of dimethylformamide; 8 ul. of ethylene diamine (120 umoles) were added at room temperature.
A precipitate formed immediately. The reaction was allowed to proceed for 40 hours at room temperature.
The precipitate was recrystallized in dimethyl-formamide, filtered and dried. The fluorescein-ethylene diamine adduct, 605 mg (14 umoles), was dissolved in 2 mls of dimethylsulfoxide; 9 mg (26 umoles) of N-hydroxysuccinimidobiotin was added and the resulting mixture allowed to stand at room temperature for several hours. The conjugate was purified by chromatographing 100 ul of the above reaction mixture on a silica gel column (20x1.5 cm, 230-400 mesh, 60A pore size), preequilibrated and eluted with methanol. Three fluorescent peaks separated with the lead fraction exhibiting maximum fluorescence polarization change upon complexation with avidin.

05 EXAMPLE 2: Use of Fluorescent Biotin Conjugate in Avidin Assay To glass or plastic tubes or disposable cuvettes was added 1.3 mls of lxlO 3 M
biotin-fluorescein in 0.1 M phosphate buffered saline, pH = 7.4. The concentration of biotin-fluorescein conju~ate essentially determines the sensitivity of the assay because the high binding constant of biotin for avidin allows virtually complete complexation oE the two to occur at equimolar concentrations. The concentration of the conjugate was chosen to yield a 20:1 sample:buffer fluorescence ratio. No special precautions were taken to reduce buffer fluorescence. To this solution was routinely added sufficient avidin to yield the concentrations shown in Figure 2. The biotin-fluorescein conjugate can be added to the assay in a volume as small as 1 ul. The remainder of the assay volume (1.35 ml. total in this assay configuration) can be the avidin sample; the sample thus experiences virtually no dilution. The assay mixture was allowed to incubate at least three minutes at room temperature. Fluorescence polari-zation of the mixture was then determined. These results were used to construct a standard curve (avidin concentration in ng/ml v. polarization) for :``
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, -3~-use in determination of avidin in samples having an unknown concentration of avidin. A representative standard curve is shown in Figure 2.

EXAMPLE 3: Use of Fluorescent~Biotin Conjugate in 05 Biotin assay Avidin was added to sample containing (known~
differing concentrations of biotin. Avidin was added at a concentration of 40 ng/ml (5xlO lO M) to the above buffer. The mixture was incubated at room temperature for three minutes; biotin-fluorescein was added to yield a final concentration of lxlO 9 M. The mixture was incubated at room temperature for an additional three minutes, and fluorescence polarization determined. The (40 ng/ml.) concen-tration of avidin used in this example resulks in approximately 50% of the maximum polarization change.

_XAMPLE 4: Homogeneous Fluorescence Polarization Immunoassay The following materials were obtained from Sigma Chemical Co.: avidin, N-hydroxysuccinimidobi-otin, fluorescein isothiocyanate, and goat anti-rabbit IgG IgG, IgG fraction. Rabbit IgG fraction was obtained from Pel~Freez Biologicals. All solvents and buffers were of reagent grade quality.
Biotin-fluorescein was synthesized as described above.
Synthesis of biotinylated rabbit IgG was carried out as follows: rabbit IgG (2 mg, 12.5 ~2~4~3r~

nmoles) was dissolved in 2 mls of pH = 7.4 phosphate buffered saline. N-hydroxysuccinimidobiotin was dissolved in N,N-dimethylformamide and 0.3 mg (880 nmoles) of ~-hydroxysuccinimidobiotin was added and 05 incubated at room temperature for two hours. The biotinylation mixture was purified by chromotography on SEPHADEX G-25 (lx25 cm), using the same buffer as eluent resulting in separation of biotinylated antibody from free biotin. The biotin: antibody ratio was 16, as determined by measuring antibody concentration by absorbance at 280 nm and measuring biotin concentration by the method of Green, N.M., Methods in EnzvmoloqY, McCormack D.B. and L.B.
Wright (ed.) 18A:418 (1970).
Polarization values were determined using an SLM fluorometer with xenon arc lamp, a monochrometer ; in the excitation beam set to 491 nm and a 10 nm b,andwidth 520 nm filter in the e~ission beams.
Polarization filters were placed in both excitation beams in order to make it possible to align the emission filter in parallel with or perpendicular to the excitation filter. An SLM SPC-822 data process-ing module was used in the photon counting mode to determine polarization values.
Antibody assaY. To glass or plastic tubes or disposable cuvettes was added various amounts of goat anti-rabbit IgG. Biotin-antigen (rabbit IgG) was added to yield a Einal assay concentration of 60 ng/ml and the solution was incubated one hour at room temperature. Avidin was then added to a ~ concentration of 80 ng/ml, followed after three :
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~2~7fl~6 minutes by sufficient biotin-fluorescein to yield a concentration of lxlO 9 M. Polarization was then measured. All materials were made up in 0.1 M
phosphate-buffered saline. Antibody sample may 05 approach the total volume of the assay as all other components may be added in very small volumes. The concentration of the other components was determined as follows.
Biotin-antigen concentration was determined by adding various concentrations of biotin-antigen to sufficient avidin to obtain a final concentration of 80 ng/ml. This combination was incubated for three minutes. Biotin-fluorescein (final concentration lxlO 9 M) was added and polarization was determined.
The level oP biotin-antigen yielding 20% of maximum polarization change was chosen for the antibody and antigen assays.
The avidin concentration was chosen by select-ing the level that yielded 80% of maximum polariza-tion change when the effect of varying concentrationof avidin on the polarization of biotin-fluorescein was determined (Figure 2).
; The biotin-fluorescein concentration was chosen to yield a 20:1 sample:buffer fluorescence ratio with no special precautions to reduce buffer flu-orescence.
Antigen assay. Various amounts of antigen, rabbit IgG, yielding the concentrations shown in Figure 5 were incubated one hour with goat anti-rabbit IgG IgG (4.4 ug/ml final concentration).Using the above concentrations, biotin-antigen was , , ~ .. , . ~ . .

then added and incubated for one hour. Avidin was added and after three minutes, biotin-f:Luorescein was added and the polarization determined.
Incubation times of one hour tantigen with 05 antibody) may be reduced to several minutes~
This assay may also be carried out with the immunological components bound to an appropriate solid phase. This requires longer reaction times, to allow for diffusion to the surface, but may yield improved sensitivity or a means of removing inter-fering sample substances.
The assay presented is a homogeneous Eluores-cence polarization immunoassay applicable to molecules regardless of their molecular weight. 'rhe strategy for this assay is outlined in Scheme 1.
The assay is useful for detecting and quantifying antigens or antibodies. It is also applicable to any binding protein and its receptor as well as small molecules and their (large or small) binding agents.

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Scheme 1 Reaction Variable Constant Products_ Polarization ' Fluorophore only . - B-F ~ low 1. ~Fig. 1) avidin B-F B-F--avidin high , 2. tFig. Z? B-~g + avidin B-F low B-F avidin--B-A

3. ~Fig. 5) Ab + B-Ag B-F--avidin high avidin B-Ag--Ab B-F

~. (Fig. 6) Ag + Ab B-F low :j B-Ag Ag--Ab avidin avidin--B-Ag B-F
, . . .
Abbreviations: B-F = biotin-fluorescein B Ag = biotin-rabbit IgG
Ab = goat anti-rabbit IgG IgG -Ag - rabbit IgG

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The assay probe (B-F) is biotin covalently coupled to a fluorophore, which in this case is fluorescein. When excited by polariz~d light, B-F
emits light which is depolariæed (polarization is 05 low) because of the rapid tumbling of this small mole_ule. When complexed to the more slowly tumbling macromolecule avidin, polarization is high (reaction 1.). Complexing of biotin and fluorescein is modulated by other components. Biotin is covalently coupled to one component ~i.e., an antigen) of a comp-ex-forming pair which consists of a ligand (i.e., the antigen) and a specific binding partner for the antigen (i.e., an antibody). The biotin-antigen is added in a concentration suffi-cient to complex with the avidin, allowing B-F to remain uncomplexed treaction 2). If antibody is added initially, antibody complexes biotin-antigen and by steric blocking prevents avidin from binding biotin-antigen. This makes it possible for avidin to complex B F resulting in high polarization (reaction 3.). When free antigen is added initi-ally, antigen complexes antibody, which makes it possible for biotin-antigen to complex avidin. This results in free B-F and low polarization.
Thus the assay for antibody consists of re-action 3 and the assay for antigen is reaction 4.
The change in polarization that occurs in each sequential reaction occurs in a manner dependent on the concentration of the variable reagent, as sh3wn in the figures.

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The effect of varying avidin (reaction 1) on the polarization of B-F is shown in Figure 2. The polarization varies from 0.06 to 0.23 in this example; more highly purified samples of biotin-05 fluoroscein show a polarization change from0.02-0.31. A concentration of lxlO 9 M avidin was chosen for use in the following reactions.
A standard curve for the assay of antibody Ireaction 3) is represented in Figure 5. Varying ; 10 concentrations of antibody were added to the above biotin-antigen and incubated for one hour. Avidin and B-F were added and polarization measured. A
typical competitive binding curve can be constructed from the data. Shorter incubation times may be used. This is IgG fraction polyclonal antibody and requires a higher concentration of antibody than biotin-antigen to neutralize the latter. This is due at least in part to its lack of purity, but may also indicate a requirement for more than a one-to-one complex to prevent access by avidin to the biotin. The assay range lies between about 0.2 and 20 ug/ml for this impure fraction. A concentration of 3xlO 8 M (4.4 ug/ml) was chosen for use in the following antigen assay.
A standard curve for the assay of antigen (reaction 4) is shown in Figure 6. The assay range is about 40 to 400 ng/ml (2xlO to 2xlO 9 M). The lower quantities of antigen required to neutralize the antibody is consistent with both explanations (see rationales above) for the amount of antibodyrequired. The near equivalence of avidin and ';

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antigen levels required is remarkable and shows that there is no significant loss of sensitivity through the several steps in the procedure.

EXAMPLE 5: Heterogeneous Fluorescence Polarization Immunoassay Polystyrene test tubes were coated with one ml.
of a solution of affinity purified goat anti-rabbit IgG at 20 ug/ml 0.005 M phosphate buffered saline ~PBS) (pH 7.4) for three days. The tubes were rinsed twice with 0.05M PBS (pH 7.4) and filled with ; 900 ul 0.05 M PBS (pH 7.4). Serial dilutions of antigen were prepared in 0.12% Triton X-100 PBS: 100 ul aliquots were incubated in antibody-coating tubes for 2.5 hours at 37C.
Antigen-biotin was added in 50 ul 0.12%
TritonTM X-100 PBS to yield a final concentration of 84 or 42 ng/ml. The tubes were incubated overnight at 37C. Avidin was then added in 50 ul PBS to give a final concentration of 69 ng/ml followed by fluoroscein-biotin at a final concentration compar-able to that in the homogeneous assay ~see Example 4~. The fluorescence polarization was then deter-mined.

EXAMPLE _: Simultaneous Assay An alternative approach to this methodology has also been shown to be effective and to result in simplification of the assay in terms of the number ; of components in the antigen assay and the number of additions required. This involves a prior combina-. .

~2 -4~-tion of components, rather than a sequential one and the elimination of the biotinylated antigen. Though an apparently minor variation in protocol, this changes the basis of the assay. The sequential 05 assays above rely on a competition for avidin between biotin-fluorophore and biotin-antigen. This is modulated by antibody, which sterically prevents the large molecule avidin from binding the biotin-antigen. In the simultaneous assay, an avidin:
biotinylated antibody complex is preformed. The addition of analyte (antigen) results in formation of larger complexes which sterically prevent small biotin-fluorophore from having access to the avidin.
Biotin-fluorophore and avidin, therefore, do not bind. Similar simultaneous assays Por antibody using a preformed avidin: biotinylated-antigen complex can be performed.
In this method, avidin is mixed with bio-tinylated antibody in a 10:1 avidin:biotin-antibody ratio (this may be varied). This serves as a reagent. This complex is used at a concentration such that avidin is in site molar equivalence to the biotin-fluorophore concentration to be used in the assay. Varying concentrations of analyte antigen are incubated for short times with the avidin:
biotin-antibody complex, biotin-fluorophore is added, and the polarization determined. Results are shown in Figure 7, which represents the effect of rabbit IgG in the presence of avidin:biotin-anti-rabbit IgG on the polarization of biotin-fluorescein.

4~i Equivalents Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific 05 embodiments of the invention described herein. Such equivalents are intended to be encompassed within the scope of this invention.
.

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Claims

1. A method of determining a ligand of interest in a liquid, comprising:
a. incubating the liquid, biotin-labeled ligand of interest, biotin-labeled fluoro-phore, specific binding partner for the ligand of interest and avidin, under conditions appropriate for binding of ligand of interest and its specific binding partner to occur; and b. measuring the change in fluorescence polarization which occurs after the addition of avidin.

2. A method of Claim 1 wherein the biotin-labeled fluorophore is biotin-labeled fluorescein.

3. A method of Claim 1 wherein the ligand of interest is an antigen or a hapten and the specific binding partner is an antibody specific for said antigen or an antibody specific for said hapten.

4. A method of Claim 1 wherein the ligand of interest is an antibody and the specific binding partner is an antigen for which the antibody is specific.

5. A method of determining a ligand of interest in a liquid, comprising:
a. incubating the liquid, biotin-labeled ligand of interest, biotin-labeled fluoro-phore, specific binding partner for the ligand of interest and avidin, under conditions appropriate for binding of ligand of interest and its specific binding partner to occur; and b. comparing fluorescence polarization after incubation with fluorescence polarization of an avidin-free control, the control comprising the liquid, biotin-labeled ligand of interest and biotin-labeled fluorophore.

6. A method of determining a ligand of interest in a liquid, comprising the steps of:
a. combining a known volume of a liquid to be analyzed for the ligand of interest;
biotin-labeled ligand of interest; biotin-labeled fluorophore; and specific binding partner for the ligand of interest, under conditions appropriate for specific binding reactions to occur between ligand of interest and its specific binding partner;
b. measuring fluorescence polarization of the resulting combination;
c. adding avidin to the resulting combina-tion, under conditions appropriate for binding of avidin and biotin; and d. measuring fluorescence polarization of the liquid.

7. A method of Claim 6 wherein the ligand of interest is an antigen or a hapten and the specific binding partner is an antibody specific for the antigen or an antibody specific for said hapten.

8. A method of determining a ligand of interest in a liquid, comprising:
a. incubating 1) a known volume of the liquid; 2) a complex comprised of biotin-labeled ligand of interest bound to a specific binding partner for said ligand of interest; and 3) avidin, under condi-tions appropriate for binding of ligand of interest and its specific binding partner to occur;
b. adding biotin-labeled fluorophore; and c. determining fluorescence polarization of the liquid.

9. A method of determining a ligand of interest in a liquid, comprising:
a. incubating a known volume of the liquid; a complex comprised of biotin-labeled ligand of interest bound to avidin; biotin-labeled fluorophore; and specific binding partner for said ligand of interest, under conditions appropriate for binding of ligand of interest and its specific binding partner to occur; and b. comparing the fluorescence polarization of the liquid after incubation with fluores-cence polarization of an avidin-free control, the control comprising the liquid, biotin-labeled fluorophore and specific binding partner for said ligand of interest.

10. A method of determining a ligand of interest in a liquid, comprising:
a. incubating an insoluble phase containing a specific binding partner for the ligand of interest, the liquid, and biotin-lableled ligand;
b. separating the insoluble phase from the soluble phase after incubation;
c. incubating the soluble phase with biotin-lableled fluorophore;
d. measuring fluorescence polarization of the soluble phase;
e. incubating the soluble phase with a known quantity of avidin; and f. determining the change in fluorescence polarization of the soluble phase after incubation.

11. A method of Claim 10 wherein the ligand of interest is an antigen, the specific binding partner is an antibody specific for the antigen and the biotin-labeled fluorophore is biotin-labeled fluoroscein.

12. A method of determining a ligand of interest in a liquid, the ligand of interest having at least two binding sites for its specific binding partner, comprising:
a. incubating an insoluble phase containing a specific binding partner for the ligand of interest, the liquid, and biotin-labeled binding partner, under conditions appropriate for binding of the ligand of interest and its specific binding partner;
b. separating the insoluble phase from the soluble phase;
c. adding avidin to the soluble phase.
d. incubating an aliquot of the avidin-containing soluble phase with biotin-labeled fluorophore; and e. measuring the fluorescence polarization of the aliquot after incubation.

13. A method of determining a ligand of interest in a liquid, comprising:
a. incubating an insoluble phase containing a specific binding partner for the ligand of interest, the ligand and biotin-labeled specific binding partner for the ligand of interest, under conditions appropriate for binding of the ligand of interest and its specific binding partner;
b. separating the insoluble phase from the soluble phase;

c. incubating avidin with the insoluble phase;
d. separating the insoluble phase from the soluble phase;
e. incubating the insoluble phase with biotin-labeled fluorophore or incubating the soluble phase with biotin-labeled fluorophore; and f. measuring the fluorescence polarization after incubation.

14. A method of Claim 13 in which the ligand of interest is an antigen, the specific binding partner is an antibody specific for the antigen and the biotin-labeled fluorophore is biotin-labeled fluorescein.

15. A method of determining an antigen of interest or a hapten of interest in a liquid, compris-ing:
a. incubating a known quantity of the liquid;
biotin-labeled antigen of interest or biotin-labeled hapten of interest; anti-body specific for the antigen of interest or antibody specific for the hapten of interest; biotin-labeled fluoroscein and avidin; and b. determining the change in fluorescence polarization.

16. A method of determining a ligand of interest in a liquid, comprising:
a. incubating a known volume of the liquid with 1) a complex comprised of avidin, biotin and biotin-labeled specific binding partner for the ligand of interest; 2) ligand of interest; and 3) biotin-labeled fluorophore, under conditions appropriate for binding reactions to occur between ligand of interest and its specific binding partner;
b. measuring fluorescence polarization after incubation; and c. comparing the measured fluorescence polarization with fluorescence polariza-tion of a control, the control comprising the liquid, ligand of interest and biotin-labeled fluorophore.

17. A method of determining an antibody in a liquid, comprising:
a. incubating a known volume of the liquid;
biotin-labeled antigen for which the antibody is specific; avidin and biotin-labeled fluorescence;
b. measuring fluorescence polarization after incubation; and c. comparing the measurement with fluores-cence polarization of an avidin-free control.

18. A method of determining the concentration of biotin in a sample of unknown biotin concentra-tion, comprising:
a. incubating the sample with avidin, under conditions appropriate for binding of biotin and avidin;
b. adding a fluorescent-biotin conjugate after incubation, and incubating under conditions appropriate for binding of avidin and biotin:
c. measuring fluorescence polarization after incubation; and d. comparing the measured fluorescence polarization with fluorescence polariza-tion measurements of samples of known biotin concentration.

19. A method of Claim 18 wherein the fluorescent-biotin conjugate is fluorescein-biotin.

20. A method of determining the concentration of avidin in a sample of unknown avidin concentra-tion, comprising:
a. incubating the sample with a fluorescent-biotin conjugate, under conditions appro-priate for binding of avidin and biotin;
b. measuring fluorescence polarization of the sample after incubation; and c. comparing the measured fluorescence polarization measurements of samples having known avidin concentration.

21. A method of Claim 20 wherein the fluorescent-biotin conjugate is fluorescein-biotin.

22. A method of Claim 21 wherein the final concen-tration of fluorescein-biotin is approximately 1x10-9M.