WO1997020213A1 - Luminescent probes for protein detection - Google Patents

Abstract

The invention relates to novel chemical complexes and compositions comprising lanthanide chelates. Methods for detecting, quantifying and isolating targets are described. Such methods comprise contacting the target with a lanthanide chelate, illuminating the resulting lanthanide-chelate-target complex with electromagnetic radiation and detecting emitted phosphorescence of the lanthanide identifying the presence and location of the complex. The chelate comprises a first domain that binds to the lanthanide, a second domain that specifically and reversibly binds to the target and a third domain that absorbs UV light. Lanthanide chelates can be safely and completely eluted from the target and the target isolated and utilized for additional applications. These lanthanide chelates or lanthanide-chelate-target complexes can be used in kits for the rapid, specific and sensitive detection of targets from samples obtained from patients, animals, cultures or the environment.

Description

LUMINESCENT PROBES FOR PROTEIN DETECTION BACKGROUND OF THE INVENTION Field ofthe Invention

The present invention relates to methods and compositions for reversible binding to and detection of targets using luminescent lanthanide chelates, and lanthanide-chelate-target complexes The targets may be proteins or nucleic acids The invention also relates to kits which comprise lanthanide chelates or lanthanide-chelate-target complexes

Description ofthe Background Routine total protein quantification is fundamental to a wide variety of scientific endeavors and applications Protein quantification is necessary, for example, in enzyme isolation procedures to determine purity and yield and for membrane receptor studies to evaluate ligand binding data Total protein is often the parameter of choice for sample standardization in electrophoretic separations Urinary protein measurement is used clinically to screen pathological conditions such as myeloma (immunoglobulin light chains) and tubular disorders (β2 microglobuhn), as well as diabetes related retinopathy and nephropathy (albumin) Nutritionists calculate total body protein in laboratory animals to determine total body energy and metabolic efficiency Proteins are quantified by a variety of methods that are based upon different physical principles Ultraviolet light spectrophotometry utilizes the intrinsic absoφtive properties of aromatic armno acid side chains for quantification Turbidimetπc methods involve protein precipitation and subsequent spectrophotometric analysis Other solution-based techniques, such as the biuret, bicinchroninic acid and Folin-Ciocalteau assays, rely upon chemical reactions with the protein, leading to the generation of a colored product

Finally, solution-based methods such as the Coomassie brilliant blue and pyrogallol red-molybdate assays measure shifts in the wavelength maximum of dye absorbance upon formation of a dye-protein complex Many solution-based colorimetric total protein labels are adaptable to solid-phase formats Since the protein is specifically bound to a membrane or other solid support, contaminants are readily washed away Therefore, solid-phase protein assays are less susceptible to interfering agents commonly found in the sample, such as 2-mercaptoethanol, Tris, salts and detergents Total protein quantification is easily achieved directly on the membrane support by densitometry using instrumentation such as a flat-bed scanner or CCD camera Alternatively, the stains can be eluted from membranes and quantified using a spectrophotometer Since the assays are performed on a solid-phase support, both small and large sample volumes are readily accommodated Very dilute protein solutions are easily concentrated on the membrane by repeated application of samples The colored product is usually stable, so there are no stringent time requirements for completion ofthe assays Analysis is rapid and a permanent visual record is generated Finally, solid-phase assays are readily amenable to automation, using 96-well plate readers All of these colorimetric methods are, however, fairly insensitive because they are limited by the molar absorptivity of the colored product An alternative technique involves labeling protein with radioactive elements Radiolabel techniques can be exquisitely sensitive and are often compatible with subsequent biochemical analyses However, regulatory bodies frown upon the use of radiolabeled materials in instruments such as protein sequencers because ofthe significant contamination problems Thus, the hazards of handling radioactive materials, coupled with limited shelf life, disposal problems and expense, have motivated the search for alternative detection methods

Chemiluminescence is a special type of fluorescence whereby light is produced as the result of a chemical reaction When this process is performed through biological means it is referred to as bioluminescence The firefly enzyme luciferase, a well-known example, produces light upon the oxidation of luciferin

Fluorescence is the emission of light as a molecule returns from an excited state to the ground state Phosphorescence is similar to fluorescence, but the excited molecule first undergoes a transition to a long-lived excited state It then returns slowly to the ground state over several seconds and light is emitted Phosphorescence and fluorescence are often not carefully distinguished in the technical literature, however, and many phosphorescing labels are referred to as fluorescent However, in reality a given molecule may both fluoresce and phosphoresce, making the distinction somewhat academic Examples of organic luminescent labels include coumarin, fluorescamine, rhodamine, erythrosine, eosine, dansyl chloride, and their derivatives

Although the measurement of light emission is intrinsically more sensitive than the measurement of light absorbance, conventional fluorescent detection methods are limited by high background signals Background is created by many components ofthe detection system, including the excitation and detection apparatus, sample holders, particulate matter and the substrates or reagents themselves Background fluorescence is particularly problematic in biological samples For example, serum has relatively strong background fluorescence and is also affected by heavy scattering, where the excitation light is dispersed by collisions rather than being absorbed

One method of increasing the sensitivity of luminescent techniques is time resolved fluorometry In this technique, the sample is excited for only a brief period of time The label phosphoresces for a longer period than the background fluorescing molecules Thus, after some period of delay, during which the background signal declines, the phosphorescence ofthe label is measured

Certain rare earth metals, alone or combined with various chelators, have the ability to emit light Fifteen ofthe rare earth elements classified in Group IIIB ofthe Periodic Table are provided in Table 1 Table 1 The Rare Earth Elements

Cerium Subgroup Yttrium Subgroup

Lanthanum La Europium Eu

Cerium Ce Gadolinium Gd

Praseodymium Pr Terbium Tb

Neodymium Nd Dysprosium Dy

Promethium Pm Holium Ho

Samarium Sm Erbium Er

Thulium Tm

Ytteribium Yb

Lutetium Lu

The rare earth elements comprise the cerium and yttrium subgroups Although yttria is not a rare earth element, it is typically found associated with the rare earths and can only be separated with difficulty Typical sources ofthe rare earths include monazite, bastnasite and related fluocarbonate materials as well as minerals ofthe yttrium group. Rare earth elements also occur as fission products of uranium and plutonium, and are the only source of promethium

The luminescence of a metal chelate is, in many ways, analogous to the process of photosynthesis. In the plant, the chlorophyll molecules and accessory pigments (carotenoids) are clustered into light-harvesting antenna complexes in the lamellae ofthe thykaloid discs ofthe chloroplasts Light is absorbed by a molecule of chlorophyll or one ofthe accessory pigments, which leads to an excited state The excited state is passed from one pigment molecule to another until it reaches the reaction center. At the reaction center, a special chlorophyll molecule converts the energy ofthe excited state to chemical energy in the form of ATP or NADPH In the case of the rare earth metal chelates, the metal ion can be thought of as the reaction center. The organic chelator molecules act as the light- harvesting antenna for the metal ion, but instead of converting the absorbed light into chemical energy, the metal ion re-emits the energy as light The chelator molecules efficiently absorb light and pass it to the metal ion This excites electrons from the F shell to the D shell and, upon subsequent decay to the ground state, longer wavelength light is released The nature of the chelate determines the efficiency and wavelength of the absorbed light, but the emission wavelength is determined by the metal ion itself and is not greatly affected by the antenna molecules Metals used in this fashion include the lanthanides, europium, terbium, samarium, gadolinium, and dysprosium The lanthanide chelates, especially those of europium and terbium, are well suited as biological labels The absorbance ofthe chelates can be very strong and varies with the chelate selected, but generally the excitation maximum is within the UV (200-360 nm), allowing the use of commercially available lamps or lasers. Long UV is preferred because short UV is hazardous to work with and many materials, such as glass and water, are opaque to it

Although the quantum yield of lanthanide chelates is often less than that of organic luminescent labels, the lanthanides emit at relatively long wavelengths where the background biological fluorescence is low The long Stokes shift makes it easy to detect emitted light without flooding the detector with excitation wavelength light and the long lifetime of the luminescence (50-1000 μsec) allows the measurement of signal after the short duration biological fluorescence has decayed to background For all of these reasons, the signal to noise ratio is high

It has recently become possible to sequence femtomolar concentrations of protein and, hence, desirable to detect extremely small concentrations of protein either in solution or immobilized on supports Although there are many protein labels available, most were not specifically developed for compatibility with direct N-terminal microsequencing or m situ digestion followed by internal protein microsequencing from membranes

One group recently compared several labeling methods for compatibility with protein microsequencing (Christiansen, J , & Houer, G (1992) Electrophoresis 13 179-83) Proteins were electroblotted to polyvinylidene difluoride membrane and labeled with Coomassie brilliant blue, mercurochrome, Amido black, Dabitc, light green, silver, and colloidal gold In that test, only Coomassie brilliant blue was fully compatible with subsequent microsequencing However, Coomassie brilliant blue is not compatible with nitrocellulose membranes because the membrane itself strongly binds the dye, seriously diminishing the sensitivity

Recently, iron chelates have been utilized as labels that produce colored complexes when complexed with proteins (Patton, W F , et al (1994) Anal. Biochem. 220 324-335) This total protein labeling technique was shown to be compatible with microsequencing and immunodetection, but increased sensitivity and a broader dynamic range is still desired Furthermore, the iron chelate technique as described is incompatible with detection of protein in semi-solid materials such as gels

Certain lanthanide chelates have been used as biological labels in limited instances A generic description of the lanthanide chelates comprises a triplet sensitizer nucleus and three heteroatom containing groups (U S Pat No 4,637,988) This structure allows for the transfer of energy from the chelate to the lanthanide

Time resolved fluorometry has been used in combination with some lanthanide chelates (U S Pat No 4,374,120) This technique utilizes an EDTA- like molecule that is covalently bound to a target molecule, such as an antibody The bifunctional EDTA-like molecule allows the binding of both the antibody and the lanthanide The resulting structure is further complexed with a di-ketone or di¬ hydroxy molecule which functions to absorb light The 1/1/1 (antibody- EDTA/Eu/diketone) complex is then detected by time resolved fluorometry The method is suitable for sandwich immunoassays where the secondary antibody is conjugated to an EDTA-like molecule which is then capable of complexing with a lanthanide chelate As the EDTA-like compound must be attached to the target protein, the technique is generally unsuitable for most biochemical analyses, including microsequencing

Chelates of europium (Eu) and terbium (Tb) have been incoφorated into latex beads to reduce the level of quenching in aqueous media These beads may be bound to antigen, to antibody or to enzymes and have been used in a variety of biological assays (U S Pat No 4,259,313) Lanthanide chelates may also be covalently bound to bioaffinity or ligand compounds which can be used in immunoassays, nucleic acid hybridizations, lectin assays and the like (WO 92/16839) Bioaffinity reactions are performed on a solid support to isolate specific proteins Phosphorescence is read after elution from the support for ease and sensitivity of detection Also, a multi-label, time resolved assay for detecting the phosphorescence of covalently bound lanthanide-chelate-nucleic acids has been used in automated DNA sequencing (WO 90/00623) These techniques, however, are of limited value due to undesirable covalent modifications of the molecule of interest or the limited availability of suitable bioaffinity-ligand pairs

It is also possible to enzymatically amplify signal detection (U S Pat No. 5,262,299, WO 89/10975) Briefly, a selected substrate is enzymatically and reversibly transformed into a compound that will complex with lanthanide and phosphoresce One example of a combination that functions in this manner is the alkaline phosphatase and 5-fluorosaIicylate phosphate compounds Alkaline phosphatase converts 5-fluorosalicylate phosphate into 5-fluorosalicylate which combines with europium and phosphoresces when excited at the appropriate wavelength Similarly, several chelates have been tested in enzymatic, time resolved, fluorometric immunoassays (U S Pat No 5,312,922) A hydroxyl group on a chelate may be converted to a phosphate ester or galactoside for use with the enzymes alkaline phosphatase or β-galactosidase Esters are enzymatically treated to be phosphorescent (or non-phosphorescent) when combined with a lanthanide Although the range of detectable molecules may be increased by conjugating the enzyme to the molecule of interest, such as a nucleic acid or an antibody, covalent attachment to an enzyme generally eliminates the possibility of subsequent analyses such as microsequencing.

Protein staining with terbium chloride has been recently described (R.A. Copeland, (1994) Anal. Biochem. 220.218-19). Terbium is precipitated in sodium dodecyl sulfate (SDS) gels to form a cloudy background As terbium does not interact with protein, the protein bands remain clear Upon illumination with UV radiation, the terbium precipitate phosphoresces and the protein bands appear dark when viewed with the appropriate filter Although the method is generally applicable to proteins, it is fairly insensitive requiring upwards of 100 ng of protein due, in large part, to the very poor chelating properties of SDS Furthermore, the SDS denatures the protein.

Specific staining of calcium binding proteins and total protein staining was achieved by time resolved fluorometry using both terbium and europium (I. Hill et al. (1994) Anal. Biochem. 216:439-43). In this assay, the lanthanide interacts with tryptophan in the calcium binding pocket of a calcium binding protein. This technique allows for detection of as little as 25 ng of a mutant calmodulin. Total protein staining occurred by weak nonspecific electrostatic attraction between the cation and the protein Binding occurred via carboxyl groups which are generally negatively charged at pH 6 5. Binding was sufficiently weak as to be effectively competed away with low molar EDTA, leaving only the specific calcium binding proteins bound to the lanthanides. Although total protein staining is completely reversible, it is also quite insensitive, being comparable with a Coomassie blue stain Summary ofthe Invention The present invention overcomes the problems and disadvantages associated with current labeling techniques and provides novel methods for the detection and isolation of molecular targets such as proteins and nucleic acids

One embodiment ofthe invention is directed to lanthanide chelate comprising a lanthanide and a ligand wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to a target, and a third domain that absorbs UV radiation Typical lanthanide chelates comprise an element of europium, terbium, samarium, gadolinium or dysprosium, and a ligand such as bathophenanthroline disulfonic acid Targets may be proteins or nucleic acids, or fragments or constituents thereof

Another embodiment of the invention is directed to lanthanide- chelate-target complexes comprising a lanthanide, a ligand and a target, wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to the target, and a third domain that absorbs UV light Targets may be, for example, proteins, peptides, amino acids, nucleic acids, nucleotides or fragments or modifications thereof

Another embodiment of the invention is directed to methods for detecting a target in a sample The sample to be screened is contacted with a lanthanide chelate wherein the lanthanide chelate reversibly binds to the target This sample is illuminated with electromagnetic radiation, such as ultraviolet radiation, and any radiation emitted is detected and can be quantified Samples which can be tested include patient and environmental samples Because the lanthanide-chelate- target interaction is a non-covalent one, the reaction is fully reversible Thus, the lanthanide chelate can be removed and the target utilized for procedures such as sequencing

Another embodiment of the invention is directed to methods for detecting protein Protein is contacted with a lanthanide chelate wherein the chelate has a first domain that binds to the lanthanide, a second domain that reversibly binds to protein, and a third domain that absorbs UV light Binding is non-covalent and fully reversible Bound protein is illuminated with UV and the emitted phosphorescence is detected

Another embodiment of the invention is directed to a multi-label immunodetection method whereby total protein and one or more target proteins can be sequentially detected A sample is contacted with a primary antibody that specifically binds to a target protein The sample is contacted with a second antibody that specifically binds to the primary antibody, wherein the second antibody has a polyamine-tag which functions to increase the stability of lanthanide chelate binding at increased pH. The sample is contacted with a lanthanide chelate at a first, acidic pH and the phosphorescence of total protein in the sample is measured. The sample is adjusted to a second, basic pH and the phosphorescence ofthe target protein is determined Additional secondary antibodies of increasing polyamine-tag length may be employed when multiple target proteins are to be detected Antibodies may be monoclonal or polyclonal, but preferably the primary antibody is monoclonal and the secondary antibody is polyclonal

Another embodiment of the invention is directed to methods for precipitating a target protein from a sample The sample containing target protein is contacted with a lanthanide chelate wherein the lanthanide chelate reversibly binds to the protein Target protein can be collected by, for example, filtration or centrifugation.

Another embodiment of the invention is directed to methods for isolating a target molecule from a sample without the requirement of covalent modification of the target. The sample is contacted with a lanthanide chelate and the lanthanide chelate reversibly binds to the target The mixture is illuminated with UV to produce phosphorescence Phosphorescing targets are identified and can be isolated from other substances.

Another embodiment of the invention is directed to kits for the detection of a target comprising a lanthanide chelate or a lanthanide-chelate-protein complex Targets may be detected in any biological samples, such as samples from patients, animals, cultures, or from the environment, such as in soil or water samples The kit may further comprise a binding buffer and an elution buffer

Other embodiments and advantages ofthe invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be learned from practice ofthe invention Description ofthe Drawings

Figure 1 Multi-Detection Immunoassay A schematic representation of a multi-detection immunoassay (A ) All proteins are detected at low pH and (B ) sequential detection of polyamine-tagged protein is achieved by increasing the pH 1 is the europium complex; 2 is the amine-labeled secondary antibody, 3 is the primary antibody, 4 is the protein, and 5 is the membrane support

Figure 2 Excitation and Emission Spectra (A ) Excitation and (B ) emission maxima of bathophenanthroline disulfonate-europium complex in solution (solid lines) and bound to bovine serum albumin immobilized on PVDF membrane (dotted lines)

Figure 3 Dynamic Range Determination of the sensitivity and dynamic linear range of bathophenanthroline disulfonate-europium labeling using serially diluted bovine serum albumin bound to polyvinyl difluoride membrane (A ) CCD image of phosphorescing protein bands (B.) Standard curve Figure 4 Bathophenanthroline Disulfonate to Europium Ratio

Optimization of the ratio of bathophenanthroline disulfonate to europium for detection of protein bound to nitrocellulose membrane

Figure 5 Protein-to-Protein Variation Response curves for different proteins immobilized on nitrocellulose membrane and labeled with bathophenanthroline disulfonate-europium

Figure 6 Specificity of Labeling Evaluation of agents that could potentially interfere with bathophenanthroline disulfonate-europium binding to protein

Figure 7 Binding to Polyamino Acids Binding of bathophenanthroline disulfonate-europium to various homopolymers and heteropolymers of amino acids to determine the effect of amino acid side chains on binding

Figure 8 Ampholyte Staining Staining of various ampholytes to determine the effect of amino and carboxyl groups on the binding of bathophenanthroline disulfonate-europium Figure 9 Resistance of Poly-amines to Elution. Bathophenanthroline disulfonate-europium is removed from protein with elution solution Description ofthe Invention

As embodied and broadly described herein, the present invention is directed to chemicals, chemical complexes and methods for labeling targets such as proteins, nucleic acid, and derivatives, and to kits which comprise these chemicals and chemical complexes

A highly sensitive, broadly applicable method for protein detection is presently needed. This method should also be compatible with typical analytical processes such as microsequencing, immunoassay, enzymatic assays, mass spectrometry, carbohydrate analysis and Western blotting Criteria for the labeling of a protein with a sensitive and specific label include high affinity for the protein, low affinity for other biological materials as well as common laboratory reagents, rapid and straightforward labeling techniques and application conditions. The label should also be compatible with a wide range of electrophoretic matrices and have a large quantum yield for maximal sensitivity An emission maximum in the 500- 600 nm region of the spectrum avoids interference from common biological fluorophores It should also have quantifiable binding over a broad range of total protein (a broad dynamic range) The label should have an ability to amplify for increased sensitivity, a reasonable cost, low toxicity and be readily available Labels should also be completely reversible so that other specific stains or biochemical assays may be employed

The lanthanide chelates of this invention meet these requirements as labeling tools for biological and other target molecules Lanthanide chelates comprise an element selected from the lanthanide series and a chelate (or ligand or chelator) that will bind to a metal ion The chelate comprises a ligand that reversibly attaches to a target Attachments may be through hydrophobic, hydrophilic, Van der Waals, and ionic or other non-covalent interactions The chelate absorbs energy from UV radiation and transmits this energy to the lanthanide which, in response, phosphoresces.

Lanthanide chelates and lanthanide-chelate-target complexes possess strong luminescence at wavelengths longer than most background fluorescence Additionally, the lanthanide emits light for a longer period of time, allowing the use of time resolved detection means. This allows for higher sensitivity for lanthanide chelates than for most organic luminescent dyes and a highly sensitive detection technique, based upon the reversible binding of lanthanide chelate to target, can be created. Further, these lanthanide chelates do not cause protein modification such as N- or C-terminal blocking, interfere with enzymatic cleavage sites, mask antigenic recognition sites or change HPLC elution profiles.

Ligands contain a first domain which binds to the lanthanide, and preferably contain side chains that are combinations of ketone, carboxyl, hydroxyl or pyridine groups. A second domain reversibly binds to protein and should consist of one or more anionic residues for electrostatic interaction with protonated amines of proteins (the negative charge of the second ligand domain interacts with the positive charge ofthe protonated amine). Groups that function in this regard include sulfonate, sulfate, phosphonate and phosphate groups. Carboxylate groups are less appropriate for this puφose Aromatic or heteroaromatic functionalities also enhance binding avidity to targets such as proteins by hydrophobic interactions. A third domain absorbs the excitation light which is then transferred to the lanthanide ion, which subsequently emits the light at a longer wavelength This domain preferably contains 5- or 6-member monocyclic or polycyclic, aromatic or heteroaromatic (containing oxygen and/or nitrogen and/or sulfur) ring structures, a necessity for chromophores. Alternatively, the chelating groups may bind and transfer the excitation energy to the surface of a lanthanide colloid to form the luminescent complex.

Chelate and/or target binding sites can be incorporated into the ring structure ofthe chromophore or, alternatively, distinct chelate, chromophore and target binding sites can be engineered, allowing for separate optimization of each domain Lanthanide chelates may also be clathrate compounds which are inclusion complexes wherein molecules of one substance are completely enclosed within another Chelates may also contain three functional domains distributed among two or more molecules For example, one molecule may contain a chromophore and lanthanide-binding domain while the other contains lanthanide-binding and protein- binding domains Resulting complexes between the lanthanide and the two chelators would contain all three functional domains

Chelates that function in this regard include pyridines, quinolines, nicotines, their derivatives and the like, which have anionic moieties attached thereto Most preferably, a sulphonate moiety is used Thus, bathophenanthroline disulfonic acid (BPDS) is preferred as a chelating agent Additional chelating agents include phthalocyanine-tetrasulfonic acid, 2,2'-biquinoline-4,4'-disulfonιc acid, 4-hydroxy-7-sulfonyl-3-quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5-sulfonyl-6-methylpyridine-3-carboxylic acid, ρyridine-2-carboxyl-3-hydroxy-4-sulfonic acid and combinations thereof

One embodiment ofthe invention is directed to lanthanide chelates that reversibly bind targets that contain protonated amines These lanthanide chelates comprise at least one rare earth element such as a lanthanide and at least one ligand Suitable lanthanides include any one or more ofthe elements from the cerium or yttrium subgroups Preferably, the lanthanide is europium, terbium, samarium, gadolinium or dysprosium and a preferred embodiment comprises europium and bathophenanthroline disulfonic acid for reversible binding to targets such as proteins Targets may be molecules such as amino acids, nucleotides, macromolecules such as proteins, peptides, polypeptides or nucleic acid, or modifications of these molecules or macromolecules Targets may be labeled while in solution or when immobilized on a solid (or semi-solid) supports such as nitrocellulose, PVDF, nylon or gels Because the procedure is fully reversible, labeled proteins may subsequently be utilized as antigens or for other biochemical techniques such as protein or carbohydrate sequencing Other useful applications include quantifying total protein in clinical assays, precipitating proteins and detecting electrophoretically separated proteins The labeling reaction can be completely and easily reversed by eluting the lanthanide-chelate from the target protein in a solution of basic pH At pH levels higher than the pKa of protonated amines, the amines will no longer be protonated (positively charged) and the electrostatic interactions that contribute to lanthanide chelate binding will be lost Thus, the lanthanide chelate will dissociate from the target molecule Also, it is possible to compete away lanthanide chelate binding to target with the addition of a chelate such as EDTA The EDTA competes for lanthanide, allowing the elution reaction to be carried out at lower pH With 20 mM EDTA, it is possible to elute lanthanide chelate from protein at a pH of 6 0 or higher. It is important to note that the order of addition of target, lanthanide or chelate can vary Lanthanide may be added first, last or simultaneously with the chelate Excess lanthanide chelate may be removed if desired Chelates are excited at a suitable wavelength of, for example, about 200 nm to about 400 nm, and preferably from about 280 nm to about 360 nm Phosphorescence is then measured at a wavelength appropriate for the lanthanide employed Emission wavelengths for each lanthanide are well known to those of ordinary skill or can be easily determined

There are applications where removal of excess lanthanide chelate before phosphorescent measurement is not required For example, in competition and other homogeneous assays, washing is generally not required In many protein detection methods, it is necessary to remove unreacted reagents before measurement ofthe label Typically, one ofthe components is immobilized in some fashion on a solid (or semi-solid) support so that unreacted components may be washed away by washing the support Target proteins, for example, may be transferred to a solid support such as nitrocellulose membrane by electroblotting or capillary blotting. Membrane is incubated with lanthanide chelate solubilized in some binding solution under conditions that favor protein-lanthanide chelate complex formation. Excess lanthanide chelate is removed by gently washing the membrane in binding solution that lacks lanthanide chelate. Alternatively, the excess lanthanide chelate may be removed by dialysis, chromatography and like means.

Another method of removing excess reagents is to take advantage ofthe protein precipitating abilities of certain lanthanide chelates. For example, the lanthanide chelate will precipitate proteins at concentrations similar to those employed for labeling. Complex formation may be performed in solution, the complex collected by centrifugation and phosphorescence measured either by first resuspending the pellet or by direct measurement of the phosphorescence of the pellet.

Lanthanide chelates may also be used in purification schemes. For example, targets such as proteins in a sample may be complexed with the lanthanide chelate and collected by centrifugation. Alternatively, targets can be separated from other substances in the sample which do not luminesce. This can be done manually or automatically by, for example, fluorescence activated cell sorting (FACS) analysis or another automated procedure. A simple assay measuring luminescence with increasing pH correlates the number of amine groups with pH stability. Binding stability is increased with an increased amino-to-carboxyl acid ratio. This fact can be used for a number of sophisticated applications. For example, a multi-detection immunoassay can be performed whereby one or more monoclonal antibodies are bound to one or more secondary antibodies as illustrated in Figure 1. The secondary antibodies are tagged with poly-amines to increase the stability of binding to lanthanide chelates at a basic pH. Initially, phosphorescence is measured at a low pH of about 3-4 which allows for the detection of all ofthe proteins in the sample The pH is raised and lanthanide chelate binding to most proteins is reversed, leaving only lanthanide chelate binding to the tagged secondary antibodies Bovine serum albumin, for example, will bind lanthanide chelate at pH 3-4, whereas a tag of poly- allylamine HCl will remain labeled until pH 10 If multiple secondary antibodies with poly-amine tags of increasing length are employed, it is possible to sequentially raise the pH and sequentially detect a variety of secondary antibodies This technique can be adapted for use in either a microtiter well or an immunoblot format

Another embodiment of the invention is directed to a kit for the reversible detection of protein which contains a lanthanide and a chelate The kit may further contain a binding solution and/or an elution solution Kits may also contain various wash, elution or incubation solutions, as well as other buffers, stabilizing and storage solutions, and antibodies

Lanthanide chelates can be utilized as readily reversible labels for targets such as protein in solution or immobilized on a solid support Reversible target binding is best achieved by an anionic moiety such as sulfonate, sulfate, phosphonate or phosphate groups Binding may also be enhanced with aromatic or heteroaromatic functionalities The procedure is simple and easy to perform, requiring reagents that are easily prepared in the laboratory, stored at room temperature for extended periods of time and can be reused several times without loss in labeling sensitivity The labeling procedure is relatively inexpensive as it does not utilize precious metals such as gold For example, unlike colloidal gold labeling, which requires up to four hours of development time, lanthanide chelate labeling requires 10-20 minutes to complete Quantitative stoichiometry of complex formation with proteins and peptides make lanthanide chelates also suitable for use in dot-blot assays for routine protein quantification Sensitivity is high, in the low nanogram range, and the dynamic range is quite broad, giving a linear response over a 250-fold range

Further, lanthanide chelates do not modify targets such as proteins and nucleic acids, and are compatible with immunoblotting and protein microsequencing reagents and procedures Lanthanide chelates do not cause N- or C-terminal blocking of proteins, interfere with the recognition of the tryptic cleavage sites, mask the antigenic recognition site for monoclonal anti-actin antibody or change the high pressure liquid chromatography (HPLC) or reverse phase (RP) HPLC elution profiles ofthe sequenced amino acids This makes the lanthanide chelates ideal for use in many applications where biochemical assays are to be performed subsequent to labeling

The following experiments are offered to illustrate embodiments of the invention and should not be viewed as limiting the scope ofthe invention Examples

Example 1 Screening Candidate Chelates

Chelates, including 3-hydroxypicolinic acid and bathophenanthroline disulfonate (BPDS) (from Sigma Chemical Co , St Louis, MO), were evaluated for their ability to complex with lanthanides and enhance phosphorescence Chelates were combined with lanthanides and phosphorescence was measured on a spectrofluorometer Measurement can be optimized as described below, but generally for terbium complexes, time delay was set to 0 4 msec, while time gate was set to 4 1 msec For europium complexes, time delay was set to 0 05 msec, while time gate was set to 1 5 msec Terbium emission was monitored at 491 and 545 nm while europium emission is monitored at 590 nm and 615 nm Excitation wavelength should be in the 250 to 370 nm range

Combinations of chelate and lanthanide that phosphoresce were further evaluated with respect to protein binding Test proteins were spotted onto nitrocellulose membranes, 0.45-μm pore size (Bio-Rad Laboratories, Hercules, CA), air dried and incubated with the lanthanide complexes Membranes were incubated in distilled water, allowed to dry and evaluated for phosphorescence upon illumination with a UV light source It is sufficient to evaluate phosphorescence by eye, but quantification can be best achieved using a CCD camera equipped with an appropriate band pass filter. Reversibility of binding was evaluated by eluting the lanthanide chelate from the membranes and requantifying the phosphorescence Chelates that reversibly label proteins were selected for further evaluation Example 2 Labeling Procedure

Membrane bound protein in a dot blot format was used to test the ability of lanthanide chelates to label protein Polyvinyl difluoride (PVDF) membrane (Immobilon-P, Oxford Glycosystems) or nitrocellulose membrane were dried and incubated in three changes of 50 mM sodium acetate, pH 4 0 for 5 minutes each while rocking on a rotary shaker (100 φ ) Membranes were incubated in 0 1% polyvinyl pyrrolidone-40 in 50 mM sodium acetate, pH 4 0 for 5 minutes, if blocking was desired Membranes were incubated in 1 5 mM BPDS, 0 1 mM europium chloride (EuCl₃, prepared in distilled water) for 10-15 minutes Membranes were rinsed twice in distilled water for 5 minute periods

To measure the amount of phosphorescence, membranes were dried completely and the front surface ofthe membrane was illuminated at 305 nm with a UV light box Bands were quantified by acquiring phosphorescence data using a Biolmage CCD camera, reflectively For europium, the lens ofthe camera was equipped with a 600 nm band pass filter so that only the red emission reached the detector Terbium required a 540 nm band pass filter The camera's shutter was left open for up to 2 minutes to collect the signal Example 3 Elution of Label

Stability of the lanthanide chelate association with nitrocellulose- bound protein was evaluated as a function of pH in a dot blot format Sodium acetate was used to prepare buffers of pH 3-5, 4-moφholine ethane sulfonic acid (Mes) was used for the pH 6 buffer, Hepes was used for the pH 7 buffer, Tris was used for the pH 8 buffer, and either AMPSO or Tris were used for pH 9 and 10 buffers All buffers were prepared at 100 mM final concentration with 20 mM EDTA added

Preliminary evaluation of various chelators indicated that EDTA and EGTA competed most efficiently for the lanthanide ion complexed to the protein (data not shown) The lanthanide chelate can easily be eluted from the protein at a pH of 6 and above in the presence of 20 mM EDTA The lanthanide chelate can also be eluted by incubating at basic pH without any EDTA. MgCl₂ (20-100 mM) was relatively ineffective at eluting the colored complexes as was NaCl (20-100 mM), 10% acetic acid and 20% methanol. We have eluted and then relabeled membranes without any significant loss in detection sensitivity, indicating that elution of the lanthanide chelates can be achieved without release of the protein from the membrane. Optimal elution conditions were determined and found to be incubation of membranes in 150 mM Tris, 20 mM EDTA, pH 8.8 for 10 minutes, followed by rinsing of membranes in distilled water for 10 minutes Example 4 Determination of Excitation and Emission Maxima

Emission and excitation maxima may be determined by spectrofluorometry. For example, 6 mM BPDS and 0.5 mM EuCl₃ were used to generate solution spectra 1.9 μg/mm² BSA was applied to the membrane and labeled with the same solution. Spectra were obtained using a Perkin-Elmer LS 50B spectrofluorometer with a quartz cuvette for the solution assay or a plate reader attachment for the solid-phase measurements. Figure 2A shows the excitation spectra, while Figure 2B shows emission spectra The spectra for the lanthanide chelate (Eu/BPDS) in solution (solid lines) and bound to BSA (Eu/BPDS/BSA) immobilized on PVDF membrane (dotted lines) are shown The data indicates that the excitation maxima for the trivalent Eu/PBDS/BSA complex lies between 285 nm and 300 nm and the emission maxima is between 605-615 nm Example 5 Determination of Dynamic Range

Dynamic range is ascertained by measuring the phosphorescence of a variety of different concentrations of protein This was done by time-resolved phosphorescence detection of different concentrations of BSA using a Perkin-Elmer LS 50B spectrofluorometer. BSA was applied to a PVDF membrane using a dot blot apparatus, the membrane dried and then labeled with 0 5 mM EuCl₃ and 6 mM BPDS. Excitation wavelength was 293 nm, while emission wavelength was 615 nm The time gate for measurement was 1 5 msec and the time delay was 0 05 msec Linear increase in signal with increasing concentration was obtained over a range of 15 to 476 ng (data not shown)

To determine the lower limits of lanthanide chelate labeling, serially diluted BSA was quantified gravitometrically and then applied to PVDF membranes as above The membranes were allowed to dry and then incubated in a solution of 6 mM BPDS, 0 5 mM EuCl₃ Florescence detection was performed using a Biolmage CCD camera system and an ultraviolet light box (302 nm) (UVP TransiUuminator, Upland, CA) The lens ofthe camera was fitted with a 600±70 nm band pass filter (Biolmage Products, Ann Arbor, MI) Exposure time was 2 minutes Figure 3 A is the CCD image of phosphorescing protein bands

The data was replotted as a standard curve in Figure 3B The X axis is the amount of protein per band divided by the square area ofthe band (ng/mm²) The Y axis is the integrated intensity in arbitrary units (intensity of all the pixels in the image that belong to a particular band) The Y-intercept was 1 5 and the slope was 0 43 The linear correlation coefficient ("r") ofthe best fit line through the data points was 0 99 From these two experiments, it was determined that the linear dynamic range was at least 1 9 ng/mm² to 476 ng/mm² (250-fold range) Example 6 Optimization ofthe Lanthanide to Chelate Ratio Optimization of the ratio of europium to BPDS for detection of protein bound to nitrocellulose membrane was performed Europium concentration was maintained at 0 5 mM and BPDS concentration was varied The different compositions were evaluated for their ability to stain BSA (24 ng/mm²) immobilized on nitrocellulose membrane The results are shown in Figure 4, where the X axis is the chelate to europium ratio and the Y axis is the integrated intensity Optimal signal was obtained at a 3 1 ratio of chelate to lanthanide

In a similar fashion, the optimal lanthanide to chelate ratio was determined for protein bound to PVDF membrane Optimal phosphorescence was obtained at a 12 1 ratio of chelate to lanthanide (data not shown) The higher ratio, relative to nitrocellulose, is likely due to nonspecific binding of BPDS to the hydrophobic membrane

Example 7 Protein to Protein Variation in Labeling

The response ofthe lanthanide chelate labels as a function of protein sequence was examined in a dot blot format. Figure 5 shows the response curves for different proteins immobilized on nitrocellulose membrane and labeled with lanthanide chelate (0 1 mM Eu/ .5 mM BPDS) The proteins evaluated are BSA (hollow circles), ovalbumin (filled diamonds), gelatin (filled triangles), phosvitin (crosses) and hemoglobin (filled circle) Phosvitin is highly phosphorylated and the negative charge of the phosphate group is expected to inhibit interaction of the protein with the sulfonate moiety of BPDS Hemoglobin is a highly colored protein and is likely to absorb emitted light from the europium Both of these proteins are refractory to lanthanide chelate labeling

However, other proteins tested are readily detected by lanthanide chelates A high linear correlation was noted between labeling intensity and protein amount in the 0.1 to 10 0 μg range for BSA and ovalbumin Gelatin binds to lanthanide chelates better than the other proteins The reason for this is not clear, though it is noted that gelatin has a high glycine content Example 8 Specificity of Labeling The specificity of labeling can be determined by comparing the binding to a variety of substrates A variety of molecules were dot blotted onto nitrocellulose membrane and examined for binding to lanthanide chelates (0 1 mM Eu/1 5 mM BPDS) Results are summarized in Figure 6 The X axis represents each polymer tested and the Y axis is the percent integrated intensity (intensity of the spot divided by the summation ofthe intensity of all the spots, times 100) The lanthanide chelate did not bind appreciably to one μg amounts of acryiamide, agarose, ATP, glycerol, MgCl₂, NaCl, PVP-40, SDS, sucrose, TEMED, Tris, Triton X-100 or Tween 20 DNA, however, appears to bind the lanthanide chelate Three amounts of DNA were applied to the membrane at 0 1 , 1 0 and 10 0 μg Labeling of DNA indicates that the lanthanide chelates described may be suitable for labeling nucleic acids and protein Nucleic acid labeling may be optimized in a manner similar to that described herein for protein labeling

As expected, the lanthanide chelate also binds to the amino acid glycine and the proteins urea and protamine sulfate (protamines are simple proteins that yield basic amino acids on hydrolysis and are found combined with nucleic acid in the sperm of fish) Primary amines seem to be required for lanthanide chelate labeling as TEMED does not generate a reaction

Example 9 Mechanism of Labeling Amino Acid Side Chains

The contribution of various amino acid side chains to lanthanide- chelate-protein complex formation was examined using a series of amino acid homopolymers and random copolymers (Aldrich Chem Co , Milwaukee, WI) Polymers were spotted onto nitrocellulose membrane, allowed to dry, and labeled with the lanthanide chelate.

A total of one μg of each polymer was spotted onto nitrocellulose membrane and labeled with 0 4 mM EuCl₃, 100 mM NaCl, and 4 0 mM BPDS Results are summarized in Figure 7 The lanthanide chelate was found to react with a wide variety of amino acid side chains Strong reactions were noted with arginine, lysine, ornithine and valine, while moderate to weak reactions were noted with aspartate, glycine, methionine, phenylalanine, proline, serine and tyrosine Among the heteropolymers evaluated, the lysine containing poly-(glu-lys-tyr) stained much better than the others, poly-(glu^»Na-ala-tyr) and poly-(glu-tyr) Poly-S-CBZ-Lys (poly-s-carboxybenzoxy-L-cysteine) stained weakly Thus, basic side chains and small aliphatic side chains appear to play a very significant role in lanthanide chelate labeling A five amino acid peptide lacking basic residues (leucine enkephalin) was also evaluated This peptide showed only moderate to weak labeling It has been previously determined that leucine enkephalin is not detectable at all by silver labeling procedures. Thus, lanthanide chelate tended to label a wider spectrum of amino acid residues than colloidal gold, ferrocyanide, silver label or the organic dyes

Example 10 Mechanism of Labeling: Carboxyl and Amino Groups Ampholytes (Oxford Glycosystems) are heterogeneous poly-amino poly-carboxylic acids. To assess the role of carboxyl and amino groups on labeling, commercially available ampholytes were fractionated by pl with a Rotofor Preparative EEF chamber (Bio-Rad Laboratories; Hercules CA) The ampholytes were then dotted onto membranes, labeled, and phosphorescence was quantified Data were fitted with a second order polynomial equation and the results are shown in Figure 8 The "y" intercept ("b") was 384; m, was -490 00; M₂ was 122.00 and "r" was 0.94. Acidic fractions (low pl) possess predominantly carboxyl moieties, while the basic fractions (high pi) contain mostly amino moieties. In general, ampholytes with a high amino to carboxyl ratio (high pl) are preferentially stained by the lanthanide chelate. The acidic fractions labeled poorly Thus, the chelates appear to interact with amino groups and not carboxyl groups on proteins. Example 1 1 Poly-amines are Resistant to Elution

The experiments described so far indicate that lanthanide chelate binds to proteins primarily via positively charged primary amines This discovery indicates that a protein with many primary amines could be made resistant to lanthanide chelate elution. To test this hypothesis, residual label present after elution was measured from a variety of poly-amines. One μg of each polymer (BSA, gelatin, poly-L-lys, poly-his, poly-allylamine HCl and poly-2-vinyl pyridine, Aldrich Chem. Co.; Milwaukee, WI) was applied to nitrocellulose membranes, labeled with Eu/BPDS and label eluted for 10 minutes in 150 mM Tris, pH 8 8 The results are shown in Figure 9. The X axis is the polymer tested and the Y axis is the percent integrated intensity normalized to the intensity ofthe poly-allylamine signal Poly-allylamine retained the complex to the greatest extent, followed by poly-L- lysine These are the polymers with the greatest number of primary amines (and thus the highest pl) and are therefore the most positively charged at pH 8 8 Thus, it will be possible to tag proteins with poly-allylamine to increase resistance to the elution of label at higher pH's

Example 12 Immunodetection of Proteins after Labeling This is a prophetic example HL-60 (human promyelocytic leukemia cells, ATCC CCL 240, American Type Culture Collection, Rockville, MD) protein extracts are prepared in 63 mM Tris, 2 0% sodium dodecyl sulfate, 10 0% glycerol, 5 0% 2-mercaptoethanol, pH 6 8 and heated to 100°C for 10 minutes Vaπous amounts ofthe cell lysate (0 5 μg to 10 0 μg) are run on 10 0% polyacrylamide gels by standard protocols After electroblotting and labeling with the lanthanide chelate, label is eluted from the membrane as described The nitrocellulose membranes are blocked in 5 0% nonfat dry milk in PBS and immunodetection performed using the standard ECL Western blotting protocol (Amersham Interna¬ tional, Buckinghamshire, England), utilizing monoclonal anti-actin antibody, clone KJ43A (Sigma Chemical Co , St Louis, MO) as the primary antibody Monoclonal antibody is detected using a chemiluminescence visualization procedure Lanthanide chelate label is expected to have no adverse effects on chemiluminescent detection ofthe actin Example 14 Protein Sequence Analysis After Labeling This is a prophetic example Labeled protein is subjected to protein sequencing to determine the compatibility ofthe lanthanide chelates with amino- terminal and internal sequencing Labeled proteins are subjected to amino terminal or internal amino acid sequence analysis after removal of the label (Patton, W F , et al (1994) Anal. Biochem 220.324-35) For N-terminal sequencing, regions of the Coomassie brilliant blue labeled or lanthanide chelate labeled PVDF membranes corresponding to the protein of interest are excised and loaded directly into the ABI 477A sequencer, equipped with a special blot reaction cartridge (Applied Biosystems) For internal microsequencing, target proteins labeled with Ponceau red or lanthanide chelate are excised from the nitrocellulose membrane, subjected to in situ proteolytic cleavage with 0.5 μg trypsin for 3 hours at 37°C and in the presence of 10% acetonitrile, 3% Tween 80 in 100 mM NH₄HCO₃, pH 8.3 Resulting fragments are separated by microbore reverse-phase HPLC. Selected peak fractions are analyzed by automated Ed an degradation The sequencability of lanthanide chelate labeled proteins is expected to be nearly identical to positive controls. Thus, the lanthanide chelate labeling method is fully compatible with current protocols for N-terminal and internal sequencing.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration ofthe specification and practice ofthe invention disclosed herein All U.S Patents cited herein are specifically incoφorated by reference. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

Claims

We Claim

1 A lanthanide chelate comprising a lanthanide and a ligand wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to a target, and a third domain that absorbs UV radiation 2 The lanthanide chelate of ciaim 1 wherein the lanthanide is selected from the group consisting of europium, terbium, samarium, gadolinium and dysprosium

3 The lanthanide chelate of claim 1 wherein the ligand is selected from the group consisting of bathophenanthroline disulfonic acid, phthalocyanine- tetrasulfonic acid, 2,2'-biquinoline-4,4'-disulfonic acid, 4-hydroxy-7-sulfonyl-3- quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5- sulfonyl-6-methylpyridine-3 -carboxylic acid, pyridine-2-carboxyl-3-hydroxy-4- sulfonic acid and combinations thereof

4 The lanthanide chelate of claim 1 wherein the second domain comprises a sulfonate, a sulphate, a phosphonate or a phosphate, and the third domain comprises an aromatic or a heteroaromatic ring

5 The lanthanide chelate of claim 4 wherein the third domain further comprises aromatic or heteroaromatic monocyclic, bicyclic or tricyclic rings

6 The lanthanide chelate of claim 1 which comprises europium and bathophenanthroline disulfonic acid 7 The lanthanide chelate of claim 1 wherein the target is protein or nucleic acid

8 The lanthanide chelate of claim 1 wherein the lanthanide phosphoresces upon absorbing energy from the chelate

9 A lanthanide-chelate-target complex comprising a lanthanide, a ligand and a target wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to the target, and a third domain that absorbs electromagnetic radiation

10 The lanthanide chelate of claim 9 wherein the target is protein or nucleic acid 11 The lanthanide chelate of claim 9 wherein the lanthanide phosphoresces upon absorbing energy from the chelate

12 A method for detecting a target in a sample comprising a) contacting the sample with a lanthanide chelate that reversibly binds to the target, b) illuminating the sample with electromagnetic radiation, and c) detecting emitted phosphorescence

13 The method of claim 12 wherein the target is an amino acid, a peptide, a protein, a nucleic acid, a nucleotide or a derivative thereof 14 The method of claim 12 wherein the sample is a biological sample from a patient

15 The method of claim 12 wherein the patient is a human

16 The method of claim 12 wherein the sample comprises biomass, soil, water, manufactured product or by-product, hazardous waste, or a combination thereof 17 The method of claim 12 wherein the lanthanide is selected from the group consisting of europium, terbium, samarium, gadolinium and dysprosium

18 The method of claim 12 wherein the chelate is selected from the group consisting of bathophenanthroline disulfonic acid, phthalocyanine-tetrasulfonic acid, 2,2'-biquinohne-4,4'-disulfonic acid, 4-hydroxy-7-sulfonyl-3-quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5-sulfonyl-6- methylpyridine-3-carboxylic acid, pyridine-2-carboxyl-3-hydroxy-4-sulfonic acid and derivatives and modifications thereof

19 The method of claim 12 wherein the reversible binding is non-covalent

20 The method of claim 12 wherein the electromagnetic radiation is in the range of between about 275 nm to about 370 nm

21 The method of claim 12 wherein the sample comprises protein immobilized on a solid support

22 The method of claim 21 wherein the solid support is nitrocellulose, nylon, or polyvinyl difluoride 23 A method for detecting a protein comprising the steps of a) contacting the protein with a lanthanide chelate comprising a chelating agent that has a first domain which binds to a lanthanide, a second domain that reversibly binds to the protein, and a third domain that absorbs elecromagnetic radiation, b) illuminating the protein with electromagnetic radiation, and c) detecting emitted phosphorescence

24 The method of claim 23 wherein the second domain comprises a sulfonate, a sulfate, a phosphonate or a phosphate and the third domain comprises an aromatic or a heteroaromatic ring

25 The method of claim 23 wherein the third domain further comprises aromatic or heteroaromatic monocyclic, bicyclic or tricyclic rings

26 The method of claim 23 wherein the lanthanide is selected from the group consisting of europium, terbium, samarium, gadolinium and dysprosium 27 The method of claim 23 wherein the chelating agent is selected from the group consisting of bathophenanthroline disulfonic acid, phthalocyanine- tetrasulfonic acid, 2,2'-biquinoline-4,4'-disulfonic acid, 4-hydroxy-7-sulfonyl-3- quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5- sulfonyl-6-methylpyridine-3-carboxylic acid, pyridine-2-carboxyl-3-hydroxy-4- sulfonic acid and combinations thereof

28 The method of claim 23 wherein the protein is immobilized on a solid support

29 A method for detecting total protein and a target protein in a sample comprising the steps of a) contacting the sample with a primary antibody that specifically binds to the target protein, b) contacting the sample with a secondary antibody that specifically binds to the primary antibody, wherein the secondary antibodies comprises a polyamine-tag, c) contacting the sample with a lanthanide chelate at a first pH and detecting phosphorescence from the total protein; and d) adjusting the sample to a second, higher pH and detecting phosphorescence from the target protein 30 The method of claim 29 wherein the first pH is from about 3 to 6 and the second pH is from about 7 to 10

31 The method of claim 29 wherein the sample comprises protein immobilized on a solid support

32 The method of claim 29 wherein the solid support is nitrocellulose, nylon or polyvinyl difluoride

33 A method for precipitating a protein from a sample that comprises the steps of a) contacting the sample with a lanthanide chelate that reversibly binds to the protein, and b) collecting the protein by centrifugation

34 The method of claim 33 wherein the lanthanide chelate comprises a lanthanide selected from the group consisting of europium, terbium, samarium, gadolinium and dysprosium

35 The method of claim 33 wherein the lanthanide chelate complex comprises a chelating agent selected from the group consisting of bathophenanthroline disulfonic acid, phthalocyanine-tetrasulfonic acid, 2,2'-biquinoline-4,4'-disulfonic acid, 4-hydroxy-7-sulfonyl-3 -quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5-sulfonyl-6-methylpyridine-3-carboxylic acid, pyridine-2-carboxyl-3-hydroxy-4-sulfonic acid and combinations thereof 36 A method for isolating a target without covalent modification ofthe target comprising the steps of a) contacting the target with a lanthanide chelate to form a lanthanide- chelate-target complex, b) illuminating complex with electromagnetic radiation to cause the complex to phosphoresce, c) isolating the phosphorescing complex, and d) eluting the lanthanide chelate from the complex 37 The method of claim 36 wherein the target is protein or nucleic acid

38 The method of claim 36 wherein the target is contacted with lanthanide chelate complex by incubation in a binding solution

39 The method of claim 36 wherein the complex is illuminated with ultraviolet radiation from about 250 nm to 390 nm 40 The method of claim 36 wherein phosphorescing target is isolated by fluorescence activated cell sorting

41 The method of claim 36 wherein the lanthanide chelate complex is removed from the mixture by dialysis

42 A kit for the detection of a target comprising a lanthanide chelate 43 The kit of claim 42 further comprising a binding solution and an elution solution