Patent 6,602,989

The detection of gallium in biological samples is required due to its role in the diagnosis of tumor and for possible treatment of malignancies. However, the use of purely instrumental techniques is unsuitable for detection of low levels of gallium in biological matrices. New protein conjugates have been synthesized based on 4-(2-pyridylazo) ligands. The conjugates detect gallium in biological matrices using a non antibody-based sandwich assay format. The recovery level is between 97-101.3 with a relative standard deviation of less than 5%. The assay results in a detection limit of 5x10^-8 M and a remarkable selectivity for gallium(III) relative to other metals investigated. The new method provides adequate accuracy for gallium applicable for animal physiology and clinical toxicology.

SUMMARY OF THE INVENTION

Having described the current state-of-the-art and associated problems that still remain, it is an important aspect of the present invention to provide processes and materials that are used to deliver therapeutic medicines and to quantify concentrations of these medicines at the target and remote sites, in particular, the detection and quantification of gallium(III) ions in body fluids to assist in determining optimal dosage for gallium(III) radiotherapy of lesions and other illnesses.

Another aspect of the invention is to provide an analytical test for quantifying the level of gallium(III) with minimal interference from other metal ions that might be present, thereby eliminating the need for prior processing for removal of ancillary metal ions.

Another aspect of the invention is to provide a simple, rapid, and inexpensive process to quantify the level of gallium(III) in body fluids such as blood (whole blood, blood serum, blood plasma, blood cell), urine, feces, saliva, breast milk, and so on. Generally speaking, in clinical examination, metals contained mainly in blood or urine are measured in vivo and in vitro.

Another aspect of this invention is to provide a therapeutic ternary coordination complex from a protein conjugate-gallium(III) ion-enzyme conjugate where the protein has an affinity for an antigen from a cancerous lesion and wherein a therapeutic dose of radioactive gallium is localized at the lesion or tumor site. The preferred protein in this case is an antibody or antibody segment that has an affinity for the antigen.

Another aspect of the invention is to provide an enzyme conjugate that is specific to binding with a gallium(III) complex of a protein conjugate in order to form a ternary complex that is easily detected and quantifiable.

Another aspect is to use derivatives of pyridylazo resorcinol (PAR) and their protein conjugates to form tridentate complexing ligands with gallium(III) ions to quantify gallium metal in body fluids.

Another aspect of this invention provides methods for the preparing conjugates of proteins or polypeptides with metal ions. The resulting conjugates are useful as radiopharmaceuticals.

Another aspect of this invention is to provide binary and ternary protein conjugate-metal complexes possessing therapeutic and diagnostic value that can be administered to a patient, transported in the body, and adsorbed at diseased sites.

Another aspect of the invention is to provide a process to form protein-conjugates using derivatives of PAR.

To achieve the above described general aspects of the invention, the following specific objectives are enumerated: (i) to prepare metal-protein conjugates using 4-(2-pyridylazo)resorcinol (PAR), and (ii) to provide an analytical basis for discrimination of the resulting conjugates for detecting gallium. PAR has been used extensively to analyze metals. It possesses a variety of useful spectroscopic and luminescence properties. To our knowledge, the use of 2-pyridylazoresorcinol and its derivatives, such as 1-(2-pyridylazo)-2-naphthol (PAN), have not been employed to prepare biological conjugates. Compared to PAN, PAR was chosen as a modifier in our work because of its relative stability and higher solubility in water.

We hereby report novel binary and tertiary complexes useful for therapeutic and diagnostic treatments using protein-modified 2-pyridylazoresorcinol ligand derivatives (PAR) as the biorecognition element. PAR was derivatized with proteins and enzymes such as ovalbumin, bovine serum albumin (BSA), and alkaline phosphatase (AP) to generate the respective conjugates. In one embodiment the synthesis was carried out using a water-soluble carbodiimide and N-hydroxy succinamide coupling techniques. The results of characterization experiments using electrospray mass spectrometry, UV/vis spectroscopy, and Fourier transform infrared (FTIR) experiments confirmed that a new class of protein conjugates has been synthesized. The conjugates were used for detecting gallium in a sandwich enzyme-linked immunosorbent assay (ELISA) format. The detection format does not require the use of antibody, contrary to conventional immunoassay techniques. This method results in a remarkable selectivity for gallium(III) relative to other metals investigated, including Fe(II), Zn(II), In(III), Hg(II), Tl(III) and Pb(II).

DESCRIPTION OF PREFERRED EMBODIMENTS

Metal Ions of Interest

Of particular interest to the present invention are metal ions that may be found in the human bloodstream. Such metals include endogenous, essential metal ions and nonphysiologic metal ions that may be present either as a result of their use as therapeutics or because of the ingestion, absorption or inhalation of metals present in the environment. Thus, virtually all metal ions from magnesium (atomic number 12) to plutonium (atomic number 94); but in particular, lead, mercury, nickel, cadmium, thallium, antimony, silver, chromium, manganese, platinum, gold, aluminum, bismuth, gallium, iron, copper, zinc, cobalt, molybdenum, selenium, and vanadium ions, are amenable for use in this process.

Diagnostic radionuclides useful in the present invention include ruthenium-95, ruthenium-97, ruthenium-103, ruthenium-105, technetium-99m, mercury-197, gallium-67, gallium-68, osmium-191, indium-111, indium 113m and lead-203. Therapeutic radionuclides include palladium-103, palladium-109, silver-111, antimony-119, actinium-225, gold-198, gold-199, copper-67, rhenium-186, rhenium-188, rhenium-189, lead-212 and bismuth-212.

Among the radionuclides and labels useful in the methods of the present invention, gamma-emitters, positron-emitters, x-ray emitters and fluorescence-emitters are suitable for localization and/or therapy, while beta- and alpha-emitters and electron- and neutron-capturing agents, such as boron and uranium, also can be used for therapy.

For diagnostic or therapeutic purposes, the metal ion should be such that minimal radiation damage is caused to healthy tissue. In order to achieve this, radioactive metal ions are selected that decompose within a short period of time for example, several hours to 4 days, and do not decay to such an extent as to generate sufficient particles that cause significant collateral damage to surrounding healthy cells. A preferred metal ion in the current invention is gallium(III) and in particular radio isotopes such as ⁶⁶ Ga(III), ⁶⁷ Ga(III), ⁷¹ Ga(III), ⁷² Ga(III), or ⁷³ Ga(III)

These characteristics, coupled with the known therapeutic properties of gallium, and the contraindication that gallium(III) has an affinity for non-target sites, is the motivation to find alternative modes of delivery for gallium that will increase its selectivity for the target area and methods of quantifying gallium(III) in non-target areas in order to determine the safest regime for administering radiotherapy using gallium(III).

According to one preferred embodiment of the present invention, proteinaceous binary and ternary complexes of gallium are provided in order to improve target selectivity and also to provide accurate quantification of gallium(III) concentrations.

Ligands

The ligand-containing molecules of the present invention consist of a central metal ion bound to a number of other molecules, termed ligands. The nature of the chemical bond formed between a ligand and a metal can be thought of as involving the donation of a pair of electrons present on the ligand molecule or, in molecular orbital terms, as a molecular orbital formed by combining a filled orbital of the ligand with a vacant orbital of the metal. Those atoms in the ligand molecule that are directly involved in forming a chemical bond to the metal ion are therefore termed the donor atoms, generally comprising elements of Groups V and VI of the periodic table, with nitrogen, oxygen, sulfur, phosphorus and arsenic being those most commonly employed.

Molecules that contain two or more donor atoms capable of binding to a single metal ion are termed chelating agents, or chelators, and the corresponding metal complexes are called chelates. The number of donor atoms present in a given chelator is termed the denticity of that chelator, ligands possessing two donor sites being called bidentate, those with three donor sites, tridentate, and so forth. In general, the higher the denticity of a chelator the more stable are the chelates formed, up to the point at which the denticity of the chelator matches the maximum coordination number attainable by the particular metal ion of interest. The maximum coordination number of a given metal ion in a given oxidation state is an intrinsic property of that metal, reflecting the number of vacant orbitals and, hence, the number of chemical bonds it is able to form with ligand donor atoms. When all of the available vacant orbitals have been used to form bonds to donor atoms in the ligand or ligands, the metal is said to be coordinatively saturated.

For any given metal ion, useful ligands according to the present invention may be selected by one skilled in the art, employing the following criteria. Ligands must possess donor atoms (or sets of donor atoms in the case of chelating ligands) that favor binding to the target metal ion. The general preference of any given metal ion for particular donor atoms (generally selected from the group consisting of carboxylic, phenolic, or ether oxygen atoms, amine, imine, or aromatic nitrogen atoms and charged or neutral sulfur atoms) is well known in the art.

It is not essential that any of the exogenous ligands bound to an antibody, be chelating ligands, according to the present invention. Though ligands suitable for use in the present invention are preferably chelating ligands, the presence of a chelating ligand is not essential, as non-chelating ligands, such as certain phosphines and sugar analogs, can also be useful. These ligands can be either monodentate or of higher density.

The ligands must also offer the prospect of forming ligand-metal linkages that are likely to remain stable in vivo. That is, the ligand-metal linkage does not dissociate during the time required to raise an immune response to or perform quantification of the complex. For many metals of interest there exists considerable art relating to the in vivo stability of particular ligand-metal linkages, which may be used to guide ligand selection. This requirement favors the selection of chelating ligands, as chelating ligands generally form coordination complexes of higher thermodynamic stability than do corresponding combinations of monodentate ligands.

Within the scope of the present invention, there are many useful ligands that have been shown to coordinately bond to metal ions; they may either be mono, bi, or tridentate, depending on the number of sites that the ligand binds to the metal ion. Most preferred are ligands that are tridentate and contain a plurality of ligand sites. Useful chelating ligands that form highly stable complexes with many metal ions include pyridylazo resorcinol derivatives, polyaminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) and phenol-containing aminopolycarboxylates such as N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), aminobenzyl derivatives of ethylenediaminetetraacetic acid derivatives (see U.S. Pat. No. 4,722,892 to Meares et al.), dihydroxydisulfonic acid derivatives (TIRON) (see U.S. Pat. No. 4,732,974 to Nicolotti, et al.). These chelators have a denticity of 3 or higher and, as most transition and main group metals have a maximum coordination number of 6, the resulting species are coordinatively saturated binary or ternary complexes (i.e., complexes consisting of a single to two ligand molecules and a single metal ion).

A further requirement is that each of the above described ligands be bifunctional. A bifunctional ligand is a molecule that contains, in addition to at least one metal binding site (donor atom), a second reactive moiety through which the ligand may be covalently linked to, for example, a protein, a solid phase or a label, without significantly affecting the metal-binding properties of the ligand. Reactive moieties include such groups as hydroxy, phenoxy, carboxy, and isothiocyanato.

One skilled in the art will readily recognize positions within a given ligand molecule where incorporation of an additional reactive moiety will not affect metal-binding properties. Such a skilled practitioner will also readily recognize reactive moieties that are useful in coupling the ligand to another molecule or solid phase. Typically, the sites for ligand complexation and covalent bonding are structurally and electronically isolated from each other so that there is no interference with their respective properties. The product of such reactions is designed to create at least one "exogenous chelating group" per polypeptide/protein chain.

Ligands should also be selected so that they contain a highly differentiated organic framework, incorporating wherever possible aromatic structures, rigid ring systems and asymmetric carbon centers. Having selected the donor atom set, denticity, bifunctional side arm and organic framework of the ligands, the stoichiometry of the resulting target metal ligand-containing complex, whether ternary or of higher molecularity is dictated by the maximum coordination number of the particular metal ion of interest.

Bifunctionality of the ligands is essential for configuring screening assays used to identify and select monoclonal antibodies that are specific for metal containing ligand complexes, for preparing affinity chromatography media used to purify such antibodies, and for preparing antigen-label tracers for use in immunoassays.

In addition to displaying bifunctionality and stable ligand-metal binding in vivo, preferred ligands also incorporate structural features that are thought to contribute to immunogenicity and differential recognition by proteins. Such structural features include aromatic ring systems (Zoller, et al, J. Nucl. Med.,33, 1366-72 (1992)), rigid cyclic structures (Kosmas, et al, Cancer Res., 52, 904-11 (1992)) and asymmetric carbon centers (Reardon, et al, Nature, 316, 265-68 (1985); Zoller, et al, J. Nucl. Med., 33, 1366-72 (1992)).

The present invention preferably employs exogenous chelators based on derivatives of aza mono- and fused heterocyclic compounds having an azo linking group adjacent to the nitrogen atom in the heterocycle. A most preferred example is the use of pyridylazoresorcinol (PAR) and its derivatives as exogenous chelators. Examples of PAR derivatives that are illustrative, but not limiting, within the present invention are:

3-amino-4-[3-(1-methyl-2-piperidyl)-2-pyridylazo]phenol;

1-(2-pyridylazo)-2,7-naphthalenediol;

6,7-dihydroxy-5-(2-pyridylazo)-2-naphthalenedisulfonic acid;

2,6-diamino-3-(pyridylazo)pyridine;

4-(2-pyridylazo)methyl salicylic acid;

3-hydroxy-4-(2-quinolinylazo)phenol;

3-hydroxy-4-(2-pyrimidylazo)phenol; and

3-hydroxy-4-(2-benzimidazolylazo)phenol.

Other Examples of These

((heterocyclyl)azo)orthohydroxyarylene (PAR) derivatives are represented in FIG. 2. Within the scope of this invention are heterocyclic rings that have at least one heteroatom, itself having a lone pair of electrons, in a position adjacent to the site of the attachment of the aza functionality to the heterocycle. Likewise, the orthohydroxyarylene functionality can be generalized to require a heteroatom such as oxygen, nitrogen or sulfur attached to the ortho position (relative to the site of the aza functionality attachment) of the aryl ring. The hetero atom on the aryl ring will contain a lone pair of electrons and will comprise such moieties as hydroxyl, amino or sulfhydryl respectively.

These PAR derivatives are preferred compared to prior art chelators since they form relatively stable complexes and have acceptable solubility in water. As discussed hereinbelow in greater detail, these derivatives, when bound to proteinaceous materials, are highly selective to the metal ions to which they coordinately bind. Unexpectedly, the exogenous ligand-containing chelating functionalities of the protein conjugates bind essentially exclusively to gallium metal ions. This unique property allows these materials to detect gallium very accurately, even in the presence of other metal ions.

The stability of these metal complexed conjugated proteinaceous materials also allows them to be used in vivo and in vitro as diagnostic and therapeutic reagents. Prior art conjugates do not have the specificity for complexation to a given metal. Therefore, they suffer from the possibility of equilibration with other metal ions found in vivo, thereby causing either a deterioration in imaging quality or loss of therapeutic activity. In the case of in vivo diagnostic or therapeutic use, it is highly desirable to have the proteinaceous material be specific to a given antigen, thereby causing the metal complexed to be localized at the site where infection or disease predominates. For this application, it is highly desirable to have the proteinaceous material be an antibody or a biologically active fragment therefrom.

Proteinaceous Materials of Interest

Proteinaceous material can be derived from synthetic as well as natural sources such as mammals, amphibians, reptiles, birds, insects and plants, as well as recombinant or transgenic sources. Examples include such materials as a peptides, polypeptides, proteins, antigens, glycoproteins, lipoproteins, or the like (e.g., hormones, lymphokines, growth factors, albumins, cytokines, enzymes, immune modulators, receptor proteins, antibodies including monoclonal antibodies, and antibody fragments or fractions thereof with at least one molecule having at least one ligand site for binding to a metal ion).

Antibody fragments may be advantageous for tissue imaging systems because these antibody fragments permeate target sites at an increased rate. Fab' fragments of IgG immunoglobulins are obtained by cleaving the antibody molecule with pepsin (resulting in a bivalent fragment, (Fab')₂ or with papain (resulting in 2 univalent fragments, 2 Fab) (see: Parham, 1983, J. Immunol. 131: 2895-2902; Lamoyi and Nisonoff, 1983, J. Immunol. Meth. 56: 235-243.) The bivalent (Fab')₂ fragment can be split by mild reduction of one or a few disulfide bonds to yield univalent Fab' fragments. The Fab and (Fab')₂ fragments are smaller than a whole antibody molecule and, therefore, permeate the target site or tissue more easily. This may offer an advantage for in vivo imaging, since conjugates more readily penetrate in vivo sites (e.g., tumor masses, infection sites, etc.). An additional advantage is obtained when using conjugates formed with antibody fragments because these fragments do not cross a placental barrier. As a result, using this embodiment of the invention, an in vivo site (such as a tumor) may be imaged in a pregnant female without exposing the fetus to the imaging compound.

In one embodiment the protein is preferably one that can cause an immunogenic response in an animal. Examples of such proteins are: bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin, immunoglobulin (IgG), etc.

Targeting proteins are known which preferentially bind marker substances that are produced by or associated with lesions. For example, antibodies can be used against cancer-associated substances, as well as against any pathological lesion that shows an increased or unique antigenic marker, such as against substances associated with cardiovascular lesions, (e.g. vascular clots including thrombi and emboli, myocardial infarctions and other organ infarcts, atherosclerotic plaques; inflammatory lesions; and infectious and parasitic agents).

The cancer states include carcinomas, melanomas, sarcomas, neuroblastomas, leukemias, lymphomas, gliomas, and myelomas.

The infectious diseases include those caused by invading microbes or parasites. As used herein, "microbe" denotes virus, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms, "parasite" denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to antibody-induced clearance or lytic or phagocytic destruction (e.g., malarial parasites, spirochetes and the like, including helminths), while "infectious agent" or "pathogen" denotes both microbes and parasites.

Preparation of Binary Conjugate Complexes

The covalent binding of the ligand-containing compound to the proteinaceous material can be achieved by direct reaction of the two materials through reactive sites on both materials. In the case of the proteinaceous material, potentially reactive pendant sulfydryl, hydroxy, phenolic, amino, and carboxy groups can be used as a binding site. When proteins have carbohydrate moieties, oxidizing agents can be used for generating aldehydic sites for subsequent covalent binding with amine derivatives such as primary amines, hydrazine derivatives, hydroxylamine derivatives, phenyl hydrazine, semicarbazide, and thiosemicarbazide groups coordinately bound to a metal ion, thereby providing a water-soluble antibody-metal ion complex.

Binary conjugate complexes are prepared by reacting a proteinaceous material such as a polypeptide, protein, antigen, lipoprotein, an antibody including monoclonal antibody, enzyme, hormone, or fractions thereof with at least one molecule having at least one ligand site. In a preferred embodiment, the proteinaceous material may have multiple sites available for attachment of a plurality of ligand-containing molecules. For in vivo therapeutic and diagnostic studies, monoclonal antibodies produced by a hybridoma technique provide distinct advantages as carriers for imaging systems employing metal chelated binary and ternary conjugate complexes. First, monoclonal antibodies bind only to one molecular site (i.e., an epitope) with specific binding constants. Second, such antibodies are homogeneous and thus are purified with relative ease. Third, monoclonal antibodies can be made in large quantities, and relatively inexpensively, by particular hybridoma lines.

Other, more complex conjugates can be used as exogenous chelators. In these cases, the conjugate must have one site for coordinate complexation with the metal ion and a second, reactive site must be present for attachment to the proteinaceous material. Typically, similar reactive functionalities can be utilized as discussed for the proteinaceous material, but in this case other functionalities that are even more reactive, such as epoxides, azides, or activated acid functionalities, such as esters or acid halides, may also be employed. Preferred in the present invention is the use of ancillary activating reagents, such as carbodiimides, which assist in condensation reactions. The carbodiimide dehydration reaction is further facilitated by the use of an ester forming compound such as N-hydroxy succinamide.

In one preferred embodiment of the invention the conjugated proteinaceous material, also known as a "capture chelator", is immobilized on a substrate that has an affinity for proteinaceous materials. Such substrates or solid phases, (e.g., cellulose or Sepharose), can be composed of materials that are hydrophilic such as polysaccharides, cellulosics; or hydrophobics such as polystyrene; or in the case of antibody proteinaceous materials, antigen-fixed compositions. The substrates can also optionally have reactive sites similar in nature to the reactive sites of the ligand containing compositions discussed supra. In this manner the proteinaceous material is covalently bound to the substrate.

Subsequent to immobilization, an effluent containing the target metal ion is placed in contact with the immobilized proteinaceous conjugate and the metal ion is coordinately bound to the conjugate. In this manner, quantification of the metal ion concentration can be determined.

A preferred conjugated proteinaceous material of the present invention comprises pyridylazoresorcinol (PAR) derivatives of antibody proteins, proteins such as ovalbumin, bovine serum albumin, or enzymes such as alkaline phosphatase. Specifically, when enzymes are utilized in the proteinaceous conjugate, rather than acting as a "capture chelator", the enzyme conjugate is utilized as a "detector chelator" due to the ability of the enzyme to catalyze a variety of reactions that can be readily monitored and thereby quantified. The preferred capture or detection conjugate metal ion complex comprises gallium(III) ions.

In an alternative embodiment to the immediately preceding one, an uncomplexed antibody is immobilized on a solid substrate. Subsequently, a known quantity of serum containing a target antigen species is applied to the immobilized antibody on the substrate causing the antigen to be adhered to the substrate. A second solution containing a complexed antibody as described herein is then administered on the substrate and allowed to associate with the antigen. Any unassociated complexed antibody is extracted, and the remaining complexed antibody is detected and quantified.

In another embodiment, the use of the capture conjugate is useful as an antidote for removal of toxic levels of gallium from animal subjects by in vivo complexation or filtration of the subject's blood through a filtering device comprising immobilized capture conjugate.

In another embodiment of the present invention, the proteinaceous conjugate can be used in an aqueous solution to determine the concentration of metals such as gallium(III), iron(III), and zinc(II). In the latter case, Zn(II) complexes with PAR conjugates are fluorescent, allowing for an assay method that is facile, sensitive, and requires no additional reagents.

In another embodiment of the present invention, a metal ion complexed antibody conjugate is used for diagnostic or therapeutic analysis in vivo in mammalian subjects by administering to the patient an effective dose of the metal ion complexed antibody conjugate via inoculation, oral uptake, or other means. As noted supra, in this case the conjugate should have an affinity for an antigen at the target area. This is typically achieved by having the proteinaceous material comprise an antibody that has specific affinity for an antigen known to be a tumor marker such as carcinoembryonic antigen (CEA), human chorionic gonadotropin or its beta subunit, colon specific antigen-p, tumor specific glycoprotein or the like.

Antibodies in the present invention may be directed against any target (e.g., bacterial, fungal, parasitic, mycoplasmal, histocompatibility, differentiation, and other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs, and other biologically active molecules). Such metal ion complexed antibody conjugate thereby concentrates locally at the disease site where they can be non-invasively detected by such means as emission tomography, nuclear magnetic resonance imaging, and in vitro spectroscopy. For therapeutic efficacy, the complexed antibody conjugate should contain a radio isotope that emits cytotoxic beta particles or alpha particles.

Ternary Complexes: Protein Conjugate₁ -Metal-Protein Conjugate₂ (PC₁ -M-PC₂)

In another embodiment of the present invention, a ternary complex can be formed by complexing the above mentioned metal complexed binary proteinaceous conjugate (i.e., the capture chelator) with a second protein conjugate, preferably an enzyme conjugate (i.e., the detector chelator) having the same or different ligands from the metal complexed binary proteinaceous conjugate. The enzyme conjugate complexes to the metal ion of the metal complexed binary proteinaceous conjugate to form a "sandwich" or ternary complex.

In one embodiment, the ligand functionality on the detector chelator is the same as on the metal complexed capture chelator. However, most preferred sandwich complexes of the present invention employ two different proteinaceous conjugates. A most preferred ligand is the tridentate pyridylazoresorcinol conjugate, and a most preferred metal ion is gallium(III). A most preferred enzyme is alkaline phosphatase. Other examples include horse-radish peroxidase, .beta.-D-galactodisase, urease, glucose oxidase, and ribonuclease. As with the binary complexes disclosed supra, the ternary complex can be used for detection or therapy.

In a preferred in vitro detection process, the detection chelator is added to the immobilized metal complexed capture chelator and allowed to form a ternary complex. This ternary complex comprises a proteinaceous conjugate, a metal ion and an enzyme conjugate. After formation of the ternary complex, an assaying reagent is added to react with the enzyme portion of the ternary complex in order to form a species that is readily detected and quantified.

In a preferred embodiment the assaying reagent is p-nitrophenylphosphate (PNPP). With the addition of PNPP, enzyme activity doubles between 25 and 37 and the hydrolysis of the PNPP assaying reagent occurs. The products of this hydrolysis reaction are colored inorganic phosphates and the corresponding alcohol, p-nitrophenol, which can be detected at very low concentrations by monitoring its absorption at 405 nm. The concentration of the p-nitrophenol can then be determined by use of the Beer-Lambert Law.

Other colored or fluorogenic assaying reagents that are applicable in the present invention include p-nitro-.beta.-D-galactoside, 4-methyl umbelliferyl-.beta.-D-galactosidase (MUG), o-phenylene diamine, 2,2'-azino-di(3-ethylbenzothiazoline sulfonato-6) (ABTS), o-toluidine, 5-aminosalicylic acid, and o-dianisidine. Other promising new assaying reagents are 3-dimethylaminobenzoic acid (DMAB), and 3-methylbenzothiazoline.

A number of other assaying reagents which yield insoluble products are available for use with alkaline phosphatase enzyme. One such assaying reagent is 5-bromo-4-chloro-3-indoyl phosphate.

The enzymes selected for the present invention can be used to facilitate detecting means by allowing reactions such as acid-base neutralization or redox to generate species that can be detected by such techniques as optical absorption, electron spin resonance, fluorescence, NMR relaxation, or if the metal ion is radioactive, radioactive emission. Many of these same techniques can also be used to detect the binary complexes discussed supra. The enzyme conjugate may optionally be tagged with a substance that fluoresces or emits radiation. In this manner an assaying reagent may not be necessary to quantify the metal ion complexed in the ternary complex.

Alternatively, the activity of the enzyme can be monitored electrochemically. This is because the action of the enzyme on the assaying reagent should result in a change in the observed electrochemistry of the system. The potential limits between which these changes must occur are determined by the medium in which the measurements are performed. This limit is between approximately -0.25V and about +0.90V versus SCE (i.e., between the potentials for the reduction of dissolved oxygen and the oxidation of water respectively).

Other possible electrochemically active assaying reagents for alkaline phosphatase are phenyl phosphate, naphthyl phosphate, and p-aminophenylphosphate (see Bard A. J., and Faulkner, L. R. (1980), Electrochemical Methods-Fundamentals and Applications, John Wiley, New York; and Heineman, W. R., Halsall H. B., (1985), Anal. Chem. 57, 1321A.) Phenyl phosphate has no electrochemistry within the potential window for aqueous solutions, but its hydrolysis product, phenol, can be oxidized at the potentials around +0.8V versus SCE. When p-aminophenyl phosphate is used, in irreversible wave in cyclic voltammetry at around +0.45V versus Ag/AgCl is usually obtained. But its hydrolysis product, p-aminophenol, has reversible electrochemistry with a half-potential wave of -0.065V versus Ag/AgCl.

Assay Principle

The detection of Ga(III) using the PAR-conjugate is based on a ternary (sandwich) chelate principle as shown in FIG. 4. In the first step in forming the ternary complex, the metal ion is captured by an immobilized PAR protein conjugate. In the second step, the protein conjugate is detected using an enzyme-labeled PAR-conjugate known as the detection chelator.

The first step of the process for assaying the sandwich is to coat a solid-phase substrate with the protein conjugate, capture chelator (i.e., PAR-ovalbumin (PAR-OVA)), and a metal analyte is incubated with the immobilized according to: ##STR1##

where S1* represents the binding sites of the capture chelator while k1 and k2 are the association and dissociation rate constants respectively. All other sample constituents are washed out and the bound metal analyte is quantitated during the second step by adding the excess detection chelator_(i.e., PAR-alkaline phosphatase (PAR-AP)). After incubation, the PAR-AP is bound to a different site on the metal ion capture chelate molecule according to: ##STR2##

where S2*_represents the binding sites of the labeled conjugate and k3, k4_are the rate constants. Any unbound detection chelator_(PAR-AP) is washed out. The signal is detected by reacting with an assaying reagent which, in one embodiment, produces a measurable absorption enhancement at 405 mn. The signal is directly related to the metal concentration in the sample. Since this assay involves 2 chelators, it provides an improved specificity over a competitive assay principle, because the cross-reactivity substances that nominally interfere with competitive assay produce no signal in the sandwich assay format.

Claim 1 of 7 Claims

We claim:

1. A protein conjugate gallium (III) complex comprising the reaction product of

a) a ((heterocyclyl)azo)orthohydroxyarylene derivative comprising a protein binding functionality wherein the azo linking group is attached ortho to the nitrogen atom of the heterocycle and wherein the hydroxy group on the arylene moiety is in a position ortho to the site of attachment of the azo linking group and

b) a protein

the conjugate being complexed with gallium (III).

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