WO1998000696A1

WO1998000696A1 - Pretreatment reagents and methods using the same

Info

Publication number: WO1998000696A1
Application number: PCT/US1997/012420
Authority: WO
Inventors: Anna P. Jaklitsch; Daniel J. Monger; Matthias Pfeiffer; Stephen H. Roth; Henry Jeong
Original assignee: Jaklitsch Anna P; Monger Daniel J; Matthias Pfeiffer; Roth Stephen H; Henry Jeong
Priority date: 1996-07-03
Filing date: 1997-07-03
Publication date: 1998-01-08
Also published as: EP0850402A4; JP2001514737A; EP0850402A1; CA2230284A1; AU3730097A

Abstract

Compositions and kits are disclosed for pretreating samples that are to be analyzed for the presence and/or amount of an associated analyte. The composition comprises a lower alkyl alcohol in an amount of about 30 % to about 40 % by volume, a glycol in an amount of about 20 % to about 40 % by volume, and an aqueous component comprising about 20 mM to about 30 mM copper salt. Additionally, the aqueous component can comprise about 0.5 mM to about 20 mM of a buffer, and about 0.005 % to about 0.2 % by weight of a non-ionic detergent and has a pH of about 2.0 to about 4.6. The kits further include one or more reagents for conducting an assay for the associated analyte. Also disclosed are improvements in assays for associated analytes wherein the improvements comprise pretreating a sample suspected of containing the associated analyte with the above composition.

Description

PRETREATMENT REAGENTS AND METHODS USING THE SAME Field of the Invention

The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials (analytes) that may be readily and accurately determined, as well as the methods for the determination. Convenient and reliable means for detecting the presence of low concentrations of materials in liquids is desired. In clinical chemistry these materials may be present in body fluids in concentrations below 10^"12 molar. The difficulty of detecting low concentrations of these materials is enhanced by the relatively small sample sizes that can be utilized and the fact that some analytes are associated with other components in the sample and are difficult to detect accurately.

The monitoring of levels of drugs, such as therapeutic drugs, in biological fluid samples is useful in the proper treatment of patients receiving such drugs. It is important to monitor drug levels to avoid toxic dose levels or dose levels that are therapeutically ineffective. Many assay techniques have been developed to determine quantitatively the level of drug in a patient sample. Such assay techniques generally involve immunoreactions and include, for example, radioimmunoassay, enzyme immunoassay, agglutination immunoassays, fluorescent polarization immunoassays, and so forth.

In developing an assay there are many considerations. One consideration is the signal response to changes in the concentration of an analyte. A second consideration is the ease with which the protocol for the assay may be carried out. A third consideration is the variation in interference from sample to sample. Ease of preparation and purification of the reagents, availability of equipment, ease of automation and interaction of the material of interest with sample components are some of the additional considerations in developing a useful assay. For example, some analytes are associated with various other components of a sample such as proteins, cells and other cellular material, thus making their detection difficult or impossible.

The body relies upon a complex immune response system to distinguish self from non-self. The proper functioning of the immune system is vital for the long term health of the body. Deficient immune response can lead to the body's inability to protect itself from non-self matter. Excessive immune response can lead to the body's over reaction to what would otherwise be innocuous matter.

At times, the body's immune system must be controlled in order to either augment a deficient response or suppress an excessive response. For example, when organs such as kidney, heart, heart-lung, bone marrow, and liver are transplanted in humans, the body will sometimes reject the transplanted tissue by a process referred to as allograft rejection.

In treating allograft rejection, the immune system is frequently suppressed in a controlled manner through drug therapy. Immunosuppressant drugs are carefully administered to transplant recipients in order to help prevent allograft rejection of non-self tissue. One such drug which finds use as an immunosuppressant in the United States and other countries is cyclosporin A (CsA) (U.S. Patent Nos. 4,117,118 (1978) and 4,396,542 (1983)). CsA may have other useful properties such as antibiotic, anti-arthritic and anti-inflammatory activities and may find use in the treatment of other conditions such as diabetes, malaria and autoimmune diseases.

Even though CsA is a highly effective immunosuppressant drug, its use must be carefully managed because the effective dose range is narrow and excessive dosage can result in serious side effects. Renal dysfunction, hypertension, cardiovascular cramps, hirsutism, acne, tremor, convulsions, headache, gum hyperplasia, diarrhea, nausea, vomiting, hepatotoxicity, abdominal discomfort, paresthesia, flushing, leukopenia, lymphoma, sinusitis and gynecomastia have been observed in kidney, heart or liver transplant patients undergoing CsA treatment. Too little CsA can lead to graft rejection.

Management of CsA dosage involves careful control of the level of the drug present in the patient. Because the distribution and metabolism of CsA varies greatly between patients, and because of the wide range and severity of adverse reactions, accurate monitoring of drug level is considered essential. CsA associates with materials present in whole blood samples and, thus, an accurate assay requires that CsA be extracted from these other materials.

The methods and compositions of the present invention relate to reagents for pretreating samples suspected of containing analytes of interest, in particular analytes that occur in samples in association with other materials.

Documents are cited in this disclosure with a full citation. These documents relate to the state-of- the-art, and each document is hereby incorporated by reference.

Brief Description of the Related Art European patent application No. 283,801 (1988) (Wang, et al . ) and its priority document U.S. Patent No. 5,239,057 describe a fluorescence polarization assay for cyclosporin A and metabolites and related immunogens and antibodies.

Quesniaux, et al . , Molecular Immunology (1987) 4(H) : 1159-1168, describe the specificity and cross- reactivity of monoclonal antibodies to cyclosporin. Ball, et al . , discuss specific radioimmunoassay with a monoclonal antibody for monitoring cyclosporine in blood in Clin. Chem. (1988) 3_4 (2) :257-260. McBride, et al . , describe the measurement of cyclosporine in plasma from patients with various transplants: HPLC and radioimmunoassay with a specific monoclonal antibody compared in Clin. Chem. (1989) 15(8) :1726-1730.

The liquid-chromatographic measurement of cyclosporin A and its metabolites in blood, bile and urine is discussed in Clin. Chem. (1988) 34 ( 1) :34-39.

Bowers, et al . , disclose studies of cyclosporine blood levels: analysis, clinical utility, pharmacokinetics, metabolites, and chronopharmacology in Transplantation Proceedings (1986) XVIII (6):137-143.

U.S. Patent No. 5,135,875 (Meucci, et al . ) discusses a protein precipitation reagent, employing a glycol, a short-chained aliphatic alcohol and zinc.

European Patent Application No. 0 471 293 A2 (Meucci, et al . ) discloses a solubilization reagent for biological test samples.

European Patent Application No. 0 473 961 A2 (Morrison, et al . ) discloses immunoassay reagents and method for determining cyclosporin.

U.S. Patent No. 4,959,303 (Milburn, et al . ) describes an assay for antigens by binding immune complexes to solid supports free of protein and non-ionic binders.

Despite the extensive teachings of the prior art regarding assays for cyclosporin and pretreatment reagents, the development of a pretreatment formulation which facilitates the detection of an analyte associated with other components of a sample, wherein the individual components of the pretreatment formulation, either alone or in combination, do not interfere with the assay medium and subsequent detection, has not been taught or suggested by the prior art. SUMMARY OF THE INVENTION

Thus, the present invention provides a novel pretreatment composition for treating samples being assayed for the presence and/or amount of an associated analyte, wherein the pretreatment composition facilitates the detection of an analyte associated with other components of a sample which would otherwise obscure detection of the analyte under conventional assay conditions, absent pretreatment. In accordance with the present invention, a sample is pretreated to render the associated analyte more readily available to one or more of the assay reagents. The inventive pretreatment compositions serve to lyse cells present in the sample, precipitate proteins which may be present, and solubilize the analyte of interest, rendering the analyte more easily detectable.

One aspect of the present invention relates to a composition comprising a lower alkyl alcohol in an amount of about 30% to about 40% weight/volume (w/v) , a glycol in an amount of about 20% to about 40% w/v, and at least about 30% w/v of an aqueous component comprising about 20 mM to about 30 mM metal salt.

Another embodiment of the present invention is a composition comprising ethanol in an amount of about 30% to about 40% w/v, propylene glycol or ethylene glycol in an amount of about 20% to about 40% w/v, and about 30% to about 40% w/v of an aqueous component comprising about 20 mM to about 30 mM copper salt, about 0.5 mM to about 20 mM of a buffer, and about 0.005% to about 0.1% w/v of a non-ionic detergent, such that the aqueous component has a pH of about 3.0 to about 4.6.

Another aspect of the present invention is an improvement in an assay for the determination of an associated analyte in a sample suspected of containing the associated analyte. The assay comprises contacting a sample suspected of containing the associated analyte with a pretreatment reagent, contacting the sample with assay reagents for detection of the analyte. The improvement comprises employing as the pretreatment reagent one of the above compositions.

Another aspect of the present invention is a kit comprising in packaged combination (a) one or more reagents for conducting a determination of an associated analyte and (b) one of the above compositions.

Another aspect of the present invention is an improvement in an assay for the determination of an immunosuppressant drug in a sample suspected of containing the drug. The assay comprises contacting the sample with a specific binding member for the drug and detecting binding of the specific binding member to the drug. The present improvement comprises contacting the sample with one of the above compositions either prior to or in conjunction with the contacting step.

Another embodiment of the present invention is an improvement in an assay for the determination of an immunosuppressant drug in a sample suspected of containing the drug. The assay comprises contacting the sample with antibodies for the drug and a conjugate of a label and a compound recognized by the antibodies, and detecting immune complexes of the label conjugate and the antibodies. The improvement comprises contacting the sample with the one of the above compositions either prior to or in conjunction with the first step of the assay.

These and other objects and embodiments are disclosed or will be obvious from the following Detailed Description.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a graphical representation of the results of an assay for tacrolimus (FK506) with the use of the inventive pretreatment composition versus with methanol alone. DETAILED DESCRIPTION

The present invention provides compositions and kits for pretreating samples, to facilitate the determination of the presence and/or the amount of an associated analyte. In accordance with the present invention, a sample is pretreated to make the associated analyte more readily available to one or more of the assay reagents. Samples to be analyzed are pretreated to lyse cells precipitate proteins, and to solubilize cyclosporin, each of which may be present in the sample to be analyzed. The pretreatment compositions are characterized in that they exhibit lower volatility than some of the known pretreatment materials. This permits the pretreated sample to be assayed by analyzers using sample cups open to the environment with greater sample stability and with reduced risk to the operator.

Before proceeding with a description of the specific embodiments, a number of terms will be defined. A sample is a biological material, usually a biological fluid, which can include human, animal or man-made samples. Typically, the sample is a natural fluid, such as, for example, urine, whole blood, serum, plasma, semen, spinal fluid, saliva, and so forth or an aqueous solution or extract thereof. An associated analyte is an analyte that is present in a sample in association with, e.g., complexed to, other components of the sample such as, for example, cellular material, phospholipids, proteins and the like. Such analytes include therapeutic drugs, such as, for example, immunosuppressant drugs, e.g., cyclosporin, mycophenolic acid, FK-506, rapamycin, azathioprine and steroids.

Cyclosporin is a natural or synthetic cyclic undecapeptide, used as an immunosuppressive drug to suppress unwanted immune response leading to organ rejection after transplantation. The exact structure of a particular cyclosporin may vary in minor ways from one to the next. Cyclosporins include cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporin H, cyclosporin I, atiocyclosporin, and so forth. Also included in the term cyclosporin are metabolites of cyclosporin which retain the undecapeptide ring such as, for example, major metabolites AM9 (Ml), AM19 (M8) , AMI (M17) and AM4N (M21) , and other metabolites such as M9, M10, M16 and M18. Lower alkyl alcohols are branched or straight chain alkyl alcohols containing from 1 to 5 carbon atoms, such as, e.g., methanol, ethanol, propanol, butanol, isopropanol, isobutanol, pentanol, isopentanol, etc., and mixtures thereof, preferably methanol. The term aliphatic relates to an acyclic hydrocarbon; an aliphatic hydrocarbon is a compound of carbon and hydrogen having an open chain.

Polyhydroxy aliphatic compounds are aliphatic compounds containing two or more hydroxyl groups such as, for example, alkylene polyols, e.g., glycols, glycerol and the like, and mixtures thereof, preferably, a glycol, more preferably, propylene glycol.

Glycols are aliphatic compounds containing two hydroxyl groups such as, for example, ethanediol (ethylene glycol) , propanediol (propylene glycol) , polyethylene glycol, polypropylene glycol, and the like.

Glycerol (glycerin) is an alkylene polyol, namely, 1,2,3-propanetriol.

A metal salt is a compound containing a metal ion and a non-metal counterion. The metal ion is one that provides for precipitation of hemoglobin (heme) , plasma proteins, and other interfering substances in a sample to be analyzed. Preferred metal ions include ions of copper, iron, cobalt and molybdenum, more preferably, copper ion. The particular metal ion used is determined in part so as to avoid interference in the wavelength of the measurement of a signal obtained in an assay for the associated analyte. The non-metal anion can be, by way of illustration and not limitation, a sulfate, a halide such as chloride, bromide, etc. , an acetate, and so forth.

A buffer is a buffering agent generally having a pKa of about 4.5 to about 8.5. The buffer is chosen so that a stable pH is obtained within the desired range of pH values for the aqueous component, and thus a composition, in accordance with the present invention. The buffer should have minimal effect on the stability of the components of the present compositions and should not have a deleterious effect on the reagents used in an assay for an associated analyte or on the assay measurement or results in general. Suitable buffers are homopiperazine-N,N' -bis-2-ethanesulfonic acid, acetate, citrate, tris-HCl, and the like.

The term detergent, in the context of the present disclosure, refers to a surfactant that is capable of rupturing red blood cells. A non-ionic detergent refers to a non-ionic surfactant such as, for example, a nonionic polyglycol detergent, for example, polyoxypropylene (available as Pluronic 25R2 from BASF Chemicals, Mount Olive, NJ) , saponins, digitonin, polyoxyethylene alcohols (e.g., Brij series and Lubrol) , polyoxyethylene p tert octylphenols (e.g., Triton X-100, Triton X-114 and Nonidet P-40) , fatty acid esters of polyoxyethylene sorbitan (e.g., Tween-20) , β-D- octylglucoside, β-D-dodecylmaltoside and alkyl-N- methylglucamides (all available from Sigma Chemical Company, St. Louis, MO, or Fluka Chemie AG, Buchs, Switzerland or Ronkonkoma, NY, or Aldrich Chemical

Company, Milwaukee, WI) , and so forth. Preferably, the non-ionic detergent is a polyoxypropylene or polyoxyethylene.

An anionic detergent is an anionic surfactant, such as, for example, alkyl-, alkyletherdialkyl esters-, alkylaryl-, and alpha olefin- sulfates, sulfonic acids, sulfonates, sulfosuccimates, and sulfosuccinic acids, polymerized alkyl naphthalene sulfonates, phosphate esters, free acid of complex organic phosphate esters, aliphatic hydroxylated phosphate esters, sulfated fatty acid esters, sulfated oils such as castor, sperm, soya bean, glycerol trioleate, neatsfoot, tallow and oleic acid, n-fatty acid acyl glutamates such as n-lauroyl, n- cocoyl, n-hydrogenated tallowyl, n-mixed fatty acid acyl, carboxylated polyelectrolytes, disproportionated resins, and so forth. Examples of common anionic detergents include sodium dodecylsulfate and sodium dodecyl-N- sarcosinate.

The term alkylene refers to an organic radical derived from an unsaturated aliphatic hydrocarbon, e.g. ethylene. An antibody is an i munoglobulin which specifically binds to, and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera from which the immunoglobulin can be separated by known techniques (polyclonal) , by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal) or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin, or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, igD, IgE, igGi, lgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like.

Antiseru containing antibodies (polyclonal) is obtained by well-established techniques involving immunization of an animal, such as a rabbit, guinea pig, or goat, with an appropriate immunogen and obtaining antisera from the blood of the immunized animal after an appropriate waiting period. State-of-the-art reviews are provided by Parker, Radioimmunoassay of Biologically Active Compounds, Prentice-Hall (Englewood Cliffs, N.J., U.S., 1976), Butler, J. Immunol. Meth. 7: 1-24 (1975); Broughton and Strong, Clin. Chem. 22: 726-732 (1976); and Playfair, et al., Br. Med. Bull. 30: 24-31 (1974).

Antibodies can also be obtained by somatic cell hybridization techniques, such antibodies being commonly referred to as monoclonal antibodies. Monoclonal antibodies may be produced according to the standard techniques of Antiseru containing antibodies (polyclonal) is obtained by well-established techniques involving immunization of an animal, such as a rabbit, guinea pig, or goat, with an appropriate immunogen and obtaining antisera from the blood of the immunized animal after an appropriate waiting period. State-of-the-art reviews are provided by Parker, Radioimmunoassay of Biologically Active Compounds, Prentice-Hall (Englewood Cliffs, N.J., U.S., 1976), Butler, J. Immunol. Meth. 7: 1-24 (1975); Broughton and Strong, Clin. Chem. 22: 726- 732 (1976); and Playfair, et al., Br. Med. Bull. 30: 24- 31 (1974).

Antibodies can also be obtained by somatic cell hybridization techniques, such antibodies being commonly referred to as monoclonal antibodies. Monoclonal antibodies may be produced according to the standard techniques of Kδhler and Milstein, Nature 265:495-497. 1975. Reviews of monoclonal antibody techniques are found in Lymphocyte Hybridomas, ed. Melchers, et al.

Springer-Verlag (New York 1978), Nature 266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73 (Part B) : 3-46 (1981) . Samples of an appropriate immunogen preparation are injected into an animal such as a mouse and, after a sufficient time, the animal is sacrificed and spleen cells obtained. Alternatively, the spleen cells of a non-immunized animal can be sensitized to the immunogen in vitro. The spleen cell chromosomes encoding the base sequences for the desired im unoglobins can be compressed by fusing the spleen cells, generally in the presence of a non-ionic detergent, for example, polyethylene glycol, with a myeloma cell line. The resulting cells, which include fused hybridomas, are allowed to grow in a selective medium, such as HAT- medium, and the surviving immortalized cells are grown in such medium using limiting dilution conditions. The cells are grown in a suitable container, e.g. , microtiter wells, and the supernatant is screened for monoclonal antibodies having the desired specificity.

Various techniques exist for enhancing yields of monoclonal antibodies, such as injection of the hybridoma cells into the peritoneal cavity of a mammalian host, which accepts the cells, and harvesting the ascites fluid. Where an insufficient amount of the monoclonal antibody collects in the ascites fluid, the antibody is harvested from the blood of the host. Alternatively, the cell producing the desired antibody can be grown in a hollow fiber cell culture device or a spinner flask device, both of which are well known in the art. Various conventional ways exist for isolation and purification of the monoclonal antibodies from other proteins and other contaminants (see Kohler and Milstein, supra) .

In another approach for the preparation of antibodies the sequence coding for antibody binding sites can be excised from the chromosome DNA and inserted into a cloning vector which can be expressed in bacteria to produce recombinant proteins having the corresponding antibody binding sites.

Reviews of monoclonal antibody techniques are found in Lymphocyte Hybridomas, ed. Melchers, et al. Springer-Verlag (New York 1978), Nature 266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73 (Part B) : 3-46 (1981). Samples of an appropriate immunogen preparation are injected into an animal such as a mouse and, after a sufficient time, the animal is sacrificed and spleen cells obtained. Alternatively, the spleen cells of a non-immunized animal can be sensitized to the immunogen in vitro. The spleen cell chromosomes encoding the base sequences for the desired immunoglobins can be compressed by fusing the spleen cells, generally in the presence of a non-ionic detergent, for example, polyethylene glycol, with a myeloma cell line. The resulting cells, which include fused hybridomas, are allowed to grow in a selective medium, such as HAT- medium, and the surviving immortalized cells are grown in such medium using limiting dilution conditions. The cells are grown in a suitable container, e.g. , microtiter wells, and the supernatant is screened for monoclonal antibodies having the desired specificity.

Various techniques exist for enhancing yields of monoclonal antibodies, such as injection of the hybridoma cells into the peritoneal cavity of a mammalian host, which accepts the cells, and harvesting the ascites fluid. Where an insufficient amount of the monoclonal antibody collects in the ascites fluid, the antibody is harvested from the blood of the host. Alternatively, the cell producing the desired antibody can be grown in a hollow fiber cell culture device or a spinner flask device, both of which are well known in the art. Various conventional ways exist for isolation and purification of the monoclonal antibodies from other proteins and other contaminants (see Kδhler and Milstein, supra) .

In another approach for the preparation of antibodies the sequence coding for antibody binding sites can be excised from the chromosome DNA and inserted into a cloning vector which can be expressed in bacteria to produce recombinant proteins having the corresponding antibody binding sites. In general, antibodies can be purified by known techniques such as chromatography, e.g., DEAE chromatography, ABx chromatography, and the like, filtration, and so forth.

Reagents for conducting the determination of an associated analyte are otherwise referred to herein as reagents used in an assay for the associated analyte. Such reagents depend on the nature of the assay, e.g., whether the assay is heterogeneous or homogeneous; on the nature of the binding reactions and the signal producing system utilized, e.g., enzyme immunoassay, fluorescent immunoassay, chemiluminescent immunoassay, agglutination assay, and so forth. In general, such reagents are those that permit an accurate determination to be conducted for the presence and/or amount of an associated analyte.

For example, for an immunoassay such reagents may include a conjugate of a label and a specific binding pair member, e.g., antibody or hapten, other members of a signal producing system of which the label is a part, other specific binding pair members, ancillary materials and the like. A label is any molecule which produces or can be induced to produce a signal. The label may be conjugated to a member of a specific binding pair such as an analyte or an antibody, or to another molecule such as a receptor or a molecule that can bind to a receptor such as a ligand, particularly a hapten. The label can be a member of the signal producing system, as defined below, that includes a signal producing means. The label may be isotopic or nonisotopic, preferably nonisotopic. By way of example and not limitation, the label can be a part of a catalytic reaction system such as enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, or catalysts; part of a chromogen system such as fluorophores, dyes, chemiluminescers, lu inescers, or sensitizers; a dispersible particle that can be non-magnetic or magnetic, a solid support, a liposome, a ligand, a receptor, a hapten, and so forth. A wide variety of non-enzymatic catalysts which may be employed are found in U.S. Patent No. 4,160,645 (1979), the appropriate portions of which are incorporated herein by reference. A large number of enzymes and coenzymes for providing such products are indicated in U.S. Patent No. 4,275,149 columns 19 to 23, and U.S. Patent No. 4,318,980, columns 10 to 14, which disclosures are incorporated herein by reference. A number of enzyme combinations are set forth in U.S. Patent no. 4,275,149, columns 23 to 28, which combinations can find use in the subject invention. This disclosure is incorporated herein by reference.

Illustrative enzymes include dehydrogenases such as malate dehydrogenase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase. Of the oxidases, glucose oxidase is exemplary. Of the peroxidases, horse radish peroxidase is illustrative. Of the hydrolases, alkaline phosphatase, b-glucosidase and lysozyme are illustrative.

A conjugate is a molecule comprised of two or more subunits bound together, optionally through a linking group, to form a single structure. The binding is a chemical interaction which can be made either by a direct connection (e.g. a chemical bond) between the subunits or by use of a linking group. Conjugation is any process wherein two subunits are linked together to form a conjugate. The conjugation process can be comprised of any number of steps.

A receptor is any compound or composition capable of recognizing a particular spatial and polar organization of a molecule. These organized areas of a molecule are referred to as epitopic or determinant sites. Illustrative naturally occurring receptors include antibodies, enzymes, FAb fragments, poly(nucleic acids) , complement component, i.e. thyroxine binding globulin, lectins, protein A, and the like. Receptors are also referred to as antiligands. Natural receptors exists that binds specifically to cyclosporin. A ligand is any organic molecule for which a receptor naturally exists or can be prepared.

A member of a specific binding pair (sbp member) is one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiligand) . These will usually be members of an immunological pair such as antigen-antibody, although other specific binding pairs, such as biotin-avidin, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like, are not immunological pairs but are specific binding pairs.

A support or surface is a porous or non-porous water insoluble material. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc. ; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride) , polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene) , polystyrene, poly ethacrylate, poly(ethylene terephthalate) , nylon, poly (vinyl butyrate) , etc.; either used by themselves or in conjunction with other materials; glass available as Bioglaεs, ceramics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed. Other materials which can be employed are described above in the definition of immunogenic carrier particles and below in the definition of a signal producing system. Binding of sbp members to the support or surface may be accomplished by well-known techniques, commonly available in the literature, and described above in the definition of immunogenic carrier particles. The surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.

The function of the signal producing system is to produce a product which provides a detectable signal related to the amount of bound and/or unbound label. The signal producing system may have one or more components, at least one component being a label. The signal producing system includes all of the reagents required to produce a measurable signal including signal producing means capable of interacting with the label to produce a signal.

The signal producing system provides a signal detectable by external means, normally by measurement of electromagnetic radiation, desirably by visual examination. In one approach, the signal producing system includes a chromophoric substrate and enzyme, where chromophoric substrates are enzymatically converted to dyes which absorb light in the ultraviolet or visible region, phosphors or fluorescers.

The signal producing means is capable of interacting with the label to produce a detectable signal. Such means include, for example, electromagnetic radiation, heat, chemical reagents, and the like. Where chemical reagents are employed, some of the chemical reagents can be included as part of a developer solution. The chemical reagents can include substrates, coenzymes, enhancers, second enzymes, activators, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, and the like. Some of the chemical reagents such as coenzymes, substances that react with enzymic products, other enzymes and catalysts, and the like can be bound to other molecules or to a support. The signal producing system including the label can include one or more particles, which are insoluble particles of at least about 50 nm and not more than about 50 microns, usually at least about 100 nm and less than about 25 microns, preferably from about 0.2 to 5 microns, diameter. The particle may be organic or inorganic, porous or non-porous, preferably of a density approximating water, generally from about 0.7 to about 1.5 g/mL, and composed of material that can be transparent, partially transparent, or opaque.

Further, ancillary materials will frequently be employed in an assay in accordance with the present invention. For example, buffers will frequently be present in the assay medium, as well as stabilizers, preservatives and/or antimicrobial agents for the assay medium and the assay components.

Quantitative, semiquantitative, and qualitative methods for determining an associated analyte are considered to be methods of measuring the amount of such analyte. For example, a method which detects the presence or absence of such analyte in a sample suspected of containing the analyte is considered to be included within the scope of the above phrase. Synonymous with the recitation of the phrase determining the amount of an associated analyte are the following, non-limiting recitations, including: detecting or measuring an associated analyte; detecting or measuring the presence of an associated analyte; and detecting or measuring the amount of such analyte. One aspect of the present invention relates to compositions comprising a lower alkyl alcohol, a polyhydroxy aliphatic compound, and an aqueous component containing a metal salt. The nature and amount of alcohol in the present composition is governed by a number of considerations. The alcohol should have minimal effect on the binding of specific binding reagents used in an assay for the associated analyte. Also, the alcohol should have minimal effect on the activity of other reagents for conducting an assay such as, for example, labels, e.g., enzymes, and so forth. The amount of alcohol is at least sufficient to provide for substantial extraction of the associated analyte from other sample components and also to minimize the amount of hemoglobin and other interfering substances in the extracted sample. The amount of alcohol should be low enough so that a substantial portion, preferably all, of the metal salt remains in solution in the composition. The amount of alcohol is not so great as to result in an unacceptable evaporation rate for the present composition when used to pretreat a sample in accordance with the present invention or to result in an unacceptable integrity for the insolubilized interfering substances that form during the present pretreatment. Generally, the insolubilized interfering substances form a pellet during the course of the present pretreatment. The integrity of the pellet should be such as to not permit any significant amount of the interfering substances to be removed when the sample solution is separated from the pellet. In general, the amount of alcohol in the composition is about 30% to about 40%, preferably about 33% to about 37%, more preferably, about 35%, w/v. In a preferred embodiment, the alcohol is methanol, and it is present in an amount of about 33% to 35% w/v.

The polyhydroxy aliphatic compound should be present in an amount that provides for rapid mixing of the present composition with the sample so that minimal overall handling time is realized. Desirably, the amount of the polyhydroxy aliphatic compound is sufficient when the viscosity of the present composition is about that of the sample to be pretreated. Where the sample is whole blood, the viscosity of the composition of the invention should ideally approximate that of whole blood. In this way, the sample and the composition are rapidly and easily mixed together. The amount of polyhydroxy aliphatic compound should not be great enough to result in the extraction of hemoglobin or other interfering substances together with the extracted analyte. Usually, the polyhydroxy aliphatic compound is present in an amount of about 20% to about 40%, preferably, about 27% to about 33.3%, more preferably, about 30% to 33.3%, w/v. In a preferred embodiment, the polyhydroxy aliphatic compound is propylene glycol, and it is present in an amount of about 30% to 33.3% w/v. The amount of the aqueous component is generally determined by default based on the amount of the lower alkyl alcohol and the polyhydroxy aliphatic compound. The amount of the aqueous component is at least that necessary to minimize precipitation of the metal salt in the present composition. Normally, the aqueous component is present in an amount of at least 30%, preferably, at about 30% to about 40%, more preferably about 33% to about 38%, more preferably, about 33.3% to 36% w/v. The amount of metal salt present in the aqueous component is at least sufficient to precipitate a substantial portion of the hemoglobin and other interfering substances in the sample to be analyzed. However, the amount of the metal ion should not be great enough to cause a significant effect on the activity of assay reagents used in the analysis. In particular, the metal ion concentration should not be so great as to have a detrimental effect on the activity of a label, e.g. , an enzyme, used as part of a signal producing system. Generally, the copper salt is at a concentration of about 20 mM to about 30 mM, preferably, about 23 mM to about 27 mM, more preferably, about 25 mM.

The aqueous component may contain one or more other reagents such as a buffer and a non-ionic or anionic detergent. The concentration of a buffer is not critical. In general, the lowest concentration is chosen that achieves a desired pH for the final composition of the invention. The concentration of the buffer should not be so high as to offset the required pH for other reagents used in an assay for the associated analyte when the extracted sample is mixed with such reagents to conduct the assay. The amount of the buffer is chosen so that a stable pH is obtained within the desired range of pH values for the aqueous component, and thus a composition, in accordance with the present invention. The concentration of the buffer should have minimal effect on the stability of the components of the present compositions and should not have a deleterious effect on the reagents used in an assay for a associated analyte or on the assay measurement or results in general. The buffer is usually present at a concentration of about 0.5 mM to about 20 mM, preferably, about 0.8 to about 19 mM, more preferably, about 1 to 18.5 mM.

The detergent concentration should be sufficient to result in a substantial extraction of the associated analyte and to bring about substantial rupturing of cells present in the sample to be analyzed. Furthermore, the nature of the detergent as well as its concentration should provide for consistency in the results obtained in an assay for the associated analyte. Usually, the detergent is present in a concentration of about 0.005% to about 0.2%, preferably, about 0.01% to about 0.1%, more preferably, about 0.01%, w/v.

The final pH of the composition should not be so low that it would have a deleterious effect on reagents used in an assay for the associated analyte, nor should the pH be great enough to cause any significant precipitation of the metal ion. The pH of the composition is dependent primarily on the pH of the aqueous component. Usually, the pH of the aqueous component is about 2.0 to about 4.6, preferably, about 3.0 to 4.6, more preferably, about 4.0 to about 4.4, more preferably, about 4.3. The amount of the buffer and its pKa generally determines the final pH, but other components of the composition should be taken into consideration in this regard. Upon combining the aqueous component with the lower alkyl alcohol and the polyhydroxy aliphatic compound, there will be a slight change in the overall pH of the combination. Generally, the pH of the combination will be lowered approximately 0.1 to 0.3, usually, 0.1 to 0.2, pH units with respect to the pH of the aqueous component.

While the addition of preservatives, antimicrobials or chelating agents to the present compositions is not necessary, such agents may be included in the pretreatment composition without interfering with the function of the inventive compositions. Examples of such ancillary agents which may be included in the inventive compositions, include, but are not limited to, sodium azide, EDTA and streptamycin.

The composition of the present invention is usually packaged in a single container for convenience of use. However, components of the composition may be provided in separate containers. In that regard the lower alkyl alcohol, the polyhydroxy aliphatic compound, and the aqueous component, respectively, may be packaged in separate containers or two of these components may be combined and packaged separately from the remaining component. Further, the compositions of the instant invention may be packaged, in one or more containers, accompanied by directions for suitable admixture and use. A preferred embodiment of the present invention is a composition comprising methanol in an amount of about 30% to about 40% w/v, propylene glycol or ethylene glycol in an amount of about 20% to about 40% w/v, and about 30% to about 40% w/v of an aqueous component comprising about 20 mM to about 30 mM copper salt, about 0.5 mM to about 20 mM of a buffer, about 0.005% to about 0.1% w/v of a non-ionic detergent and a pH of about 3.0 to about 4.6. Another aspect of the present invention is an improvement in an assay for the determination of an associated analyte such as an immunosuppressant drug, e.g., cyclosporin, in a sample suspected of containing the associated analyte. The assay comprises contacting a sample suspected of containing the associated analyte with a pretreatment reagent, contacting the sample with reagents for conducting a determination of the analyte and analyzing for the results of the determination. The improvement comprises employing as the pretreatment reagent one of the above compositions. The inventive compositions are particularly compatable with the EMIT® assay format, due to the use of copper ion as the metal in the pretreatment composition. It has been surprisingly found that the use of copper is particularly compatable with the EMIT® assay system, whereas other metal ions tend to interfere with the detection of analyte by the EMIT® assay. The EMIT® assay system may be used for the detection of a variety of analytes, including, but not limited to, cyclosporin. The EMIT® assay format or Enzyme Multiplied Immunoassay Technique, is a homogeneous competitive enzyme immunoassay introduced by Rubenstein, K.E., Schneider, R.S., Ullman, E.F. "Homogeneous" enzyme immunoassay. A new immunochemical technique. Biochem. Biophys, Res. Commun. 47: 846, 1972. This techique avoids the necessity to separate labeled antigen bound to antibody from that which is unbound. It depends upon a change in the specific enzyme activity when antibody is bound to enzyme labeled antigen or conjugate. The activity of the unseparatd assay mixture is proportional to the amount of conjugate to which antibody is bound.

One broad category of techniques for conducting an assay involves the use of a receptor for the analyte. The observed effect of binding by the receptor will depend upon a label used. In some instances the binding of the receptor merely provides for a differentiation in molecular weight between bound and unbound labeled ligand. In other instances the binding of the receptor will facilitate separation of bound labeled ligand from free labeled ligand or it may affect the nature of the signal obtained from the label so that the signal varies with the amount of receptor bound to labeled ligand. A further variation is that the receptor is labeled and the ligand unlabeled. Alternatively, both the receptor and ligand are labeled or different receptors are labeled with two different labels, whereupon the labels interact when in close proximity and the amount of ligand present affects the degree to which the labels of the receptor may interact. The analysis for the results of the determination is then based on the nature of the label. Accordingly, for enzyme labels, enzyme activity is measured; for fluorescent labels, the amount of fluorescence is measured, and so forth.

One particular embodiment of an assay involving a receptor is an immunoassay where the receptor is an immunoreactant such as an antibody and immune complexes are formed in relation to the presence or amount of analyte present in the sample. The analysis for the results of the determination generally involves the detection of the immune complexes. The immune complexes are detected directly, for example, where an immune reagent such as an antibody employed is conjugated to a label. The immune complex is detected indirectly by examining for the effect of immune complex formation in an assay medium on a signal producing system or by employing a labeled receptor that specifically binds to an immune reagent used in the assay.

The assay of the invention has application to all immunoassays for associated analytes. The assay can be performed either without separation (homogeneous) or with separation (heterogeneous) of any of the assay components or products. Exemplary of heterogeneous assays are enzyme linked immunoassays such as the enzyme linked immunosorbant assay (ELISA) , see "Enzyme-Immunoassay" by Edward T. Maggio, CRC Press Incorporated, Boca Raton, Florida, 1980. Homogeneous immunoassays are exemplified by enzyme multiplied immunoassay techniques (e.g. see U.S. Patent No.

3,817,837), immunofluorescence methods such as those disclosed in U.S. Patent No. 3,993,345, enzyme channeling techniques such as those disclosed in U.S. Patent No. 4,233,402, and other enzyme immunoassays as discussed in Maggio, supra. The disclosures of the above patents are incorporated herein by reference in their entirety as to the description of the particular assay methods mentioned.

The sample to be analyzed is treated with the present composition. Accordingly, the sample is combined with a composition in accordance with the present invention. Usually, the sample is combined with an excess amount of the present composition, normally, about 100 to 500 μL, preferably, about 200 to 400 μL, more preferably, about 250 to 350 μL, of the composition per about 100 μL of the sample. The pretreatment is conducted at a temperature of about 15 to 30°C, preferably, about 20 to 25°C, more preferably, at ambient temperature. The sample and the present composition are incubated for a period of about 10 seconds to about 5 minutes, preferably, about 0.5 to 3 minutes, more preferably, about 1 to 2 minutes. Generally, the pretreatment is carried out prior to the addition of reagents for conducting an assay. However, there may be circumstances where one or more of the reagents for conducting an assay are present with the pretreatment composition where the nature of the reagent or an assay permits. Next, the treated sample is subjected to centrifugation, usually at ambient temperature for a period sufficient and force to form any solids in the medium into a mass, usually for a period of about 2 to 6 minutes at about 10,000 to 25,000 relative centrifugal force. The medium is then separated from the solid mass.

After, or coincident with subjecting the sample to pretreatment in accordance with the present invention, an assay for the associated analyte is conducted. One or more reagents are added to an aqueous medium containing the pretreated sample. The assay for the analyte will normally be carried out in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity. The aqueous assay medium may be solely water or may include from 0 to 40 volume percent of a cosolvent. The pH for the medium will usually be in the range of about 4 to 11, more usually in the range of about 5 to 10, and preferably in the range of about 6.5 to 9.5. The pH will usually be a compromise between optimum binding of the binding members of any specific binding pairs and the pH optimum for other reagents of the assay such as members of the signal producing system. Various buffers may be used to achieve the desired pH and maintain the pH of the assay medium during the determination. Illustrative buffers include borate, phosphate, carbonate, tris, barbital and the like. The particular buffer employed is not critical to this invention, but in an individual assay one or another buffer may be preferred.

Moderate temperatures are normally employed for carrying out the assay and usually constant temperatures during the period of the measurement, particularly for rate determinations. Incubation temperatures will normally range from about 5° to 45°C, more usually from about 15° to 40°C. Temperatures during measurements will generally range from about 10° to 50°C, more usually from about 15° to 40°C.

The concentration of analyte that may be assayed will generally vary from about 10~⁵ to 10^-13 M, more usually from about 10^"6 to 10^~8 M. Considerations, such as whether the assay is qualitative, semiquantitative or quantitative (relative to the amount of cyclosporin present in the sample) , the particular detection technique and the concentration of the analyte will normally determine the concentrations of the various reagents. Further, the kit may optionally contain directions for suitable admixture and use.

While the concentrations of the various reagents in the assay medium will generally be determined by the concentration range of interest of the analyte, the final concentration of each of the reagents will normally be determined empirically to optimize the sensitivity of the assay over the range. That is, a variation in concentration of analyte which is of significance should provide an accurately measurable signal difference.

While the order of addition of the reagents for conducting an assay may be varied widely, there will be certain preferences depending on the nature of the assay. The simplest order of addition is to add all the assay reagents simultaneously and to analyze for the results by determining the effect that the assay medium has on the signal as in a homogeneous assay. Alternatively, the reagents can be combined sequentially. Optionally, an incubation step may be involved subsequent to each addition, generally ranging from about 30 seconds to 6 hours, more usually from about 1 minute to 1 hour.

Another aspect of the present invention is an improvement in an assay for the determination of an immunosuppressant drug in a sample suspected of containing the drug. The assay comprises contacting the sample with a specific binding member for the drug and detecting the binding of the specific binding member to the drug. The present improvement comprises contacting the sample with one of the above compositions either prior to or in conjunction with the contacting step.

Another embodiment of the present invention is an improvement in an assay for the determination of an immunosuppressant drug in a sample suspected of containing such drug. The assay comprises contacting the sample with antibodies for the drug and a conjugate of a label and a compound recognized by the antibodies and detecting immune complexes of the label conjugate and the antibodies. The improvement comprises contacting the sample with a composition in accordance with the present invention.

Another aspect of the present invention is a kit comprising in packaged combination (a) one or more reagents for conducting a determination of a associated analyte and (b) a composition in accordance with the present invention. To enhance the versatility of the subject invention, the reagents can be provided in packaged combination, in the same or separate containers, so that the ratio of the reagents provides for substantial optimization of the method and assay. The reagents may each be in separate containers or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents.

The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents which substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the assay. Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention. The kit can further include a written description of a method in accordance with the present invention as described above. EXAMPLES The invention is demonstrated further by the following illustrative examples. Parts and percentages are by weight/volume (w/v) unless indicated otherwise. Temperatures are in degrees centigrade (°C) unless otherwise indicated.

Reagents

Methanol & Ethylene glycol - Mallinckrodt Chemical, Inc.,

St. Louis, MO Propylene glycol - Aldrich Chemical Co., Milwaukee, WI

Homopipes® buffer - homopiperazine-N,N' -bis-2- ethanesulfonic acid from Research Organics, Inc.,

Cleveland, OH

Cupric sulfate pentahydrate & Tris-HCl - Fluka Chemical Corporation, Ronkonkoma, NY

Pluronic 25R2® - BASF Chemicals, Mount Olive, NJ

Example 1 Assay for Cyclosporine Assays for cyclosporine were conducted using the EMIT® Cyclosporine Specific Assay kit manufactured by Behring Diagnostics Inc. , San Jose, California, Part No. 6R019UL, containing Reagents A and B. Calibrators used were manufactured by Behring Diagnostics Inc., San Jose, California, Part No. 6R119UL. Manufacturer' s instructions were followed. Assays were performed on the COBAS MIRA analyzer.

Prior to conducting the assays, samples were pretreated in accordance with the present invention. Accordingly, one hundred microliters (lOOμL) of a whole blood sample and 6 calibrators were each separately treated with 300μL of the following pretreatment composition: methanol (35%) , propylene glycol (29%) , an aqueous component (36%) containing l mM Homopipes® biological buffer (pKa=4.6), 25.0 mM cupric sulfate (pentahydrate) and 0.01% Pluronic 25R2® nonionic detergent; pH=4.3. Each sample or calibrator together with the pretreatment composition were treated by vortex at 500 to 1000 rpm at ambient temperature for a period of greater than or equal to 10 seconds. After the above treatment, the mixture was allowed to incubate for greater than or equal to 2 minutes at ambient temperature and then the mixture was centrifuged for greater than or equal to 2 minutes at 20,800 relative centrifugal force (rcf) . On the analyzer, 27.5μL of the resulting pretreated sample was incubated for 75 seconds with 155μL of Reagent A of the above EMIT assay kit. Subsequently, 75 μL of Reagent B of the EMIT assay kit was added. After a 175 second incubation, enzyme activity, (a function of drug concentration) was monitored by following the production of NADH spectrophotometrically at 340 nm for 100 seconds. A total of 60 samples and six calibrators were pretreated and assayed as described above using the EMIT, assay kits. Assay parameters on the COBAS MIRA analyzer Assay Temperature 37°C

Wavelength 340 nm

Volume of Pretreated Sample 27.5μL

Diluent Volume (water) 47.5μL

Reagent A Volume 155 μL Incubation Time (sample + Reagent A) 75 sec Reagent B Volume 75 μL

Delay Time (sample + Reagent A + Reagent B) 175 sec Read Time 100 sec

The assay standard curve range extended to 500 ng/mL. Analytical recovery within the curve range varied from 90 to 103%. Within run precision with trilevel controls ranged from 3.2 to 3.9% CV.

The results are summarized in the following table: Table 1

CYCLOSPORIN CONCENTRATION EXTRACTION HATE EXTRACTION RATE |

(ng/ml) INVENTION* METHANOL

0 89.5 89.5

50 98.4 92.1

100 98.9 95.2

200 106.3 102.1

350 113.4 108.8

500 117.7 114.0

*Nβt absorbance adjusted; extraction rate reflects the change in absorbance which is related o the amount of analyte extracted from a specimen.

Table 2

CYCLOSPORIN CONCENTRATION EXTRACTION RATE* (ng/ml) % RECOVERY

(ng/ml)

0 0 -

50 44.8 89.6

100 95.4 95.4

200 205.0 102.5

350 348.8 99.7

500 474.3 94.9

* Pretreatment in accordance with the present invention, quantitated from methanol extraction curve; extraction rate is the quantity of analyte extracted.

As a control, the above assay method was repeated using methanol pretreatment. One hundred microliters of a whole blood sample and 6 calibrators were each separately vortexed with 200 mL methanol. Incubation and centrifugation were as described above. The supernatant was used as the pretreated sample for conducting the remainder of the assay. The assay standard curve range extended to 500 ng/mL. Analytical recovery within the curve range varied from 89 to 102.5%. Within run precision with trilevel controls ranged from 2.8 to 7.5% CV. Between run precision with the same controls ranged from 5.3 to 15.8% CV.

Example 2 Assay for Tacrolimus (FK506) Assays for tacrolimus were conducted using a method similar to that described above in Example 1 for cyclosporine. Assays were performed on the COBAS MIRA analyzer.

Tacrolimus samples were prepared at 25 to 400 ng/mL blood hemolysate spiked with a stock solution of tacrolimus in methanol. After mixing of each sample, aliguots were taken and subjected to extraction. In addition, tacrolimus calibrators were prepared at 6.25 to 100 ng/mL (concentrations same as above plus the appropriate dilution factor) using methanol (no hemolysate) , spiked directly with a stock a solution of tacrolimus in methanol.

Prior to conducting the assays, tacrolimus spiked samples were pretreated in accordance with the present invention. Accordingly, one hundred microliters (100 μL) of the tacrolimus spiked hemolysate and 6 calibrators were each separately treated with 300μL of the following pretreatment composition: methanol (35%) , propylene glycol (29%) , an aqueous component (36%) containing 1 mM Homopipes® biological buffer (pKa=4.6), 25.0 mM cupric sulfate (pentahydrate) and 0.01% Pluronic 25R2® nonionic detergent; pH=4.3. In addition, the reference extraction was performed using methanol. Accordingly, one hundred microliters (100 μL) of the tacrolimus spiked hemolysate were each separately treated with 300 μL of methanol. The remainder of the assay was as described above in Example 1. Enzyme conjugates were prepared in a manner similar to that described in U.S. Patent No. 4,727,022, the relevant disclosure of which is incorporated herein by reference. Antibodies used were monoclonal antibodies for tacrolimus prepared by standard hybrid cell technology (see, for example, Kδhler and Milstein, Nature. supra) . The results are summarized in the following tables:

Table 3

TACROLIMUS CONCENTRATION (ng/ml) EXTRACTION RATE EXTRACTION RATE I INVENTION* NETNAN0L

0 178.7 178.7

6.25 185.2 186.6

12.5 190.7 190.0

25 196.8 197.2

50 207.2 208.2

100 217.1 216.4

*Net absorbance adjusted; extraction rate reflects the change in absorbance which is related to the amount of analyte extracted from a specimen.

Table 4

TACROLIMUS CONCENTRATION (ng/ml ) EXTRACTION RATE* (ng/ml ) X RECOVERY (no/Ml >

0 0 -

6 . 25 5. 8 93 . 4

12 . 5 12 . 6 100. 8

25 25 89. 9

50 49. 4 98 . 7

100 102 . 7 102 . 7

*Pretreatment in accordance with the present invention, quantitated from methanol extraction curve,

The results of the assay for tacrolimus are shown graphically in Fig. 1.

Example 3 Assay for Cyclosporine

Assays for cyclosporine were conducted using the EMIT® Cyclosporine Specific Assay kit, described hereinabove

Prior to conducting the assays, samples were pretreated in accordance with the present invention. Accordingly, one hundred microliters (lOOμL) of a whole blood sample and 6 calibrators were each separately treated with 300μL of the following pretreatment composition: methanol (33.3%), ethylene glycol (33.3%), an aqueous component (33.3%) containing 18.3 mM Tris-HCl (pKa=7.8), 25.0 mM cupric sulfate (pentahydrate), 0.01% Pluronic 25R2® nonionic detergent; 0.33 mM sodium EDTA, sodium azide (0.033%) and streptomycin (0.0017%); pH=4.5.

Each sample or calibrator together with the pretreatment composition were treated by vortex at 500 to 1000 rpm at ambient temperature for a period of greater than or equal to 10 seconds. After the above treatment, the mixture was allowed to incubate for greater than or equal to 2 minutes at ambient temperature and then the mixture was centrifuged for greater than or equal to 2 minutes at 20,800 relative centrifugal force (rcf). On the analyzer, 27.5μL of the resulting pretreated sample was incubated for 75 seconds with 155μL of Reagent A of the above EMIT assay kit. Subsequently, 75 μL of Reagent B of the EMIT assay kit was added. After a 175 second incubation, enzyme activity, (a function of drug concentration) was monitored by following the production of NADH spectrophotometrically at 340 nm for 100 seconds.

A total of 10 curves each derived from six calibrators were generated with invention and methanol as reference. These samples were pretreated and assayed as described above using the EMIT assay. Assay parameters on the COBAS MIRA analyzer Assay Temperature 37°C

Wavelength 340 nm Volume of Pretreated Sample 27.5μL

Diluent Volume (water) 47.5μL

Reagent A Volume 155 μL

Incubation Time (sample + Reagent A) 75 sec Reagent B Volume 75 μL Delay Time (sample + Reagent A + Reagent B) 175 sec Read Time 100 sec

The results are summarized in the following table:

Table 5

CYCLOSPORIN CONCENTRATION EXTRACTION RATE EXTRACTION RATE

(ng/ml) INVENTION* METHANOL

0 93.5 93.5

50 97.8 96.6

100 102.5 100.7 200 110.4 108.1

350 117.9 116.0

500 121.8 120.6

*Net absorbance adjusted ; extraction rate reflects the change in absorbance which is related to the amount of analyte extracted from the specimen

Table 6

CYCLOSPORIN CONCENTRATION EXTRACTION RATE* % RECOVERY

(ng/ml) (ng/ml)

0 0.4 —

50 64.1 128.2

100 119.2 119.2

200 229.7 114.3

350 398.8 113.9

500 563.9 112.8

•Pretreatment in accordance with the present invention, quantitated from methanol extraction curve; extraction rate is the quantity of analyte extracted.

As a control, the above assay method was repeated using methanol pretreatment. One hundred microliters of a whole blood sample and 6 calibrators were each separately vortexed with 200 mL methanol.

Incubation and centrifugation were as described above. The supernatant was used as the pretreated sample for conducting the remainder of the assay. The assay standard curve range extended to 500 ng/mL. Analytical recovery within the curve range varied from 112 to 128%. Within run precision with trilevel controls ranged from 2.8 to 7.5% CV. Between run precision with the same controls ranged from 5.3 to 15.8% CV. Example 4 Assay for Tacrolimus (FK506) Assays for tacrolimus were conducted using a method similar to that described above in Example 3 for cyclosporine. Assays were performed on the COBAS MIRA analyzer.

Tacrolimus samples were prepared at 25 to 400 mg/mL using blood hemolysate spiked with a stock solution of tacrolimus in methanol. After mixing of each sample, aliquots were taken and subjected to extraction. In addition, tacrolimus calibrators were prepared at 6.25 to 100 ng/mL (concentrations same as above plus the appropriate dilution factor) using methanol (no hemolysate) , spiked directly with a stock a solution of tacrolimus in methanol.

Prior to conducting the assays, tacrolimus spiked samples were pretreated in accordance with the present invention. Accordingly, one hundred microliters (100 μL) of the tacrolimus spiked hemolysate and 6 calibrators were each separately treated with 300μL of the following pretreatment composition: methanol (33.3%), ethylene glycol (33.3%), an aqueous component (33.3%) containing 18.6 mM Tris-HCl (pKa=7.8), 25.0 mM cupric sulfate (pentahydrate), 0.01% Pluronic 25R2® nonionic detergent, 0.33 mM sodium EDTA, sodium azide

(0.033%) and streptomycin (0.0017%; pH=4.5. In addition, the reference extraction was performed using methanol. Accordingly, one hundred microliters (lOOμL) of the tacrolimus spiked hemolysate were each separately treated with 300μl of methanol. The remainder of the assay was as described above in Example 3. Enzyme conjugates were prepared in a manner similar to that described in U.S. Patent No. 4,727,022, the relevant disclosure of which is incorporated herein by reference. Antibodies used were monoclonal antibodies for tacrolimus prepared by standard hybrid cell technology (see, for example, Kδhler and Milstein, Nature. supra.. The results are summarized in the following tables:

Table 7

•Net absorbance adjusted; extraction rate reflects the change in absorbance which is related to the amount of analyte extracted from the specimen.

Table 8

TACROLIMUS CONCENTRATION (ng/ml) EXTRACTION RATE* (ng/ml) % RECOVERY (ng/πtf)

0 0 -

6.25 4.7 75.2

12.5 11.4 90.8

25 22.4 89.6

50 46.7 83.4

100 92.8 92.8

While the present invention has been described with reference to the specific embodiments thereof, it will be understood by and obvious to those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising:

(a) a lower alkyl alcohol in an amount of about 30% to about 40% w/v, (b) a polyhydroxy aliphatic compound in an amount of about 20% to about 40% w/v, and

(c) an aqueous component in an amount of about 30% to about 40% w/v, wherein said aqueous component comprises about 20 mM to about 30 mM of a metal salt.

2. The composition of Claim 1 wherein said alcohol is methanol or ethanol.

3. The composition of Claim 1 wherein said alcohol is in an amount of about 31% to about 37% w/v. 4. The composition of Claim 1 wherein said polyhydroxy aliphatic compound is a glycol.

5. The composition of Claim 4 wherein said glycol is selected from the group consisting of propylene glycol and ethylene glycol. 6. The composition of Claim l wherein said glycol is in an amount of about 27% to about 34% w/v.

7. The composition of Claim 1 wherein said metal salt is selected from the group consisting of copper salts, cobalt salts, iron salts and molybdenum salts.

8. The composition of Claim 1 wherein said metal salt is about 20 mM to about 30 mM.

9. The composition of Claim 1 wherein said aqueous component comprises a buffer of pKa about 3.3 to about 8.0 at about 0.5 mM to about 20 mM.

10. The composition of Claim 8 wherein said buffer is selected from the group consisting of homopiperazine-N,N' -bis-2-ethanesulfonic acid, tris-HCl, acetate and citrate. li. The composition of Claim 1 wherein said aqueous component comprises a detergent in an amount of about 0.001% to about 0.1% w/v.

12. The composition of Claim 10 wherein said detergent is a non-ionic or an anionic detergent.

13. The composition of Claim 1 wherein the pH of the aqueous component is about 3.0 to about 4.6. 14. The composition of Claim 1 wherein the pH of the aqueous component is about 4.0 to about 4.6.

15. A composition comprising:

(a) methanol in an amount of about 30% to about 40% w/v, (b) propylene glycol in an amount of about 20% to about 40% w/v, and

(c) about 30% to about 40% w/v of an aqueous component comprising about 20 mM to about 30 mM copper salt, about 0.5 mM to about 1.5 mM of a buffer pKa about 3.3 to about 5.3, about 0.01% to about 0.1% w/v of a non-ionic detergent, said aqueous component having a pH of about 3.0 to about 4.6.

16. The composition of Claim 15 wherein said methanol is in an amount of about 33% to about 37% w/v. 17. The composition of Claim 15 wherein said propylene glycol is in an amount of about 27% to about 33% w/v.

18. The composition of Claim 15 wherein said copper salt is about 20 mM to about 30 mM. 19. The composition of Claim 15 wherein said buffer comprises homopiperazine-N,N' -bis-2-ethanesulfonic acid.

20. The composition of Claim 15 wherein the pH of the aqueous component is about 4.0 to about 4.4. 21. In an assay for the determination of a associated analyte in a sample suspected of containing said associated analyte wherein the assay comprises (1) contacting a sample suspected of containing said associated analyte with a pretreatment reagent, (2) contacting said sample with reagents for conducting a determination of said analyte and (3) analyzing for the results of said determination, the improvement comprising employing as said pretreatment reagent the composition of Claim 1.

22. The assay of Claim 21 wherein said associated analyte is a cyclosporin. 23. In an assay for the determination of a associated analyte in a sample suspected of containing a associated analyte wherein the assay comprises (1) contacting a sample suspected of containing said associated analyte with a pretreatment reagent, (2) contacting said sample with reagents for conducting a determination of said analyte and (3) analyzing for the results of said determination, the improvement comprising employing as said pretreatment reagent the composition of Claim 15. 24. The assay of Claim 23 wherein said associated analyte is a cyclosporin.

25. A kit comprising in packaged combination: (a) one or more reagents for conducting an assay for a associated analyte and (b) the composition of Claim 1.

26. A kit comprising in packaged combination:

(a) one or more reagents for conducting an assay for a associated analyte and

(b) the composition of Claim 15. 27. In an assay for the determination of an immunosuppressant drug in a sample suspected of containing said drug, said assay comprising (1) contacting the sample with a specific binding member for said drug and (2) detecting the binding of said specific binding member to said drug, the improvement which comprises contacting said sample with the composition of Claim 1 either prior to or in conjunction with step (1) .

28. The assay of Claim 27 wherein said drug is cyclosporin. 29. In an assay for the determination of an immunosuppressant drug in a sample suspected of containing said drug, said assay comprising (1) contacting the sample with antibodies for said drug and a conjugate of a label and a compound recognized by said antibodies and (2) detecting immune complexes of said label conjugate and said antibodies, the improvement which comprises contacting said sample with the composition of Claim 15 either prior to or in conjunction with step (l) .

30. The assay of Claim 29 wherein said drug is cyclosporin.