Patent Application
20110311990
The present invention generally relates to immunogens and to the antibodies they generate that specifically bind tetranor-prostaglandin E2 metabolite (tetranor-PGEM) and tetranor-prostaglandin A2 metabolite (tetranor-PGAM), the methods for their manufacture, and their uses in assay kits for quantification of tetranor-PGEM or tetranor-PGAM in biological fluids. The present invention also generally relates to the manufacture and use of tracers in the associated assay kits.
Prostaglandins are found in virtually all tissues and glands and are extremely potent mediators of a diverse group of physiological processes (Funk, C. D. Science, 2001, 294, 1871-1875). Prostaglandins can participate in a wide range of body functions, such as the contraction and relaxation of smooth muscle (Andersson, K. E., Forman, A. Acta Pharmacol. Toxicol., 1978, 43 (Suppl. 2), 90-95), the dilation and constriction of blood vessels (Abramovich, D. R., Page, K. R., Parkin, A. M. L. Br. J. Pharmac., 1984, 81, 19-21), control of blood pressure (Anderson, R. J., Berl, T., McDonald, K. M., Schrier, R. W. Kidney International, 1976, 10, 205-215), and modulation of inflammation and immunity (Hata, A. N., Breyer, R. M. Pharmacol. Ther., 2004, 103(2), 147-166). In general, prostaglandins and related compounds are transported out of the cells that synthesize them and affect other target cells close to their site of formation, mainly by interacting with the target cell's prostaglandin receptors to stimulate or inhibit some target cell function. They also alter the activities of the cells in which they are synthesized. The nature of these effects may vary from one cell type to another, and from the target cell type.
Prostaglandin E2 (PGE2) is involved in various biological activities including human parturition, respiratory function, blood pressure regulation, gastric mucosal integrity, and macrophage function (The Immunology Handbook Third Edition, David Wild (Ed.), Elsevier Ltd., Oxford, UK, 2005, p. 864). PGE2 is biosynthesized from the cyclooxygenase (COX) product of arachidonic acid and common prostanoid precursor prostaglandin H2 (PGH2) by the catalytic action of either cytosolic prostaglandin E synthase (cPGES) or membrane-bound prostaglandin E synthases (mPGES-1 and mPGES-2) (Helliwell, R., Adams, L., and Mitchell, M., Prostaglandins, Leukotrienes and Essential Fatty Acids, 70, 2004, 101-113; Murakami, M., Nakashima, K., Kamei, D. et al. J. Biol. Chem., 278, 2003, 37937-37947). Increased production of PGE2 along with synchronized upregulation of COX-2 and mPGES in a number of biological systems has been connected to proinflammatory stimuli (Murakami, M., Naraba, T., Tanioka, T. et al. J. Biol. Chem., 275, 2000, 32783-32792; Stichtenoth, D., Thoren, S., Bian, H. et al. J. Immunol., 167, 2001, 469-474; Mancini, J., Blood, K., Guay, J. et al. J. Biol. Chem., 276, 2001, 4469-4475; Thoren S. and Jakobsson, P. J. Biochem., 267, 2000, 6428-6434). The overexpression of COX-2 occurs in many colorectal tumors and is believed to play a significant role in colorectal cancer cell development (Sinicrope, F. and Gill, S. Cancer Metastasis Rev., 23, 2004, 63-75; Greenhough, A., Smartt, H., Moore, A. et al. Carcinogenesis, 30, 2009, 377-386).
As is the general case for all primary prostaglandins, direct quantitative measurements of PGE1 and PGE2 formation have been limited due to their rapid biosyntheses, metabolisms, and inherent instabilities. PGE2 is readily dehydrated to electrophilic cyclopentenone derivatives PGA2 and PGB2. The more-stable PGE metabolites therefore are potentially useful indicators of PGE biosynthesis in humans (Hamberg, M., Biochem. Biophys. Res. Commun., 49, 1972, 720-726; Ramberg, M. and Samuelsson, B., J. Biol. Chem., 246, 1971, 6713-6721). Tetranor-PGEM, or 11α-hydroxy-9,15-dioxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid, is the major metabolite of PGE1 and PGE2 found in human urine (Ramberg, M. and Samuelsson, B., J. Biol. Chem., 246, 1971, 6713-6721; Ramberg, M. and Samuelsson, B., J. Amer. Chem. Soc., 91, 1969, 2177-2178; Granstrom, E. and Samuelsson, B., J. Amer. Chem. Soc., 91, 1969, 3398-3400). Tetranor-PGEM is a urinary marker of PGE2 biosynthesis (Honda, H., Fukawa, K., and Sawabe, T., Prostaglandins, 19, 1980, 259-269; Ramberg, M., Biochem. Biophys. Res. Comm., 49, 1972, 720-726). About 15% of an infused dose of PGE2 appears as tetranor-PGEM in the urine of humans. Normal healthy males excrete 7-40 μg of tetranor-PGEM over a 24-hour period.
Quantification of PGE2 metabolites in urine based on mass spectral analyses has been reported (Seyberth, H., Sweetman, B., Frolich, J., and Oates, J., Prostaglandins, 11, 1976, 381-397; Hamberg, M. and Samuelsson, B., J. Biol. Chem., 247, 1972, 3495-3502). The quantification of tetranor-PGEM in the urine of lung cancer patients versus healthy humans using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been reported to be a facile and accurate means for assessing PGE2 biosynthesis in human physiological and pathophysiological processes (Murphey, L., Williams, M., Sanchez, S. et al. Analytical Biochemistry, 334, 2004, 266-275).
Direct PGE2 immunoassays are known and are available from multiple commercial sources. Given its instability, direct measurement of PGE2 in readily obtained biological fluids such as urine is not practical for assessing accurate levels of PGE2 production. A need exists in the art for a specific tetranor-PGEM immunoassay that measures this relatively abundant PGE2 biomarker in biological fluids, especially urine. The present invention meets this need. As will be evident elsewhere in this disclosure, an immunoassay that is capable of detecting and measuring both tetranor-PGEM and the 10,11-dehydrate tetranor-PGE2 metabolite, tetranor-PGAM, with high specificity may be used to accurately assess tetranor-PGEM concentrations in biological fluids and thus provide an indirect measure of PGE2 biosynthesis.
The present invention comprises tetranor-PGEM-carrier protein conjugates and tetranor-PGAM-carrier protein conjugates and methods for preparing them.
The present invention also comprises the use of tetranor-PGEM-carrier protein conjugates or tetranor-PGAM-carrier protein conjugates, acting as immunogens, for generating antibodies specific for tetranor-PGEM and/or tetranor-PGAM, as well as to the respective antibodies themselves.
The present invention comprises tetranor-PGEM-carrier protein conjugates and tetranor-PGAM-carrier protein conjugates and methods for preparing them.
The present invention also comprises tetranor-PGEM-molecular tag and tetranor-PGAM-molecular tag conjugates, each acting as a tracer that may be used in an assay for measuring concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample.
The present invention also comprises assay kits used for measuring tetranor-PGEM and tetranor-PGAM metabolite levels in biological samples, wherein the assay kits comprise antibodies specific for tetranor-PGEM and tetranor-PGAM and a tracer comprising tetranor-PGEM and/or tetranor-PGAM covalently bonded to a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample.
The present invention also comprises a method for measuring tetranor-PGEM and tetranor-PGAM metabolite levels in biological samples utilizing assay kits comprising antibodies specific for tetranor-PGEM and tetranor-PGAM and a tracer comprising tetranor-PGEM and/or tetranor-PGAM covalently bonded to a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample.
Other exemplary embodiments of the invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while disclosing exemplary embodiments of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
For purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements.
Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.
In this application, the use of the singular includes the plural and plural encompasses singular, unless specifically stated otherwise. In addition, in this application, the use of “or” means “and/or” unless specifically stated otherwise, even though “and/or” may be explicitly used in certain instances.
Unless otherwise defined herein, scientific and technical terms used in connection with the exemplary embodiments shall have the meanings that are commonly understood by those of ordinary skill in the art.
Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of chemistry and biology described herein are those well known and commonly used in the art.
The term “alkyl,” alone or in combination, means an acyclic or cyclic radical, linear or branched, preferably containing from 1 to about 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, teat-butyl, pentyl, iso-amyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
The term “tertiary amine base,” as used herein, refers to an amine comprising a nitrogen atom bearing a lone pair of electrons and three organic groups (substituting for the three hydrogen atoms of ammonia) that allow the nitrogen atom sufficient basicity to react with acidic hydrogen atoms of reactants or free solvated protons in a reaction mixture to form an ammonium salt comprising the nitrogen atom bearing a positive charge after forming a covalent bond with said acidic hydrogen or proton, the acidic hydrogen or proton, and the three organic groups. In certain embodiments, the organic groups comprise equivalent or various alkyl radicals as described above. In certain embodiments, the alkyl radicals are ethyl or isopropyl groups. In certain embodiments, the tertiary amine base is N,N-diisopropylethylamine, triethylamine, or triisopropylamine.
The term “biological fluids,” as used herein, refers to fluids that have human or animal origin, including but not limited to urine, whole blood, plasma, mucus, perspiration, saliva, semen, and vaginal fluid.
The term “Ellman's Reagent,” as used herein, refers to a product sold by Cayman Chemical Company, Incorporated (Catalog No. 400050) comprising 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and acetylthiocholine. The reagent is sold as a solid mixture and may be reconstituted into a solution by dissolving in a solvent such as water as described elsewhere in this disclosure.
The term “blank,” as used herein, refers to background absorbance caused by Ellman's Reagent. The blank absorbance should be subtracted from the absorbance readings of all other wells.
The term “total activity,” as used herein, refers to total enzymatic activity of an enzymatic tracer. This is analogous to the specific activity of a radioactive tracer.
The term “non-specific binding (NSB),” as used herein, refers to non-immunological binding of the tracer to the well. Even in the absence of specific antibody a very small amount of tracer still binds to the well; the NSB is a measure of this low binding.
The term “maximum binding (B0),” as used herein, refers to the maximum amount of the tracer that the antibody can bind in the absence of free analyte.
The term “% bound/maximum bound (% B/B0),” as used herein, refers to the ratio of the absorbance of a particular sample or standard well to that of the maximum binding (B0) well.
The term “standard curve,” as used herein, refers to a plot of the % B/B0 values versus concentration of a series of wells containing various known amounts of analyte.
The term “determination (dtn),” as used herein, refers to refers to an amount of reagent, where one dtn is the amount of reagent used per well.
The term “immunogen,” as used herein, refers to an antigen that induces adaptive immunity.
The term “antigen,” as used herein, refers to any molecule recognized by the immune system.
The term “adaptive immunity,” as used herein, refers to antigen-specific immune response.
The term “carrier protein,” as used herein, refers to a protein to which PGE2 metabolite tetranor-PGEM or PGE2 metabolite tetranor-PGAM is covalently attached to form a metabolite-carrier protein conjugate such that an immune response to tetranor-PGEM or tetranor-PGAM is generated when the conjugate is injected into a host organism. Exemplary carrier proteins include but are not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, and thyroglobulin.
The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the exemplary embodiments may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab, and F(ab)2, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow, E. and Lane, D., Editors, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow, E. and Lane, D., Editors, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Huston, J., Levinson, D., Mudgett-Hunter, M. et al. Proc. Natl. Acad. Sci. USA, 85, 1988, 5879-5883; Bird, R., Hardman, K., Jacobson, J. et al. Science, 242, 1988, 423-426.).
As used herein, the term “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies. The term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of VH (variable heavy chain immunoglobulin) genes from an animal.
The term “specifically binds,” as used herein with respect to an antibody, means an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, in a sample containing tetranor-PGEM, an antibody that specifically binds to tetranor-PGEM recognizes and binds to tetranor-PGEM but does not substantially recognize or bind to other molecules in the sample. Furthermore, as used herein with respect to an antibody, the term “specifically binds” may also mean an antibody which recognizes a metabolite and a closely-related molecule derived from said metabolite. For example, an antibody may be considered to recognize and “specifically bind” to tetranor-PGEM and its closely-related dehydrate tetranor-PGAM while not substantially recognizing or binding to other molecules.
The term “detection analyte” as used herein, may be used interchangeably with the term “tracer” and refers to any entity comprising tetranor-PGEM or tetranor-PGAM and a molecular tag covalently linked to tetranor-PGEM or tetranor-PGAM, which produces a readable signal that may be measured to calculate concentration of the respective tetranor-PGEM or tetranor-PGAM in a test sample.
The term “molecular tag,” as used herein, is a molecule or molecular moiety such as, but not limited to, a fluorophore moiety, a chemiluminescent moiety, a biotin-avidin system, or a protein that catalyzes a conversion of a substrate of the protein into a product for which a measured readable signal or property of the test sample is changed by said conversion.
The exemplary embodiments described herein are useful in various applications, including but not limited to, research, diagnostic, and clinical.
The exemplary embodiments described herein may be based on the discovery of an antibody that specifically binds to tetranor-PGEM (I) and tetranor-PGAM (II).
Tetranor-PGAM may be formed by dehydration of the p-hydroxyketone tetranor-PGEM, which involves the elimination of a water equivalent consisting of the 11R-hydroxy group at the keto β-position and an adjacent proton on the keto α-position, to afford the cyclopentenone tetranor-PGAM scaffold. Dehydration may occur spontaneously under certain conditions in an essentially irreversible manner. Dehydration may also proceed in an acid- or base-catalyzed manner under certain conditions.
Tetranor-PGAM may also be formed by metabolism of PGA2, a dehydrated metabolite of PGE2, to tetranor-PGAM in a metabolic pathway similar to that of the metabolism of PGE2 to tetranor-PGEM.
The α,β-unsaturated ketone (enone), tetranor-PGAM, is an electrophilic molecule (electrophile) that possesses the propensity to, by a Michael addition mechanism, chemically react with certain nucleophilic functional groups of nucleophilic molecules (nucleophiles), such as the sulfhydryl groups of glutathione (GSH) and protein cysteine (Cys) residues, to form tetranor-PGAM-derived PG metabolite moiety-nucleophile conjugates (IIIA) and (IIIB), also referred to herein as “tetranor-PGAM-nucleophile Michael adducts” as shown in the following schemes:
Exemplary embodiments may involve a sulfhydryl-bearing nucleophile comprising a carrier protein forming a Michael adduct with tetranor-PGAM to make an immunogen. Other exemplary embodiments may involve a sulfhydryl-bearing nucleophile comprising a molecular tag forming a Michael adduct with tetranor-PGAM for making a tracer. Exemplary tetranor-PGAM-nucleophile Michael adducts include but are not limited to tetranor-PGAM-carrier protein Michael adducts, tetranor-PGAM-enzyme Michael adducts, tetranor-PGAM-fluorophore Michael adducts, tetranor-PGAM-chemiluminescent moiety Michael adducts, and tetranor-PGAM-biotin-avidin system Michael adducts.
In certain embodiments, an immunogen or tracer wherein the prostaglandin metabolite is linked to the carrier protein or molecular tag, respectively, through one of the side chain terminal carboxyl moieties of the PG metabolite is produced by a conjugation reaction.
In particular, the present invention may first be directed to methods for preparing a tetranor-PGEM-carrier protein conjugate, a tetranor-PGAM-carrier protein conjugate, or a tetranor-PGAM-carrier protein Michael adduct. A tetranor-PGEM-carrier protein conjugate, as defined herein, is an immunogen that is capable of inducing the production of antibodies that specifically bind to tetranor-PGEM or that bind only to tetranor-PGEM and tetranor-PGAM when injected into a biological sample. Similarly, a tetranor-PGAM-carrier protein conjugate, as defined herein, is an immunogen that is capable of inducing the production of antibodies that specifically bind to tetranor-PGAM or that bind only to tetranor-PGEM and tetranor-PGAM when injected into a biological sample. Furthermore, a tetranor-PGAM-carrier protein Michael adduct, as defined herein, is an immunogen that is capable of inducing the production of antibodies that specifically bind to tetranor-PGEM, specifically bind to tetranor-PGAM, or that bind only to tetranor-PGEM and tetranor-PGAM when injected into a biological sample. The present invention also comprises the tetranor-PGEM-carrier protein conjugates, the tetranor-PGAM-carrier protein conjugates, and the tetranor-PGAM-carrier protein Michael adducts themselves.
In certain embodiments, the present invention may be directed to a method for preparing immunogens comprising tetranor-PGEM-KLH (“keyhole limpet hemocyanin”), tetranor-PGAM-KLH, tetranor-PGEM-BSA (“bovine serum albumin”), or tetranor-PGAM-BSA protein conjugates. The present invention also comprises tetranor-PGEM-KLH, tetranor-PGAM-KLH, tetranor-PGEM-BSA and tetranor-PGAM-BSA protein conjugates themselves.
In certain embodiments directed to methods for preparing a tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate, the method comprises preparing a reaction mixture by contacting tetranor-PGEM or tetranor-PGAM with alkyl chloroformate and with a tertiary amine base. In certain of these embodiments, the alkyl group of the alky chloroformate comprises an acyclic or cyclic radical, linear or branched, preferably containing from 1 to about 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tent-butyl, pentyl, iso-amyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. In certain of these embodiments, the alkyl chloroformate comprises isobutyl chloroformate. In certain of these embodiments, the tertiary amine base comprises N,N-diisopropylethylamine, triethylamine, or triisopropylamine.
In certain of these embodiments directed to directed to methods for preparing a tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate, the molar ratio of alkyl chloroformate with respect to tetranor-PGEM or tetranor-PGAM in the reaction mixture used to form the tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate is from 10 to 200 mole percent, such as from 10 to 30 mole percent, such as 20 mole percent.
In certain of these embodiments directed to directed to methods for preparing a tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate, the molar ratio of tertiary amine base with respect to tetranor-PGEM or tetranor-PGAM in the reaction mixture used to form the tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate is from 20 to 500 mole percent, such as from 300 to 400 mole percent, such as 380 mole percent.
In certain of these embodiments directed to methods for preparing a tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate, the molar ratio of tetranor-PGEM or tetranor-PGAM to alkyl chloroformate (or isobutyl chloroformate) to tertiary amine base (i.e. tetranor-PGEM or tetranor-PGAM:alkyl chloroformate:tertiary amine base) in the reaction mixture used to form the tetranor-PGEM-carrier protein conjugate or a tetranor-PGAM-carrier protein conjugate is from 1:0.1:0.2 to 1:2:5, such as 1:1:1 or 1:0.2:3.8.
In one exemplary embodiment, a tetranor-PGEM-carrier protein conjugate or tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting an organic solution comprising 0.001-0.1 M tetranor-PGEM (limiting reagent) in a suitable organic solvent with an organic solution comprising alkyl chloroformate in said suitable organic solvent (10-200 mole % alkyl chloroformate with respect to the limiting reagent, 0.2-1 weight % alkyl chloroformate solute with respect to total mass of said organic solution comprising alkyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at a temperature range of −20° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of −5° C. to +25° C. for 1-4 hours;
3. Removing the solvent of said reaction mixture to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (0.001-0.2 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and an aqueous buffer solution at pH range 7.2-7.6 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 1-24 hours at +1 to +10° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 6-10 hours against an aqueous buffer solution comprising 0.05-0.2 M buffer, pH range 7.2-7.6 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −15° C. to −40° C.
In another exemplary embodiment, a tetranor-PGEM-carrier protein conjugate or tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM (limiting reagent) in acetonitrile with a solution comprising isobutyl chloroformate in acetonitrile (10-200 mole % isobutyl chloroformate with respect to the limiting reagent, 0.2-1 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at a temperature range of −5° C. to +5° C.;
2. Mixing said reaction mixture at a temperature range of −5° C. to +5° C. for 1-4 hours;
3. Removing the solvent of said reaction mixture to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (0.001-0.1 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 1-24 hours at +1 to +5° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 7-9 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGEM-carrier protein conjugate or tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-200 mole % isobutyl chloroformate with respect to the limiting reagent, 0.2-1 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGEM-carrier protein conjugate or tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGEM-carrier protein conjugate or tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGEM) comprising KLH, BSA, ovalbumin, or thyroglobulin, and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGEM-KLH conjugate or tetranor-PGAM-KLH conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising KLH (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stiffing said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGEM-BSA conjugate or tetranor-PGAM-BSA conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGEM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising BSA (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGEM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting an organic solution comprising 0.001-0.1 M tetranor-PGAM (limiting reagent) in a suitable organic solvent with an organic solution comprising alkyl chloroformate in said suitable organic solvent (10-200 mole % alkyl chlorofonnate with respect to the limiting reagent, 0.2-1 weight % alkyl chloroformate solute with respect to total mass of said organic solution comprising alkyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at a temperature range of −20° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of −5° C. to +25° C. for 1-4 hours;
3. Removing the solvent of said reaction mixture to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (0.001-0.2 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and an aqueous buffer solution at pH range 7.2-7.6 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 1-24 hours at +1 to +10° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 6-10 hours against an aqueous buffer solution comprising 0.05-0.2 M buffer, pH range 7.2-7.6 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −15° C. to −40° C.
In another exemplary embodiment, a tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM (limiting reagent) in acetonitrile with a solution comprising isobutyl chloroformate in acetonitrile (10-200 mole % isobutyl chloroformate with respect to the limiting reagent, 0.2-1 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at a temperature range of −5° C. to +5° C.;
2. Mixing said reaction mixture at a temperature range of −5° C. to +5° C. for 1-4 hours;
3. Removing the solvent of said reaction mixture to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (0.001-0.1 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 1-24 hours at +1 to +5° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 7-9 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-200 mole % isobutyl chloroformate with respect to the limiting reagent, 0.2-1 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (20-500 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-carrier protein conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising a carrier protein (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGAM) comprising KLH, BSA, ovalbumin or thyro globulin, and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-KLH conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate;
4. Adding a mixture comprising KLH (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In another exemplary embodiment, a tetranor-PGAM-BSA conjugate may be formed according to a method comprising the following steps:
1. Preparing a reaction mixture by contacting a solution comprising 0.01-0.02 M tetranor-PGAM in acetonitrile (limiting reagent) with a solution comprising isobutyl chloroformate in acetonitrile (10-30 mole % isobutyl chloroformate with respect to the limiting reagent, 0.5-6 weight % isobutyl chloroformate solute with respect to total mass of said organic solution comprising isobutyl chloroformate) and with a tertiary amine base (300-400 mole % with respect to the limiting reagent) at 0° C.;
2. Mixing said reaction mixture at 0° C. for 2 hours;
3. Removing the solvent of said reaction mixture under a stream of nitrogen to provide a reaction mixture concentrate; 4. Adding a mixture comprising BSA (about 0.01 mole % with respect to the limiting reagent, starting material tetranor-PGAM) and a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to said reaction mixture concentrate to provide a second reaction mixture;
5. Stirring said second reaction mixture for 12-18 hours at +4° C. in the dark;
6. Dialyzing said second reaction mixture through a 10,000 molecular weight (MW) cut-off membrane for 8 hours against a 0.1 M aqueous potassium phosphate buffer solution at pH 7.4 to provide a conjugate solution; and
7. Freezing aliquots of said conjugate solution at −20° C.
In certain embodiments, the present invention may be directed to a method for preparing immunogens comprising tetranor-PGAM-KLH or tetranor-PGAM-BSA Michael adducts. The present invention also comprises tetranor-PGAM-KLH and tetranor-PGAM-BSA Michael adducts themselves.
In an exemplary embodiment, a tetranor-PGAM-carrier protein Michael adduct may be formed according to a method comprising the following steps:
1. Contacting a carrier protein in a suitable aqueous buffer with an excess of N-succinimidyl-S-acetylthioacetate in said aqueous buffer at a temperature range of +15° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of +15° C. to +25° C. for 0.25-2 hours to produce a derivatized carrier protein mixture;
3. Purifying said derivatized carrier protein mixture to prepare a purified derivatized carrier protein mixture;
4. Treating said purified derivatized carrier protein mixture with hydroxylamine to produce a sulfhydryl-deprotected carrier protein mixture;
5. Purifying said sulfhydryl-deprotected carrier protein mixture to prepare a purified sulfhydryl-deprotected carrier protein mixture;
6. Treating said purified sulfhydryl-deprotected carrier protein mixture with tetranor-P GAM to produce a tetranor-PGAM-carrier protein Michael adduct; and
7. Purifying said tetranor-PGAM-carrier protein Michael adduct to prepare a purified tetranor-PGAM-carrier protein Michael adduct.
In another exemplary embodiment, a tetranor-PGAM-KLH Michael adduct may be formed according to a method comprising the following steps:
1. Contacting KLH in a suitable aqueous buffer with an excess of N-succinimidyl-S-acetylthioacetate in said aqueous buffer at a temperature range of +15° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of +15° C. to +25° C. for 0.25-2 hours to produce a derivatized KLH mixture;
3. Purifying said derivatized KLH mixture to prepare a purified derivatized KLH mixture;
4. Treating said purified derivatized KLH mixture with hydroxylamine to produce a sulfhydryl-deprotected KLH mixture;
5. Purifying said sulfhydryl-deprotected KLH mixture to prepare a purified sulfhydryl-deprotected KT mixture;
6. Treating said purified sulfhydryl-deprotected KLH mixture with tetranor-PGAM to produce a tetranor-PGAM-KLH Michael adduct; and
7. Purifying said tetranor-PGAM-KLH Michael adduct to prepare a purified tetranor-PGAM-KLH Michael adduct.
In another exemplary embodiment, a tetranor-PGAM-BSA Michael adduct may be formed according to a method comprising the following steps:
1. Contacting BSA in a suitable aqueous buffer with an excess of N-succinimidyl-S-acetylthioacetate in said aqueous buffer at a temperature range of +15° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of +15° C. to +25° C. for 0.25-2 hours to produce a derivatized BSA mixture;
3. Purifying said derivatized BSA mixture to prepare a purified derivatized BSA mixture;
4. Treating said purified derivatized BSA mixture with hydroxylamine to produce a sulfhydryl-deprotected BSA mixture;
5. Purifying said sulfhydryl-deprotected BSA mixture to prepare a purified sulfhydryl-deprotected BSA mixture;
6. Treating said purified sulfhydryl-deprotected BSA mixture with tetranor-PGAM to produce a tetranor-PGAM-BSA Michael adduct; and
7. Purifying said tetranor-PGAM-BSA Michael adduct to prepare a purified tetranor-PGAM-BSA Michael adduct.
The present invention may also be directed to the method of generating antibodies specific for tetranor-PGEM and tetranor-PGAM by immunizing a biological species (for example a mammal such as a mouse or rabbit), with a respective one of the tetranor-PGEM-carrier protein conjugates, tetranor-PGAM-carrier protein conjugates, or tetranor-PGAM-carrier protein Michael adducts of the present invention described above. The protocols and methods for immunizing the biological species to generate the antibodies are done by methods well known to those of ordinary skill in the art in the fields of biochemistry and/or immunology. The tetranor-PGEM-carrier protein conjugate, tetranor-PGAM-carrier protein conjugate, or tetranor tetranor-PGAM-carrier protein Michael adduct is recognized by the biological species' adaptive immune system, thereby inducing the production of antibodies that specifically bind to tetranor-PGEM and tetranor-PGAM. The present invention also comprises the antibodies themselves, which may be monoclonal antibodies or polyclonal antibodies, depending upon the method utilized.
An exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mammal is injected with an immunogen comprising a tetranor-PGEM-KLH conjugate or a tetranor-PGAM-KLH conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mammal is injected with an immunogen comprising a tetranor-PGAM-KLH Michael adduct.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mammal is injected with an immunogen comprising a tetranor-PGEM-BSA conjugate or a tetranor-PGAM-BSA conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mammal is injected with an immunogen comprising a tetranor-PGAM-BSA Michael adduct.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mouse is injected with an immunogen comprising a tetranor-PGEM-KLH conjugate or a tetranor-PGAM-KLH conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mouse is injected with an immunogen comprising a tetranor-PGAM-KLH Michael adduct.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mouse is injected with an immunogen comprising a tetranor-PGEM-BSA conjugate or a tetranor-PGAM-BSA conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a mouse is injected with an immunogen comprising a tetranor-PGAM-BSA Michael adduct.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a rabbit is injected with an immunogen comprising a tetranor-PGEM-KLH conjugate or a tetranor-PGAM-KLH conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a rabbit is injected with an immunogen comprising a tetranor-PGAM-KLH Michael adduct.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a rabbit is injected with an immunogen comprising a tetranor-PGEM-BSA conjugate or a tetranor-PGAM-BSA conjugate.
Another exemplary embodiment may be directed to a method for preparing antibodies specific for tetranor-PGEM and tetranor-PGAM comprising an immunization step wherein a rabbit is injected with an immunogen comprising a tetranor-PGAM-BSA Michael adduct.
In any of the above exemplary embodiments, the antibodies formed may be gathered from the biological sample for subsequent utilization in assay kits as described further below. The methods for gathering the antibodies are well known to those of ordinary skill in the art in the fields of biochemistry and/or immunology.
Still other exemplary embodiments may be directed methods for assessing biosynthesis of PGE2 in a subject by measuring tetranor-PGEM and tetranor-PGAM levels derived from biological fluids taken from a subject. In certain embodiments, the assessment may be accomplished by measuring tetranor-PGEM metabolite levels and tetranor-PGAM metabolite levels in urine. In other embodiments, the assessment may be accomplished by measuring tetranor-PGEM metabolite levels and tetranor-PGAM metabolite levels in plasma.
In particular, the exemplary embodiments may be directed to competitive enzyme immunoassay (EIA) kits (assay kits) in which the competition between tetranor-PGEM or tetranor PGAM derived from the biological fluid of a subject and a constant concentration of detection analyte comprising tetranor-PGEM-molecular tag conjugate (tetranor-PGEM tracer), tetranor-PGAM-molecular tag conjugate (tetranor-PGAM tracer), or tetranor-PGAM-molecular tag Michael adduct tracer for a limited amount of tetranor-PGEM/tetranor-PGAM-specific antibody binding sites is measured. Kits may include a tracer comprising tetranor-PGEM or tetranor-PGAM covalently bound to a molecular tag such as acetylcholinesterase (AChE), horseradish peroxidase (HRP), alkaline phosphatase (AP), rhodamine, or fluorescein. Kits may further include a monoclonal or polyclonal antibody having reactivity specifically with tetranor-PGEM and tetranor-PGAM, including any of the antibodies formed in accordance with exemplary embodiments described above.
In an exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Chemically coupling tetranor-PGEM or tetranor-PGAM with an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to produce a substance comprising a tracer; and
2. Purifying the substance comprising tracer to produce a substance comprising purified tracer.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a chemically-activated tetranor-PGEM or tetranor-PGAM species;
2. Chemically coupling the activated species with an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to produce a substance comprising a tracer; and
3. Purifying the substance comprising tracer to produce a substance comprising purified tracer.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising tetranor-PGEM, a tertiary amine base, and a suitable solvent;
2. Contacting the mixture with a second mixture comprising alkyl chloroformate and a suitable solvent to form a reaction mixture;
3. Removal of the solvent from the reaction mixture to obtain an activated tetranor-PGEM concentrate;
4. Reconstitution of the activated tetranor-PGEM concentrate in a solvent suitable for coupling the activated tetranor-PGEM with a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to form an activated tetranor-PGEM solution;
5. Contacting the activated tetranor-PGEM solution with a mixture comprising an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to produce a tracer mixture; and
6. Purifying the tracer mixture to produce a purified tracer mixture.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising a suitable solvent, tetranor-PGEM, and 20-500 mole percent tertiary amine base with respect to the molar amount of the tetranor-PGEM;
2. Contacting the mixture with a second mixture comprising 10-200 mole percent alkyl chloroformate with respect to the molar amount of the tetranor-PGEM and a suitable solvent to form a reaction mixture;
3. Removal of the solvent from the reaction mixture to obtain an activated tetranor-PGEM concentrate;
4. Reconstitution of the activated tetranor-PGEM concentrate in a solvent suitable for coupling the activated tetranor-PGEM with a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to form an activated tetranor-PGEM solution;
5. Contacting the activated tetranor-PGEM solution with a mixture comprising an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to produce a tracer mixture; and
6. Purifying the tracer mixture to produce a purified tracer mixture.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising a suitable solvent, tetranor-PGEM, and an equimolar amount of tertiary amine base with respect to the molar amount of the tetranor-PGEM;
2. Contacting the mixture with a second mixture comprising an equimolar amount of alkyl chlorofonnate with respect to the molar amount of the tetranor-PGEM and a suitable solvent to form a reaction mixture;
3. Removal of the solvent from the reaction mixture to obtain an activated tetranor-PGEM concentrate;
4. Reconstitution of the activated tetranor-PGEM concentrate in a solvent suitable for coupling the activated tetranor-PGEM with a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to form an activated tetranor-PGEM solution;
5. Contacting the activated tetranor-PGEM solution with a mixture comprising an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample to produce a tracer mixture; and
6. Purifying the tracer mixture to produce a purified tracer mixture.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising tetranor-PGEM, N,N-diisopropylethylamine, and acetonitrile;
2. Contacting the mixture with a second mixture comprising isobutyl chloroformate and acetonitrile to form a reaction mixture;
3. Removal of the acetonitrile from the reaction mixture to obtain an activated tetranor-PGEM concentrate;
4. Reconstitution of the activated tetranor-PGEM concentrate in N,N-dimethylformamide to form an activated tetranor-PGEM solution;
5. Contacting the activated tetranor-PGEM solution with a mixture comprising AChE and a suitable aqueous buffer solution to produce a tracer mixture; and
6. Purifying the tracer mixture on a size exclusion column to produce a purified tracer mixture.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising acetonitrile and equimolar amounts of tetranor-PGEM and N,N-diisopropylethylamine;
2. Contacting the mixture with a second mixture comprising acetonitrile and an equimolar amount of isobutyl chloroformate with respect to the molar amount of the tetranor-PGEM to form a reaction mixture;
3. Removal of the acetonitrile from the reaction mixture to obtain an activated tetranor-PGEM concentrate;
4. Reconstitution of the activated tetranor-PGEM concentrate in N,N-dimethylformamide to form an activated tetranor-PGEM solution;
5. Contacting the activated tetranor-PGEM solution with a mixture comprising AChE and a suitable aqueous buffer solution to produce a tracer mixture; and
6. Purifying the tracer mixture on a size exclusion column to produce a purified tracer mixture.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising tetranor-PGAM and a suitable solvent;
2. Forming a second mixture comprising an entity comprising a molecular tag that produces a readable signal that may be measured to calculate concentration of tetranor-PGEM and/or tetranor-PGAM in a test sample and a suitable solvent;
3. Combining the mixture with the second mixture to produce a reaction mixture; and
4. Purifying the reaction mixture to produce a purified tracer solution.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Forming a mixture comprising tetranor-PGAM and N,N-dimethylformamide;
2. Forming a second mixture comprising AChE and a suitable aqueous buffer solution;
3. Combining the mixture with the second mixture to produce a reaction mixture; and
4. Purifying the reaction mixture on a size exclusion column to produce a purified tracer solution.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Contacting AChE in a suitable aqueous buffer with an excess of N-succinimidyl-S-acetylthioacetate (SATA) in said aqueous buffer at a temperature range of +15° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of +15° C. to +25° C. for 0.25-2 hours to produce a derivatized AChE mixture;
3. Purifying said derivatized carrier protein mixture to prepare a purified derivatized AChE mixture;
4. Treating said purified derivatized carrier protein mixture with hydroxylamine to produce a sulfhydryl-deprotected AChE mixture;
5. Purifying said sulfhydryl-deprotected AChE mixture to prepare a purified sulfhydryl-deprotected AChE mixture;
6. Treating said purified sulfhydryl-deprotected AChE mixture with tetranor-PGAM to produce a tetranor-PGAM-AChE Michael adduct; and
7. Purifying said tetranor-PGAM-AChE Michael adduct to prepare a purified tetranor-PGAM-AChE Michael adduct.
In another exemplary embodiment, a tracer may be formed according to a method comprising the following steps:
1. Contacting AChE in a suitable aqueous buffer with an excess of N-succinimidyl-S-acetylthiopropionate (SATP) in said aqueous buffer at a temperature range of +15° C. to +25° C.;
2. Mixing said reaction mixture at a temperature range of +15° C. to +25° C. for 0.25-2 hours to produce a derivatized AChE mixture;
3. Purifying said derivatized carrier protein mixture to prepare a purified derivatized AChE mixture;
4. Treating said purified derivatized carrier protein mixture with hydroxylamine to produce a sulfhydryl-deprotected AChE mixture;
5. Purifying said sulfhydryl-deprotected AChE mixture to prepare a purified sulfhydryl-deprotected AChE mixture;
6. Treating said purified sulfhydryl-deprotected AChE mixture with tetranor-PGAM to produce a tetranor-PGAM-AChE Michael adduct; and
7. Purifying said tetranor-PGAM-AChE Michael adduct to prepare a purified tetranor-PGAM-AChE Michael adduct.
The above description of exemplary embodiments, and examples provided below, are merely exemplary in nature and, thus, variations thereof are not to be regarded as a departure from the spirit and scope of the invention.
The Examples provided herein describe embodiments directed to tetranor-PGEM-specific antibodies and immunogens used for generating antibodies specific for tetranor-PGEM and tetranor-PGAM. The Examples further provide methods for preparing antibodies that specifically bind tetranor-PGEM and tetranor-PGAM and immunogens used for generating antibodies specific for tetranor-PGEM and tetranor-PGAM. The Examples further describe tetranor-PGEM/tetranor-PGAM-molecular tag conjugate tracers, specifically tetranor-PGEM/tetranor-PGAM-enzyme (AChE) conjugate tracers, and provide methods for their preparation and use for quantifying tetranor-PGEM and tetranor-PGAM in test samples. The Examples further describe immunoassay kits and their use of tetranor-PGEM/tetranor-PGAM-specific antibodies for detecting and measuring quantities of tetranor-PGEM and tetranor-PGAM in a biological fluid.
Procedure 1: Preparation of Tetranor-PGEM-Keyhole Limpet Hemocyanin (KLH) Immunogen (Immunogen 1)
Tetranor-POEM (compound) was prepared by Cayman Chemical using a proprietary method. The identity of the compound was verified by mass spectrometry (MS) and by nuclear magnetic resonance (NMR) spectrometry. The compound was purified to >98% purity by preparatory thin layer chromatography (TLC), and purified compound (3 mg, 0.009 mmole) was dissolved in acetonitrile (600 μl) to provide a tetranor-PGEM solution (0.015 M). N,N-Diisopropylethylamine (density=0.742 g/mL, 60) and isobutyl chloroformate in acetonitrile solution (0.54 weight %, density=1.044 g/mL, 50 were added to the tetranor-PGEM solution. This solution was mixed at 0° C. for two hours and was subsequently dried under a stream of nitrogen. Keyhole limpet hemocyanin (KLH) (5 mg, 0.01 mol % versus tetranor-PGEM with KLH MW 5,000,000) was dissolved in 0.1 M potassium phosphate (pH 7.4) (1 mL) and stirred overnight at 4° C. in the dark. The contents where then dialyzed through a 10,000 MW cut-off membrane for eight hours against 0.1 M potassium phosphate buffer, pH 7.4 (4 L) to provide an aqueous buffered conjugate solution. Aliquots of this solution were frozen at −20° C. for immunizations.
The method of Procedure 1 immediately above was used, except that equimolar amounts of tetranor-PGEM, isobutyl chloroformate, and N,N-diisopropylethylamine were combined, to form Immunogen 1 A.
Bovine serum albumin (BSA, 3 mg) is mixed with a large molar excess of N-succinimidyl-S-acetylthioacetate (SATA, Pierce Protein Research Products/Thermo Scientific, Catalog No. 26102, 3 mg; alternatively, N-succinimidyl-S-acetylthiopropionate, or SATP, Thermo Scientific Catalog No. 26100, may be used with number of moles essentially equivalent to that of moles of SATA used) in aqueous potassium phosphate (KPhos) buffer pH 7.4 (100 mM, approximately 1 mL) and the mixture is incubated at room temperature for thirty minutes. The mixture is purified over a Sephadex G-25 size exclusion column equilibrated and run with an equilibrium mixture comprising KPhos buffer pH 7.4 (50 mM) and ethylenediaminetetraacetic acid (EDTA, 5 mM) to produce a purified acetylated BSA mixture. The purified acetylated BSA mixture is treated with hydroxylamine to a 50 mM concentration and the treated mixture is incubated at room temperature for two hours to produce a sulfhydryl-deprotected BSA mixture. The sulfhydryl-deprotected BSA mixture is purified over a Sephadex G-25 size exclusion column equilibrated and run with an equilibrium mixture comprising KPhos buffer pH 6 (50 mM) and EDTA (5 mM) to produce a set of fractions. Fractions are analyzed by absorbance at 280 nm or by the Bradford Method for protein quantification and fractions containing sufficient protein are combined to produce a purified sulfhydryl-deprotected BSA solution. A portion of the purified sulfhydryl-deprotected BSA solution containing 1 mg of the sulfhydryl-deprotected BSA solute is treated with tetranor-PGAM (1 mg) and the resulting mixture is incubated at 37° C. overnight and is purified over a Sephadex G-25 size exclusion column to produce a tetranor-PGAM-BSA Michael adduct conjugate (Immunogen 2). Conjugation efficiency is determined via immunoassay. A negative control is prepared by treating a portion of the purified sulfhydryl-deprotected BSA solution with N-ethylmaleimide (NEM) to block the sulfhydryl groups followed by incubation with tetranor-PGAM at 37° C. overnight.
To a solution comprising tetranor-PGEM (50 □g) and acetonitrile (400 μL) chilled over ice was added a solution comprising N,N-diisopropylethylamine (19.7 μg) brought to 26.6 μL volume with acetonitrile. A solution comprising isobutylchloroforrnate (20.8 μg) brought to 20 μL volume with acetonitrile was subsequently added and the reaction mixture was incubated on ice for two hours. The acetonitrile was blown off by a nitrogen stream while maintaining the mixture over ice and the concentrate was reconstituted with N,N-dimethylformamide (DMF, 200 μL). The reconstituted mixture was added to a mixture comprising acetylcholinesterase (AChE) (500 Units) and borate buffer pH 8.5 (100 mM, 1 mL) and the resulting combined mixture was incubated at 4° C. overnight. The mixture was purified on a G-25 Sephadex column eluting with 0.1 M potassium phosphate buffer, pH 7.4 and collecting 1-mL fractions. An aliquot (2 μL) of each fraction was added to a well of a 96-well plate, and each well was diluted with Ellman's Reagent (200 μL). Each diluted aliquot was incubated for about 30 seconds at room temperature and read at wavelength 414 nm. All fractions from which their corresponding aliquot-Ellman's mixtures produced greater than 10% of the maximum absorbance were combined. The combined fractions comprised concentrated bulk tracer solution, which was titered before use.
Anhydrous DMF is prepared by distillation and storage over molecular sieves. The dry DMF is used to prepare 10 mM solutions of N-hydroxysuccinimide (NHS), dicyclohexyldicarbodiimide (DCC), and tetranor-PGEM, each in a separate 10 mL reactivial that is oven dried and stored in a dessicator. In a new, dry 5 mL V-bottom reactivial, each of the prepared solutions (5 μl) is added, vortexed, briefly sealed with a septum cap and allowed to incubate at ambient temperature overnight. The next day, 0.1 M borate buffer (pH 8.5) (250 μl) is added, along with AChE (500 Units). The resulting mixture is incubated in the dark for 30 minutes at ambient temperature, then purified over a 30×1.5 cm Sephadex G-25 medium column and eluted with 0.1 M potassium phosphate buffer, pH 7.4. One-milliliter fractions are collected and those fractions with a positive Ellman's reaction are pooled. The tracer is diluted 1:1000 and tracer solution (50 μl) is used to detect specific antibody in 96-well microplate coated with mouse anti-rabbit immunoglobulin G (IgG).
Four to six week-old BALB/C mice (Charles River) were immunized by injecting intraperioneal (i.p.) with equal volumes of Immunogen 1 (100 μg; prepared in Step A, Procedure 1 of this Example; alternatively, Immunogen 1A, which was prepared in Step A, Procedure 1A of this Example, has been used to produce antiserum according to this immunization method) and Complete Freund's adjuvant followed by a boost 14 days later with an equivalent amount of immunogen in Incomplete Freund's adjuvant. Serum was collected on day 24 (10 days after the second immunization) and the titers determined based on immunoreactivity to tetranor-PGEM tracer (as described above). Mice with high serum titers to tetranor-PGEM were given a second boost i.p with antigen (100 μg) in Incomplete Freund's adjuvant on day 34 and serum was collected again and tested on day 44. Mice with high titers were given a final intravenous (i.v.) injection with immunogen (10 μg) in sterile saline. Three days later the mice were euthanized by carbon dioxide inhalation, their spleens removed under sterile conditions and prepared for fusion as described below.
The fusion reagent was prepared by autoclaving PEG4000 (4.2 g) in a glass bottle and before the PEG4000 solidifies, add DMSO (1.5 ml) and bring the volume up to 10 ml with Dulbecco's PBS containing 0.1 mg/ml anhydrous CaCl2 and 0.1 mg/ml MgCl2 (hexahydrate). The reagent was stored at 4° C. Hypoxanthine Aminopterin Thymidine (HAT) selection medium was prepared by combining fetal bovine serum (50 ml), NCTS-109 (25 ml), Hypoxanthine/Thymidine (HT) solution (2.5 ml) (1.36 mg/ml hypoxanthine, 3.88 mg/ml thymidine), aminopterin (0.0018 mg/ml) (2.5 ml), 1-glutamine (2.5 ml), penicillin-streptomycin (2.5 ml), BM Condimed H1 (Roche) (25 ml) and bring the volume to 500 ml with RPMI-1640. HT growth medium was prepared similar to HAT medium with the omission of aminopterin, and the concentration of all other additives was doubled, except BM Condimed H1, which was reduced to 10 ml.
Immediately prior to fusion, approximately 3×107 myeloma fusion partner cells (Ag8.653) were harvested and washed three times with RPMI-1640 by repeated centrifugation at 200×g and resuspension in RPMI-1640. The final pellet was resuspended in RPMI-1640 (10 ml) and the final cell number determined. A minimum of 20×107 was required for fusion.
Spleens were aseptically excised from each mouse, rinsed with sterile RPMI 1640 medium (5 ml) in a P100 Petri dish, transferred to a second sterile dish, perfused with RPMI-1640 then minced with sterile forceps. The minced spleen suspension was transferred to a 15 ml conical tube to allow debris to settle. The cell suspension was transferred to a clean 15 ml tube, centrifuged at 200×g, the cell pellet resuspended in cold RPMI-1640 (10 ml) and the cell number determined.
For the fusion, the fusion ratio of splenocyte:myeloma cells was 5:1. (i.e. 10×107 splenocytes to 2×107 myeloma cells). The appropriate volumes of splenocytes and myeloma cells were transferred to a sterile 50 ml conical tube, centrifuged and the supernatant removed. The tube was heated in a 37° C. water bath for approximately one minute, and with continuous gentle swirling of the tube, fusion reagent (1.5 ml) was slowly added over the course of one minute. Over the course of an additional minute, warm RPMI-1640 (10 ml) was added, followed by centrifugation at 200×g to pellet the cells. The cell pellet was resuspended in HAT selection medium (125 ml) and 100 μl/well plated into each of twenty 96-well plates. The cells were incubated at 37° C. in a 5% CO2 humidified atmosphere incubator. After 5-7 days, an additional HAT medium (100 μl) was added to each well. The plates were monitored until cell growth could be seen by direct observation. At this point the supernatants were harvested from wells with cell growth and screened for immunoreactivity with tetranor-PGEM tracer (see below).
Fusion supernatants were screened by transferring of supernatant (100 □1) into a well of a goat anti-mouse coated plate from Cayman Chemical Company, Incorporated [Cat# 400009] followed by addition of tetranor-PGEM-AChE Tracer (Cayman Cat# 401000) (100 μl). A negative control well contained HAT selection medium (100 μl) and a positive control well contained diluted immune mouse serum. The plates were incubated overnight at room temperature, washed, and Ellman's reagent (200 μl) added to each well. The ODs were read at 415 nm at 30, 60, and 90 minute intervals. Wells with an elevated absorbance are considered positive for antibody production, and will be expanded for further characterization.
Cells from wells that test positive in the first round of screening are transferred to HAT selection medium (3 ml) in wells of a 6-well dish or into HAT medium (5 ml) in a T25 flask. After vigorous cell growth is observed, a small volume of supernatant is harvested and re-screened as described above to eliminate any original false positives. Parental cultures that test positive two or more times are expanded in T25 flasks and a portion cyropreserved (1-2 vials per parental) in liquid nitrogen. The remaining cells are maintained in culture for further screening and subcloning. In the next screen the parental are titered based on signal. The next screen consists of a sensitivity screen selecting for antibody (Ab) with a suitable detection limit. The next screen excluded Ab with unacceptably high cross-reactivity (experiment described below) with parent metabolites, tetranor-PGFM, and tetranor-PGDM. The final screen consists of urinary measurements to confirm normal biological levels of tetranor-PGEM, and recovery experiments.
Step A: Preparation of PGE Metabolite-Carrier Protein Immunogen
Procedure 1: Preparation of Tetranor-PGEM-Keyhole Limpet Hemocyanin KLH immunogen (Immunogen 1)
Tetranor-PGEM (compound) was prepared by Cayman Chemical using a proprietary method. The identity of the compound was verified by mass spectrometry (MS) and by nuclear magnetic resonance (NMR) spectrometry. The compound was purified to >98% purity by preparatory thin layer chromatography (TLC), and purified compound (3 mg, 0.009 mmole) was dissolved in acetonitrile (600 0) to provide a tetranor-PGEM solution (0.015 M). N,N-Diisopropylethylamine (density=0.742 g/mL, 6 μl) and isobutyl chloroformate in acetonitrile solution (0.54 weight %, density=1.044 g/mL, 50 μl) were added to the tetranor-PGEM solution. This solution was mixed at 0° C. for two hours and was subsequently dried under a stream of nitrogen. Keyhole limpet hemocyanin (KLH) (5 mg, 0.01 mol % versus tetranor-PGEM with KLH MW 5,000,000) was dissolved in 0.1 M potassium phosphate (pH 7.4) (1 mL) and stirred overnight at 4° C. in the dark. The contents where then dialyzed through a 10,000 MW cut-off membrane for eight hours against 0.1 M potassium phosphate buffer, pH 7.4 (4 L) to provide an aqueous buffered conjugate solution. Aliquots of this solution were frozen at −20° C. for immunizations.
The method of Procedure 1 immediately above was used, except that equimolar amounts of tetranor-PGEM, isobutyl chloroforinate, and N,N-diisopropylethylamine were combined, to form Immunogen 1A.
Bovine serum albumin (BSA, 3 mg) is mixed with a large molar excess of N-succinimidyl-S-acetylthioacetate (SATA, Pierce Protein Research Products/Thermo Scientific, Catalog No. 26102, 3 mg; alternatively, N-succinimidyl-S-acetylthiopropionate, or SATP, Thermo Scientific Catalog No. 26100, may be used with number of moles essentially equivalent to that of moles of SATA used) in aqueous potassium phosphate (KPhos) buffer pH 7.4 (100 mM, approximately 1 mL) and the mixture is incubated at room temperature for thirty minutes. The mixture is purified over a Sephadex G-25 size exclusion column equilibrated and run with an equilibrium mixture comprising KPhos buffer pH 7.4 (50 mM) and ethylenediaminetetraacetic acid (EDTA, 5 mM) to produce a purified acetylated BSA mixture. The purified acetylated BSA mixture is treated with hydroxylamine to a 50 mM concentration and the treated mixture is incubated at room temperature for two hours to produce a sulfhydryl-deprotected BSA mixture. The sulfhydryl-deprotected BSA mixture is purified over a Sephadex G-25 size exclusion column equilibrated and run with an equilibrium mixture comprising KPhos buffer pH 6 (50 mM) and EDTA (5 mM) to produce a set of fractions. Fractions are analyzed by absorbance at 280 nm or by the Bradford Method for protein quantification and fractions containing sufficient protein are combined to produce a purified sulfhydryl-deprotected BSA solution. A portion of the purified sulfhydryl-deprotected BSA solution containing 1 mg of the sulfhydryl-deprotected BSA solute is treated with tetranor-PGAM (1 mg) and the resulting mixture is incubated at 37° C. overnight and is purified over a Sephadex G-25 size exclusion column to produce a tetranor-PGAM-BSA Michael adduct conjugate (Immunogen 2). Conjugation efficiency is determined via immunoassay. A negative control is prepared by treating a portion of the purified sulfhydryl-deprotected BSA solution with N-ethylmaleimide (NEM) to block the sulfhydryl groups followed by incubation with tetranor-PGAM at 37° C. overnight.
To a solution comprising tetranor-PGEM (50 μg) and acetonitrile (400 μL) chilled over ice was added a solution comprising N,N-diisopropylethylamine (19.7 μg) brought to 26.6 μL volume with acetonitrile. A solution comprising isobutylchloroformate (20.8 μg) brought to 20 μL volume with acetonitrile was subsequently added and the reaction mixture was incubated on ice for two hours. The acetonitrile was blown off by a nitrogen stream while maintaining the mixture over ice and the concentrate was reconstituted with N,N-dimethylformamide (DMF, 200 μL). The reconstituted mixture was added to a mixture comprising acetylcholinesterase (AChE) (500 Units) and borate buffer pH 8.5 (100 mM, 1 mL) and the resulting combined mixture was incubated at 4° C. overnight. The mixture was purified on a G-25 Sephadex column eluting with 0.1 M potassium phosphate buffer, pH 7.4 and collecting 1-mL fractions. An aliquot (2 μL) of each fraction was added to a well of a 96-well plate, and each well was diluted with Ellman's Reagent (200 μL). Each diluted aliquot was incubated for about 30 seconds at room temperature and read at wavelength 414 nm. All fractions from which their corresponding aliquot-Ellman's mixtures produced greater than 10% of the maximum absorbance were combined. The combined fractions comprised concentrated bulk tracer solution, which was titered before use.
Anhydrous DMF is prepared by distillation and storage over molecular sieves. The dry DMF is used to prepare 10 mM solutions of N-hydroxysuccinimide (NHS), dicyclohexyldicarbodinnide (DCC), and tetranor-PGEM, each in a separate 10 mL reactivial that is oven dried and stored in a dessicator. In a new, dry 5 mL V-bottom reactivial, each of the prepared solutions (5 μl) is added, vortexed, briefly sealed with a septum cap and allowed to incubate at ambient temperature overnight. The next day, 0.1 M borate buffer (pH 8.5) (250 μl) is added, along with AChE (500 Units). The resulting mixture is incubated in the dark for 30 minutes at ambient temperature, then purified over a 30×1.5 cm Sephadex G-25 medium column and eluted with 0.1 M potassium phosphate buffer, pH 7.4. One-milliliter fractions are collected and those fractions with a positive Ellman's reaction are pooled. The tracer is diluted 1:1000 and tracer solution (500 is used to detect specific antibody in 96-well microplate coated with mouse anti-rabbit immunoglobulin G (IgG).
Rabbit immunizations were performed by Robert Sargeant Antibodies (655 Ash Street, Ramona, Calif. 92065) as follows: Male New Zealand White Rabbits 9-10 weeks of age, were immunized with Complete Freund's Adjuvant (CFA) initially, followed by Incomplete Freund's Adjuvant (IFA) for all subsequent injections. Immunogen 1 (200 μg; prepared in Step A, Procedure 1 of this Example; alternatively, Immunogen 1 A, which was prepared in Step A, Procedure 1A of this Example, has been used to produce antiserum according to this immunization method) was injected for the first immunization and 100 μg for boosts. The immunogen was diluted to one milliliter with sterile saline and combined with one milliliter of the appropriate adjuvant. The antigen and adjuvant were mixed thoroughly to form a stable emulsion which is injected subcutaneously. Blood was collected from the central ear artery and allowed to clot and retract. The serum was decanted and clarified by centrifugation before freezing.
Antisera (AS) from multiple bleeds from multiple rabbits were screened by titration on a 96-well microplate with detection of specific Ab based on its ability to bind the tetranor-PGEM-AChE conjugate (diluted 1:1000). The next screen consisted of a sensitivity screen selecting for AS with a suitable detection limit. The next screen excluded AS with unacceptably high cross-reactivity (experiment described below) with parent metabolites, tetranor-PGFM, and tetranor-PGDM. The final screen consisted of urinary measurements to confirm normal biological levels of tetranor-PGEM, and recovery experiments.
To determine cross-reactivity of an antibody, set up the standard curve using optimal tracer and Ab dilutions according the kit instructions. Tetranor-PGEM was diluted to 10 ng/ml for the first point in the standard curve and diluted 2.5-fold serially 7 times. First standard dilutions (10 μg/ml) of tetranor-PGDM, tetranor-PGJM, tetranor-PGAM, and tetranor-PGFM were prepared and 7 additional 6-fold serial dilutions were performed. IC50s of all compounds were determined and each percent cross-reactivity (% XR) values of the potentially cross-reactive compounds was determined by dividing the IC50 of tetranor-PGEM by the IC50 of the test compound and multiplying by 100. See the chart below for determined % XR values.
Tetranor-PGAM was prepared by Cayman Chemical Company, Incorporated using a proprietary method. The identity of the compound was verified by mass spectrometry (MS) and by nuclear magnetic resonance (NMR) spectrometry. The compound was purified to >98% purity by preparatory thin layer chromatography (TLC), and purified compound (3 mg) was dissolved in acetonitrile (600 μl) to provide a tetranor-PGAM solution. N,N-Diisopropylethylamine (6 μl) and 0.54 weight % isobutyl chloroformate in acetonitrile solution (50 μl) were added to the tetranor-PGAM solution. This solution was mixed at 0° C. for two hours and was subsequently dried under a stream of nitrogen. Keyhole limpet hemocyanin (KLH) (5 mg, 0.01 mol % versus tetranor-PGAM with KLH MW 5,000,000) was dissolved in 100 mM potassium phosphate (pH 7.4) (1 mL) and stirred overnight at 4° C. in the dark. The contents where then dialyzed through a 10,000 MW cut-off membrane for eight hours against 0.1 M potassium phosphate buffer, pH 7.4 (4 L). Aliquots were frozen at −20° C. for immunizations.
To a solution comprising tetranor-PGAM (50 μg) and acetonitrile (400 μL) chilled over ice is added a solution comprising N,N-diisopropylethylamine (equimolar with tetranor-PGAM) brought to 26.6 μL volume with acetonitrile. A solution comprising isobutylchlorofonnate (equimolar with tetranor-PGAM) brought to 20 μL volume with acetonitrile is subsequently added and the reaction mixture is incubated on ice for two hours. The acetonitrile is blown off by a nitrogen stream while maintaining the mixture over ice and the concentrate is reconstituted with N,N-dimethylformamide (DMF, 200 μL). The reconstituted mixture is added to a mixture comprising acetylcholinesterase (AChE) (500 Units) and borate buffer pH 8.5 (100 mM, 1 mL) and the resulting combined mixture is incubated at 4° C. overnight. The mixture is purified on a G-25 Sephadex column eluting with 0.1 M potassium phosphate buffer, pH 7.4 and collecting 1-mL fractions. An aliquot (2 μL) of each fraction is added to a well of a 96-well plate, and each well is diluted with Ellman's Reagent (200 μL). Each diluted aliquot is incubated for about 30 seconds at room temperature and read at wavelength 414 nm. All fractions from which their corresponding aliquot-Eliman's mixtures produce greater than 10% of the maximum absorbance are combined. The combined fractions comprise concentrated bulk tracer solution, which is titered before use.
Anhydrous DMF was prepared by distillation and storage over molecular sieves. The dry DMF was used to prepare 10 mM solutions of N-hydroxysuccinimide (NHS), dicyclohexyldicarbodiimide (DCC), and tetranor-PGEM, each in a separate 10 mL reactivial that was oven dried and stored in a dessicator. In a new, dry 5 mL V-bottom reactivial, each of the prepared solutions (5 μl) was added, vortexed, briefly sealed with a septum cap and allowed to incubate at ambient temperature overnight. The next day, 0.1 M borate buffer (pH 8.5) (250 μl) was added, along with AChE (500 Units). The resulting mixture was incubated in the dark for 30 minutes at ambient temperature, then purified over a 30×1.5 cm Sephadex G-25 medium column and eluted with 0.1 M potassium phosphate buffer, pH 7.4. One-milliliter fractions were collected and those fractions with a positive Ellman's reaction were pooled. The tracer was diluted 1:1000 and Tracer 4 solution (50 μl) was used to detect specific antibody in 96-well microplate coated with mouse anti-rabbit immunoglobulin G (IgG).
Procedure 3 (Michael addition method): Preparation of tetranor-PGAM-AChE Michael adduct (Tracer 5)
A mixture comprising tetranor-PGAM (50 μg) and DMF (200 μL) is added to a mixture comprising AChE* (500 Units) and KPhos buffer pH 7.4 (50 mM, 1 mL) and the combined mixtures are incubated at 37° C. overnight. The mixture is purified on a G-25 Sephadex column eluting with 0.1 M potassium phosphate buffer, pH 7.4 and collecting 1-mL fractions. An aliquot (2 μL) of each fraction is added to a well of a 96-well plate, and each well is diluted with Ellman's Reagent (200 μL). Each diluted aliquot is incubated for about 30 seconds at room temperature and read at wavelength 414 nm. All fractions from which their corresponding aliquot-Ellman's mixtures produced greater than 10% of the maximum absorbance are combined. The combined fractions comprise concentrated bulk tracer solution, which is titered before use.
*AChE contains 8 free thiols (sulfhydryls)/mole of tetramer if a higher level of conjugation is required more free thiols can be introduced via SATA or SATP modification according to embodiments described herein.
Rabbit immunizations were performed by Robert Sargeant Antibodies (655 Ash Street, Ramona, Calif. 92065) as follows: Male New Zealand White Rabbits 9-10 weeks of age, were immunized with Complete Freund's Adjuvant (CFA) initially, followed by Incomplete Freund's Adjuvant (IFA) for all subsequent injections. Immunogen 3 (200 μg; prepared according to Step A of this Example) was injected for the first immunization and 100 μg for boosts. The immunogen was diluted to one milliliter with sterile saline and combined with one milliliter of the appropriate adjuvant. The antigen and adjuvant were mixed thoroughly to form a stable emulsion which is injected subcutaneously. Blood was collected from the central ear artery and allowed to clot and retract. The serum was decanted and clarified by centrifugation before freezing.
Antisera (AS) from multiple bleeds from multiple rabbits were screened by titration on a 96-well microplate with detection of specific Ab based on its ability to bind the tetranor-PGAM-AChE conjugate (Tracer 4, diluted 1:1000). The next screen consisted of a sensitivity screen selecting for AS with a suitable detection limit. The next screen excluded AS with unacceptably high cross-reactivity (experiment described below) with parent metabolites, tetranor-PGFM, and tetranor-PGDM. The final screen consisted of urinary measurements to confirm normal biological levels of tetranor-PGAM, and recovery experiments. Only one AS passed all screens.
To determine cross-reactivity of an antibody, set up the standard curve using optimal tracer and Ab dilutions according the kit instructions. Tetranor-PGAM was diluted to 10 ng/ml for the first point in the standard curve and diluted 2.5-fold serially 7 times. First standard dilutions (10 μg/ml) of tetranor-PGDM, tetranor-PGEM, and tetranor-PGFM were prepared and 7 additional 6-fold serial dilutions were performed. IC50s of all compounds were determined and each percent cross-reactivity (% XR) values of the potentially cross-reactive compounds was determined by dividing the IC50 of tetranor-PGAM by the IC50 of the test compound and multiplying by 100. See the chart below for determined % XR values.
1. EIA Buffer Preparation
The contents of one vial of EIA Buffer Concentrate (10×) (Cayman Chemical Company Catalog No. 400060) is diluted with UltraPure water (Cayman Chemical Company, Incorporated Catalog No. 400000) The vial is rinsed to remove any salts that may have precipitated.
2. Wash Buffer Preparation
Wash Buffer Concentrate (400×) (5 ml, 96-well kit; Cayman Chemical Company Catalog No. 400062) is diluted with UltraPure water to a total volume of 2 liters and Tween 20 (1 ml, Cayman Chemical Company, Incorporated Catalog No. 400035). Alternatively Wash Buffer Concentrate (400×) (12.5 ml, 480-well kit; Cayman Chemical Company Catalog No. 400062) is diluted with UltraPure water to a total volume of 5 liters and Tween 20 (2.5 ml, Cayman Chemical Company, Incorporated Catalog No. 400035).
Smaller volumes of Wash Buffer can be prepared by diluting the Wash Buffer Concentrate 1:400 and adding Tween 20 (0.5 ml/liter of Wash Buffer).
This assay is validated for urine samples (diluted at least 1:2).
Proper sample storage and preparation are essential for consistent and accurate results. PGE2 is chemically unstable in biological samples, especially those containing albumin (Fitzpatrick, F. and Wynalda, M. J Biol. Chem., 258, 1983, 11713-11718).
All samples must be free of organic solvents prior to assay.
Samples should be assayed immediately after collection; samples that cannot be assayed immediately should be stored at −80° C.
Samples of rabbit origin may contain antibodies which interfere with the assay by binding to the mouse anti-rabbit plate. All rabbit samples should be purified prior to use in the assay.
Urinary concentrations of tetranor-PGEM vary considerably and, as with any urinary marker, the values obtained by EIA should be standardized to creatinine levels.
Step C1a: tetranor-PGEM EIA Standard
The tetranor-PGEM EIA Standard (100 μl) is transferred into a clean test tube and diluted with UltraPure water (900 μl). The concentration of this solution (the bulk standard) is 100 ng/ml. (If assaying culture medium samples that have not been diluted with EIA Buffer, culture medium should be used in place of EIA Buffer for dilution of the standard curve.
Eight clean test tubes are numbered #1 through #8. EIA Buffer (900 d) is aliquoted to tube #1 and 600 μl of EIA Buffer to tubes #2-8. Bulk standard (100 μl) is transferred to tube #1 and the contents of the tube are mixed thoroughly. The standard is serially diluted by removing 400 μl from tube #1 and placing in tube #2; the contents of tube #2 are subsequently mixed thoroughly. Contents from tube #2 (400 μl) is transferred to tube #3; the contents of tube #3 are subsequently mixed thoroughly. This process is repeated for tubes #4-8. (These diluted standards should not be stored for more than 24 hours).
Step C1b: tetranor-PGEM AChE Tracer
The Tetranor-PGEM AChE Tracer is Reconstituted as Follows: First, Tetranor-PGEM AChE Tracer (100 dtn, 96-well kit) is reconstituted with EIA Buffer (6 ml) or Tetranor-PGEM AChE Tracer (500 dtn, 480-well kit) is reconstituted with EIA Buffer (30 ml). The reconstituted tetranor-PGEM AChE Tracer should be stored at 4° C. (do not freeze) and used within four weeks. A tracer dye may be added to the tracer to aid in visualization of tracer-containing wells (not required). The dye is added to the reconstituted tracer at a final dilution of 1:100 (60 μl of dye is added to 6 ml of tracer, or 300 μl of dye is added to 30 ml of tracer).
Step C1c: Tetranor-PGEM EIA Antiserum
The tetranor-PGEM EIA Antiserum is reconstituted as follows: First, Tetranor-PGEM EIA Antiserum (100 dtn, 96-well kit) is reconstituted with EIA Buffer (6 ml) or Tetranor-PGEM EIA Antiserum (500 dtn, 480-well kit) is reconstituted with EIA Buffer (30 ml). The reconstituted tetranor-PGEM EIA Antiserum should be stored at 4° C. and used within four weeks. An antiserum dye may be added to the antiserum to aid in visualization of antiserum-containing wells (not required). The dye is added to the reconstituted antiserum at a final dilution of 1:100 (60 μl of dye is added to 6 ml of antiserum or 300 μl of dye is added to 30 ml of antiserum).
Step C2: Plate Setup
Each 96-well plate or set of strips contain a minimum of two blanks (Blk), two non-specific binding wells (NSB), two maximum binding wells (B0), and an eight point standard curve run in duplicate. Each assay is assayed at two dilutions and each dilution is assayed in duplicate or triplicate.
Step C3: Performing the Assay
Step C3a: Addition of the Reagents:
1. EIA Buffer: EIA Buffer is added to NSB wells (100 □1) and B0 wells_(50 μl). If culture medium is used to dilute the standard curve, culture medium (50 μl) is substituted for EIA Buffer in the NSB and B0 wells (i.e. 50 μl of culture medium is added to NSB and B0 wells and 50 μl of EIA Buffer to NSB wells.)
2. Tetranor-PGEM EIA Standard: 50 μl from tube #8 is added to both of the lowest standard wells (S8). 50 μl from tube #7 is added to each of the next two standard wells (S7). This procedure is continued until all of the standards are aliquoted.
3. Samples: 50 μl of sample is added per well. Each sample is assayed at a minimum of two dilutions. Each dilution is assayed in duplicate or triplicate.
4. Tetranor-PGEM AChE Tracer: 50 μl is added to each well except the total activity (TA) and the Blank (Bik) wells.
5. Tetranor-PGEM EIA Antiserum: 50 μl is added to each well except the TA, the NSB, and the Bik wells.
Step C3b: Incubation of the Plate: Each plate is covered with plastic film and incubated overnight at 4 C.
Step C3c: Development of the Plate
1. Ellman's Reagent (100 dtn vial for 96-well kit) is reconstituted with UltraPure water (20 ml),
-or-
Ellman's Reagent (250 dtn vial for 480-well kit) is reconstituted with UltraPure water (50 ml).
2. The wells are emptied and rinsed five times with Wash Buffer.
3. Ellman's Reagent (200 μl) is added to each well.
4. Tracer (5 μl) is added to the Total Activity wells.
5. The plate is covered with plastic film. Optimum development is obtained by using an orbital shaker equipped with a large, flat cover to allow the plates to develop in the dark for 60-90 minutes.
Step C3d: Reading the Plate
1. The bottom of the plate is wiped with a clean tissue.
2. The plate cover is removed.
3. The plate is read at a wavelength between 405 and 420 nm.
The absorbance is checked periodically until the B0 wells reach a minimum of 0.3 A.U. (blank subtracted). The plate is read when the absorbance of the B0 wells are in the range of 0.3-1.0 A.U. (blank subtracted).
The data is plotted as % B/B0 versus log concentration using either a 4-parameter logistic or log-logit curve fit.
Step D1a: Preparation of the Data (absorbance reading of the blank wells are subtracted from the absorbance readings of the rest of the plate if not done automatically be the plate reader).
1. Absorbance readings from the NSB wells are averaged.
2. Absorbance readings from the B0 wells are averaged.
3. The NSB average is subtracted from the B0 average. This is the corrected B0 or corrected maximum binding.
4. The % B/B0 is calculated for the remaining wells. (The average NSB absorbance is subtracted from the S1 absorbance and divided by the corrected B0 from step 3 immediately above. This value is multiplied by 100 to obtain % B/B0 and the calculation is repeated for S2-S8 and all sample wells.)
Step D1b: Plot of the Standard Curve
The % B/B0 is plotted for standards S1-S8 versus tetranor-PGEM concentration using linear (y) and log (x) axes and the data is fit to a 4-parameter logistic equation.
Alternative plot: the data may also be linearized using a logit transformation. The equation for this conversion is:
logit(B/B0)=In[B/B0/(1-B/B0)]
The data is plotted as logit (B/B0) versus log concentrations and a linear regression fit is performed.
Step D1c: Determination of the Sample Concentration
The % B/B0 is calculated for each sample. The concentration of each sample is determined using the equation obtained from the standard curve plot. Samples with % B/B0 values greater than 80% or less than 20% should be re-assayed as they generally fall outside the range of the standard curve. A 20% or greater disparity between the apparent concentrations of two different dilutions of the same sample indicate interference which is eliminated by purification.
Step D2a: Sample Data
Results vary from assay run to assay run; therefore, a new standard curve must be run with each new assay performed.
The intra- and interassay CV's are determined at multiple points on the standard curve.
Step D2c: Specificity of the tetranor-PGEM EIA Antiserum
It is understood for purposes of this disclosure that various changes and modifications may be made to the invention that are well within the scope of the invention. Numerous other changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the invention disclosed herein.
The list of citations to patents, patent application publications, and publications cited herein are each hereby incorporated by reference in its entirety for all purposes.
The present application claims priority from U.S. Provisional Application Ser. No. 61/354,491, filed Jun. 14, 2010, which is herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61354491 | Jun 2010 | US |
