HYDROXYCHOLESTEROL IMMUNOASSAY

CROSS-REFERENCE TO REL ATED APPLICATION

This international application is a continuation of prior Application No. 12/927,206, filed November 9, 2010.

BACKGROUND OF THE INVENTION

( ) Field of the Invention

The present application generally relates to antibody detection of metabolites. More specifically, methods and compositions are provided for preparing and using antibodies to detect and quantify 24S-hydroxycholesterol.

(2) Description of the Related Art

24S-hydroxychoiesterol (241 IC) (structure below) is one of several mono-oxygenated metabolites of cholesterol.

Excess cholesterol in the brain is converted to 24IIC by cholesterol 24-hydroxylase (CYP46). Owing to its ability to cross the blood-brain barrier, 24HC is present, in peripheral circulation. Because CYP46 is predominantly expressed in the brain, 24HC in plasma originates almost exclusively from the brain (Lutjohann et al., 1996). Interestingly, certain CYP46 single nucleotide polymorphisms are associated with Alzheimer's disease ("AD ^M )(Garcia et al., 2009). Plasma levels of 24HC correlate with brain and hippocarapal size (Bretillon et al., 2000;

oschack et al., 2009). 24HC in plasma or cerebrospinal fluid is an indicator of brain cholesterol turnover, particularly from myelin, and demyelination caused by neuronal degeneration results in a increased flux of 24HC across the blood-brain barrier and into plasma. Thus, active neuronal cells are the major s urce of 24HC in human circulation and the concentration of 24HC in plasma is considered a surrogate marker for brain cholesterol homeostasis. Additionally, 24HC is a .signaling molecule in the brain, inducing ApoE-raediaied cholesterol efflux from astrocytes by a direct effect on ApoE transcription, protein synthesis and secretion Leoni et al., 2010). Direct toxic and inflammatory gene expression-inducing effects of 24HC may also be involved in the pathology of AD or other cognitive dysfunctions (Aiexandrov et al., 2005; Kfllsch et al., 2004; Zhao et at, 2009). Further, neurodegeneration and the resulting loss of neurons has been found in some studies to result in the reduction of 24HC in plasma in AD, Huntington's disease, and multiple sclerosis (Bjftrkhem et al., 2009; Irizarry, 2004; Leoni, 2009; Leoni et al,, 2008; Masterman et al., 2002; Solomon et al, 2009; Teunissen et al, 2003, 2005; Valen/a et al., 2007). However, other studies have found increased 24MC plasma levels in AD patients (LQtjohann and von Bergmann, 2003; Lutjohami et al., 2000). In cerebrospinal fluid, 24S-hydroxyehoieslero1 levels increase, and appear to reflect the rate of neurodegeneration (Leoni et al., 2010; Papassotiropoulos et al., 2002; Shafaati et al., 2007). Reduced plasma levels of 24HC are also induced by statin treatment (Lutjohann and von Bergmann, 2003; Vega and Weiner, 2007). In a further association with neurodegenerative disease, gene therapy with CYP46 in a mouse model of AD, administered before the onset of amyloid deposits, reduced Αβ peptides, amyloid deposits and trimcric oligomers, and improved spatial memory in those mice (liudry et al., 2010). Additionally, ablation of acyi-CoA:cholesterol acetyltransferase 1

(AC'AT l) in the brain leads to a reduction of AD-associated forms of amyloid precursor protein and increases in brain 24HC (Bryleva et al., 20 0).

There is a close relationship between the biotransformation of drugs and normal biochemical processes occurring in the human body. The metabolism of drugs involves many pathways associated with the synthesis of endogenous substrates such as steroid hormones, cholesterol and bile acids, it should be recognized that many of the enzymes involved in drug metabolism are principally designed tor the metabolism of these endogenous compounds. These enzymes metabolize drugs only because the drugs resemble the natural substrate.

During phase II metabolism, a substrate is rendered more hydrophilic through the covalent. attachment of an endogenous molecule. The eytosolic sulfotransferase (SU.LT) and t'DP-glucuronosyliransierase (l!GT) families of enzymes account for the majority of phase II metabolism in humans and animals. Sulibnation and glucuronidalion are generally considered competin pathways. Typically, sulfonation predominates at low substrate concentrations, while glucuronidation predominates at. higher concentrations. In both brain and liver, 24HC serves as a liver-X receptor (l.XR) agonist and has an important role in cholesterol homeostasis. Conversion of this metabolite to bile acids accounts for only 40-50% of the metabolite's elimination, leaving a large percentage of 24HC metabolism and excretion occurring by other pathways. The SUI T and UG enzymes represent a highly responsive defense system against the mutagenicity of carcinogenic environmental chemicals and the toxicity of xenobiolics and endogenous metabolic intermediates. Conjugation with either sulfonate or glucuronic acid has been implicated as important for biliary excretion of 24HC.

Cytosolic SULTs are involved with the conjugation of therapeutic drugs, xenobiotics and small endogenous compounds includin hydroxysteroids, thyroid hormones, estrogens, bile acids, cholesterol and oxysterols. 241 IC can be conjugated by at least three isot rms of human cytosolic SULTs, but others (e.g. SULTs IA1 , 1 A3, IBl, Id ) display no discernable activity (Cook et al„ 2009). SULTs 2A.I and 1E1. sulfonate both the 3- and 24-hydroxyl groups to form 24-hydroxycholesterok3,24-disulfate. SULT2Blb forms only 24-hydroxyeholesterol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate can be hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate is resistant to its actions. Both the 24-hydroxycholesterol-3- sulfate and 24-sulfate are antagonists of LXR activation.

Current methods for determination of 24I1C generally involve gas ehromatography-mass spectrometry, or liquid ehromatography-tandem mass spectrometry (see, e.g., Berkard et al„ 2004; DeBarber et al., 2008; Leoni et al., 2008). Because the importance of 24HC in human physiology and disease is being increasingly recognized, and because the current methods tor identifying 24HC are time consuming and require expensive equipment, there is a need for a simpler assay for 24HC. The present invention addresses that need.

BRIEF SUMMARY OF THE INVENTION

The inventors have developed methods for preparing antibodies to 24S- hydroxycholesierol (24HC). The prepared antibodies have low cross-reactivity to related metabolites, and are useful reagents lor specific and sensitive immunoassays for 24HC.

Thus, in some embodiments, a derivative of 22-hydroxycholesterol, 24$- hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. The derivative comprises a moiety comprising a free amino, carboxylic acid, or sulfhydryl group cova.lent.ly attached to the 3-OI1 position of the hydroxycholesterol.

Also provided is a protein conjugated to the above derivative. Additionally, a composition is provided comprising the above protein combined with an adjuvant that can improve an antibody immune response to the protein in a vertebrate injected with the composition.

Further provided is an antibody composition comprising antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxyeholesterol, 25-hydroxycholesterol, 26- hydroxycholesterol or 27-hydroxycholesterol.

A method of making the above-described antibodies is also provided. The method comprises: (a) preparing an imimtnogen by conjugating the above-described derivative to a carrier protein; (b) immunizing a vertebrate with, the immunogen under conditions such that the immune system f the vertebrate makes the antibodies; and (c) taking the antibodies from the vertebrate.

Additionally provided is a method of assaying for 22-hydfoxycholesterol, 24S- bydroxycholesterot, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholestero! in a fluid or tissue sample from a mammal. The method comprises combining the sample with the above-described antibodies, then determining whether the antibodies specifically bind to 22- hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol from the sample. In these methods, specific antibody binding to hydroxycholesterol from the sample indicates that the hydroxycholesterol is present in the sample.

A kit for detecting and/or quantifying 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is further provided. The kit comprises the above-described antibodies.

The present invention is also directed to a method of detecting an enzyme or enzymes utilized in phase 11 drug metabolism in a sample, where the enzyme or enzymes are capable of altering 22-hydroxyeholesterol, 24S-hydroxycholesierol, 25-hydroxycholesterol, 26- hydroxycholesterol or 27-hydroxycholesterol. The method comprises combining the sample with 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26- hydroxycholesterol or 27-hydroxycholesterol for a time sufficient for the enzyme or enzymes to modify the 22-hydroxycholesterol, 24S-hydroxycholesteroi, 25-hydroxycholesleroi, 26- hydroxycholesterol or 27-hydroxycholesterol, then assaying the sample using the above immunoassay, wherein a reduction in the amount of the 22-hydroxycholesterol, 24S- hydroxycholesterol, 25-hydroxyeholesierol,, 26-hydroxycholesierol or 27-bydroxycholeslero1 detected by the assay indicates that the sample comprises the enzyme or enzymes.

Additionally provided is a method of detecting an enzyme that synthesizes 22- hydroxycholesterol, 24S-hydroxycholesteroI, 25-hydroxycholesierol., 26~hydroxycholes-erol or 27-hydroxyeholesterol in a sample. The method comprises combining the sample with cholesterol for a time sufficient for the enzyme to modify the cholesterol, then assaying the sample using the method of claim 43, wherein an increase in the amount of 22- hydroxycholesterol, 24S-hydroxycholesteix>l, 25-hydroxycholesierol, 26-hydjroxyeholesterol or 27-hydroxycholesterol detected by the assay indicates that the sample comprises the enzyme.

Also provided is a method of evaluating progression of multiple sclerosis i a patient. The method comprises measuring 24S-hydroxycholesterol in blood plasma or serum of the patient over ti me by the above-described assay method. In this method, progression of multiple sclerosis is indicated by decreasing levels of 24S-hydroxycholesterol in the blood plasma or serum over time.

A method of determining whether a treatment for multiple sclerosis in a patient is effective is also provided. The method comprises measuring 24S-hydroxycholesterol in blood plasma or serum of the patient over time by the above-described assay method. In this method, the treatment is effective if the 24S-hydroxycholesterol levels do not decrease over time.

Further provided is a method of evaluating progression of Huntington's disease in a patient. The method comprises measuring 24S-hydroxycholesterol in blood plasma or serum of the patient over time by the alx>ve-described assay method. In this method, progression of Huntington's disease is indicated by decreasing levels of 24S-hydroxycholesterol in the blood plasma or serum over time.

A method of determining whether a treatment for Huntington's disease in a patient is effective is additionally provided. The method comprises measuring 24S-hydroxyeholesterol in blood plasma or serum of the pat ient over time by the above-described assay method, in this method, the treatment is effective if the 24S-hydroxycholesterol levels do not decrease over time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a standard curve of 24S-hydroxycholest.erol in an EI JSA using an antiserum elicited against 3-0-succinoyl-24S-hydroxycholesterol. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular f rms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of "or" is intended to include "and/or", unless the context clearly indicates otherwise.

The inventors have prepared antibodies to 24S-hydroxyeholesterol (24H ) that have low cross-reactivity to related metabolites. The antibodies are useful reagents in rapid, specific and sensitive immunoassays for 24H that can be utilized with standard immunoassay protocols. Based on these results, the inventors believe that effective antibodies with low cross-reactivity could also be made to other hydroxycholesterol metabolites, for example 22-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxyeholesterol. -llius, die inventors envision that any of the methods relating to 24HC that are discussed herein could also be utilized wit 22-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27- hydroxycholesterol As such, while most of the discussion below addresses only compositions and methods relating to 241 iC for the sole purpose of simplifying the readability of this application, the inventors believe that any of the compositions and methods could be equally applied to 22-hy&Oxyeholester l, 25-hydroxycholesterol, 26-hydroxycholesterol and 27- hydroxycholesleiOl without undue experimentation. The inventors therefore do not disclaim, and specifically include, 22-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxychoiesteroJ and 27-hydroxyeholesterol as envisioned for any of the compositions and methods below thai discuss 24HC.

Since 24HC is a hapten that is too small to illicit an antibody immune response by itself, it must be covalently coupled to a carrier protein. The 24HC*protem conjugate, when injected into a vertebrate, presents 24HC to the vertebrate's immune system as an epitope on the protein, such that antibodies are generated to the 24HC epitope. However, 24HC does not have a moiety to which it could be conveniently conjugated to carrier protein. Therefore, a derivative of 24HC is preferably prepared that has a reacti ve moiety for conjugation to proteins or other reagents.

Antibodies against the closely related compound cholesterol have been prepared

(Yinsong et al., 2007; Biro et al., 2007; Sato et al., 1976; Dijkstra et al., 1996). However, antibodies made by conjugation of a carrier protein to the cholesterol 3-OH moiety showed significantly diminished cholesterol binding activity when compared to antibodies made by injection of cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A (Dijkstra et al., J 996). Thus, the excellent results achieved herein in producing effective antisera against 24HC, as described in the Example below, were surprising and unexpected.

Any derivative providing a reactive moiety thai can be conjugated to a protein can be utilized herein. As is known in the art, moieties comprising a free amino group, a free carboxylie acid group, or a free sulihydryl group provide useful reactive groups tor protein conjugation (See. e.g., the website at piercenetXOriv¾tx)wse ^¾ fm?ndID ^::: 4EOI 8AA6-5056-8A76^E57- 3IiC84C88A328). For example, a tree amino group can be conjugated to proteins via glutaraldehyde cross-linking, or via carbodiimide cross-linking to available carboxy moieties o the protein. Also, a hapten with a free sulilrydryl group can be conjugated to proteins via maleimide activation of the protein, e.g., using 8ulfosuccinimidyl-4-(N- maleimidometliyl)cyc1ohexane-1-carboxylate (Sulfo-SMCC), then linkage to the sulihydryl group.

Because the 24-hydroxyl group is the feature that distinguishes 24HC from cholesterol and other naturally occurring hydroxycholesterols (e.g., 22-hydroxycholesterol, 25- hydroxycholesterol, 26-hydjtoxycholesterol, 27-hydroxycholesterol), the inventors reasoned that, in spite of the poor results achieved with cholesterol in similar schemes, conjugation of the carrier protein to the 3-OIJ moiety would present the branched hydroxyalkane end of t he 24I1C molecule as an epitope of the carrier molecule to the immune system, increasing the likelihood that antibodies elicited to the 24HC would have low cross-reactivity with cholesterol and other hydroxycholestero Is ,

Thus, in some embodiments, a derivative of 24S ^« hydroxycholesterol is provided. The derivative comprises a moiety comprising a free amino, carboxylie acid, or sulihydryl group covalently attached to the 3-OH.

In some embodiments, the moiety is a carboxylie acid. A carboxylie acid moiety can be conjugated to the 3-OH of 24HC by any method known in the art, including any method resulting in the following linkages:

In some embodiments, the derivative is conjugated through an ester linkage, for example via the conjugation of a diearboxylic acid to the 24HC. Any diearboxylic acid can be utilized, here. Nonlimiting examples include oxalic acid, raalonic acid, succinic acid, glutaric acid, adipie acid, pimelic acid, suberic acid, aze!aic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, and a phthalic acid. .Methods lor conjugating diearboxylic acid derivatives to hydroxy! moieties are known in the art See, e.g.. Example. In some embodiments, the derivative consists of 3-0-succinoyl-24S-hydroxycholesierol:

y y y

In other embodiments, the derivative comprises 3-0-succinoyl-24$-hydroxychoJesteroL Such derivatives can further encompass any other useiul moiety, e.g., a moiety that further assists in conjugation to a protein. An example of the latter is an N-hydroxysuccinimide ester moiety . Thus, in some embodiments, the 3-0-succinoyl-24S-hydroxycholesterol further comprises an N- hydroxysuceinimide ester conjugated to the carboxyl group on the succmoyl moiety. In some of these embodiments, the compound is

Another useful group that can be conjugated to the 3-0-succinoyI-24S- hydroxycho!esterol is a moiety that can be utilized for signaling. Nonlimiting examples of signaling moieties include chromophores, fiuorophores, and luminescent moieties. Dcrivaii/ed cholesterol comprising fiuorophores an? known in the art. See, e.g., Sparrow et al. (1999) and Jennings et al. (1999). Any iluorophore can be utilized in these compositions. Additionally, such fluorescent, hydroxycholesterols can utilize hydroxyeholesterol derivatives other than the 3- O-succinoyl derivative. For example, a hydroxyeholesterol ester-BODIPY dye, analogous to the cholesterol ester-BODIPY dye described in Jennings et al., 1 99 can be utilized. Further, besides the various positions along the ring structure of 24S-hydroxycholeslerol other than the 3ji-hydroxyi position can be derivatized with a variety of tags to enable the described assay formats, so long as this attachment does not interfere with the binding of the described antibodies to the 24-OH moiety. As an example, N,N-dimethyl amino naphthalene sulfonate (dansyl) hydroxyeholesterol can be utilized. Such a compound is analogous to the dansyl-cholesterol described in Mcintosh et al. (2008). Compare with the compounds described in U.S. Patent Application Publication US 2008/0177059, describing BODiPY-eholesterol derivatives where the BODIPY dye is conjugated to the 22 position of cholesterol. An analogous derivative of a hydroxyeholesterol would not bind to the anti-hydix>xycholesteroi antibodies described herein, since the antibodies arc designed to bind where the BODIPY dye would be conjugated in such compounds.

Another exemplary group for signaling purposes is biotin. Thus, in some embodiments, the compound further comprises a biotin conjugated to the carbox l group on the succinoyl moiety. Such a compound can also usefully comprise a linkage between the carboxy group, e.g., on the succinoy l moiety, and the biotin, to provide a space between the two groups, preventing steric hindrance of, e.g., a streplavidin-enzyme conjugate that binds to the biotin. The spacer can be of any length or construction known in the art. In some such embodiments, the compound is

Also provided herewith is a protein conjugated to any of the above derivatives, tor example the derivative 3«0-succinoyl-24S«hydroxycholesterol. The protein can be conjugated to the derivative using any of methods known in the art, for example as discussed above, e.g., glutaraldehyde, a maleimide reagent, a carbodiimide, or an activated ester, e.g., an N- hydroxysueeinimide ester. The protein can be any protein that can usefully be utilized with the derivative. For example, the protein can be an enzyme, e.g., as a signaling molecule in enzyme immunoassays. Nonlimiting examples include horseradish peroxidase and alkaline phosphatase. The protei can also be a fluorescent protein, e.g. tor the same purpose.

In other embodiments, the protein is a carrier protein for immunization of a vertebrate, to make antibodies against the derivative. Many such proteins are known in the art, e.g., keyhole limpet hemoeyanin, bovine serum albumin, ovalbumin, etc. In some embodiments, a

composition is provided comprising the above protein-derivative conjugate combined with an adjuvant that can improve an antibody immune response to the protein in a vertebrate injected with the composition. In certain specific embodiments the derivative is 3-0-suceinoyl-24S- hydroxycholesterol.

Further provided herewith is an antibody composition comprising antibodies that specifically bind to 22-hydroxychoiesterol, 24HC, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. In some embodiments, the antibodies specifically bind to 24HC ("241 IC antibodies"). In some of these embodiments, the 24HC antibodies have less thai 1% cross-reactivity to cholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 26- hydroxycholesterol, 27-hydroxycholesterol and DHKA when compared to the binding of the antibodies to 24MC In other embodiments, the antibodies have less than 0.2% cross-reactivity to 22-hydi ^, oxycbolesterol, cholesterol and DHEA when compared to the binding of the antibodies to 248-hydroxycholesterol. In additional embodiments, the antibodies have less than 0.01% cross-reactivity to cholesterol when compared to the binding of the antibodies to 24S- hydroxycholesterol .

These antibodies can be immunoglobulins of any vertebrate species, e.g., rabbit, goat, mouse, sheep, chicken, etc. and can be polyclonal or monoclonal. They can include the Fc region or they can be Fab or Fab2 fragments. Additionally, they can be from any source, e.g., from the serum of an animal injected with an immunogen such as any of the immunogens described above, or they can be from culture or ascites as is known in the art of hybridoma technology. Alternatively, the can be from recombinant sources, e.g., as described in Winter et al., 1 94, or Charlton and Porter, 2002.

A method of making 24HC antibodies is also provided. The method comprises: (a) preparing a 24HC immunogen by conjugating any of the above-described 24HC derivatives to a carrier protein; (b) immunizing a vertebrate with the immunogen under conditions such that the immune system of the vertebrate makes antibodies to 24HC; and (c) taking the 241-IC antibodies from the vertebrate. In some embodiments, the derivative is 3-0-succinoyl-24S- hydroxycholesierol. The carrier protein can he any of those discussed above. In some of these embodiments, the carrier protein is keyhole limpet heinocyanin, bovine serum albumin (BSA) or ovalbumin.

These methods are not narrowly limited to any particular set of vertebrates. In some embodiments, the vertebrate is a rabbit, a goat, a mouse, a chicken, or a sheep. Additionally, the antibodies taken from the vertebrate can be polyclonal antibodies or monoclonal antibodies.

The 24-iC antibodies described above can be utilized to detect 24IIC by any

immunoassay known in the art. Thus, additionally provided herein is a method of assaying for 24HC i a fluid or tissue sample from a mammal. The method comprises combining the sample with 24HC antibodies, then determinin whether the antibodies specifically bind to 24HC from the sample. In these methods, specific antibody binding to 24HC from the sample indicates that 24HC is present in the sample.

The sample can be from any fluid or tissue from the mammal. In some embodiments, the sample is from cerebrospinal fluid, blood (e.g., whole blood, serum or plasma), or brain tissue, since those samples are known to contain 24HC.

The sample can be from any mammal. In some embodiments, the sample is from a human, e.g., a human without, any apparent disease, or a human that has, or is suspected of having, cognitive impairment, Huntington's disease, Alzheimer's disease, or multiple sclerosis, which are known to affect the amount of 24HC in cerebrospinal fluid, plasma, and/or brain tissue.

Depending on the assay and sample, the sample can be used directly in the assay, for example after dilution in a butter, or the sample can be at least partly purified to eliminate substances that might interfere with the assay. Numerous such purification procedures are known in the art. For example, the sample could be sol vent-extracted or subjected to

chromatography, e.g., through a C18 column, before use in the assay. In some protocols, the eluate from the chromatographic procedure or extraction is dried and resuspended in a buffer that does not interfere in the assay. See, e.g., Axelson (1991). Such a drying and resuspension procedure can also be used to concentrate the sample if there is concern that the amount of the 24HC is below the level of immunoassay detection. These assay methods can comprise any immunoassay known in the art. I some embodiments, the assay is performed in a liquid phase. In other embodiments, the assay is performed on a solid phase, e.g., on a bead or a microplate, for example a 96 well microliter plate. Nonlimiting examples of immunoassays useful in these methods are a radioimmunoassay (see, e.g., U.S. Patent 4,081,525), a Luminex$ assay (see, e.g., Wong et al., 2008), a microarray assay, a fluorescence polarization immunoassay (see, e.g., U.S. Patent 4.585,862), an

immunoassay comprising a Fftrster resonance energy transfer (FRET) signaling system (see, e.g., B!omberg el al., 1999; Mayilo et a!., 2009), and enzyme immunoassay (a.k.a. enzyme linked immunosorbent assay (ELISA]). As is well known in the art, in ELISA, an enzyme combined with a substrate that becomes colored upon reaction with the enzyme provides the signal to quantify the antigen in the sample. See, e.g., O'Beime and Cooper, 1979.

Table 1 provides a summary of various immunoassays that can be utilized for detection of the hydroxychoiesterols to which antibodies are provided herewith. The general features of these assays are known in the art.

Any ELISA known in the art as useful for hapten detection can be utilized for the instant assays. ELISA for haptens generally utilize a competitive format, i.e., where the hapten (here, 24MC) in the sample competes with a labeled hapten (e.g., a biotin-hapten or enzyme-hapten conjugate) for anti-hapten antibody binding sites such that less labeled hapten is bound when there is more hapten in the sample. Thus, in these competitive assays, an increasing amount of hapten in the sample results in less enzyme bound to the solid phase, and consequently less colored signal. I such competitive assays, as defined herein, the sample can be added with the labeled hapten to compete directly for antibody binding sites, or the sample and labeled hapten can be added sequentially such that the labeled hapten simply binds where the sample hapten is not bound.

in some embodiments, the ELISA is a direct competitive EI.JSA, defined herein as where the 24HC hapten is directly bound to the signaling enz m , or an indirect competitive ELISA, where the enzyme is bound to another molecule, e.g., a second antibody, or streptavidin.

The ELISA assays provided herein can take any format known in the art. In some embodiments, a 24HC conjugate (e.g., 24HC-BSA or 24MC-polylysine) is bound to the solid phase. In. these assays, the 24HC antibody is added with the sample. Here, the 24_:1C in the sample competes with the solid phase-bound 24L1C conjugate for antibody binding sites such that less antibody binds to the solid phase when there is more 24HC in the sample. After washing, the amount of 241 IC antibody bound to the solid phase is measured, e.g., by utilizing in the competitive step a 24HC antibody-eir/yme conjugate, or by adding a second antibody- enzyme conjugate that binds to the bound 241IC antibody. A myriad of particular assays for 241 IC with this configuration can be devised without undue experimentation.

In other embodiments, the 241 IC antibodies arc bound to the solid phase, either directly or indirectly, the latter being where the solid phase is coated with an anti-antibody (tor example goat antibodies that bind to rabbit IgG antibodies (goat anti-rabbit IgG j) and the 24HC antibodies are bound to the anti-antibody. The ami -antibodies arc also known as "second antibodies". In these assays, the sample and labeled hapten is added to the solid phase to compete with antibody binding sites on the coated solid phase. After washing, the signal is generated, which measures the amount of labeled hapten that is bound to the solid phase. Numerous particular assays for 24I IC with this configuration can be devised without undue experimentation.

An illustration of the latter assay, where the 24HC antibodies are bound to the solid phase, is provided in the Example below. That assay comprises:

a. noncovalenily binding the second antibodies to the solid phase;

b. adding the 24S-hydroxycholestert>l antibodies to the solid phase under conditions such that the second antibodies specifically bind to the 24S-hydroxycholesterol antibodies;

c. adding the sample and a biol nylated 24S-hydroxycholesterol to the solid phase under conditions such that the biotinylated 24S-hydroxyeholesterol binds to binding sites on the 24S- hydroxycholesleroJ antibodies where 24S~hydroxycholesterol in the sample do not bind; d. adding an avidin-enzyme conjugate to the solid phase under conditions such that the avidin-enzyme conjugate specifically binds to the biotmylated 24S«hydroxycholesterol that bound in step c;

e. adding a substrate to the enzyme in the avidin-enzyme conjugate, wherein the substrate is converted to a colored product thai absorbs light at a specified wavelength in proportion to the amount of the enzyme bound to the solid phase;

f. measuring the absorbance of light at the specified wavelength, wherein the absorbance is inversely proportional to the amount of 24S-hydroxycholesterol present in the sample.

^" With any of the above-described immunoassays, 24MC in the sample is quantified by comparing the signal generated (e.g., fluorescence in the FRET and fluorescence polarization assays, radioactivity in radioimmunoassays, absorbance in KLISAs) in the assay where the sample is added to the signal generated in assays where a known amount of 24HC is added, i.e., comparing the sample signal to a standard curve.

The present invention is also directed to a kit for detecting and quantifying 24HC. The kit comprises 241 fC antibodies. In some embodiments, the kit further comprises a protein conjugate or a biotin conjugate of 24S-hydroxycholesterol. In other embodiments, the kit further comprises a solid phase, e.g., beads or a microtiter plate. In these embodiments, the solid phase comprises a noncovalently bound protein, where the bound protein is an antibody or a protein conjugate of 24S-hydroxycholesterol. In various embodiments, the kit further comprises an enzyme. Examples of such enzymes are alkaline phosphatase or horseradish peroxidase, covalentiy bound to 2411C or an antibody (e.g., a 24H antibody or a second antibody). In additional embodiments, the kit further comprises 24HC, for example for use in a standard curve.

As discussed in the Background section of this application, 24HC is a substrate for various eytosolic sullo.ransierases (SUI..T) and UDP-glueuronosy! transferases (IJGT). Sulfation involves the transfer of the sulfonate group of the obligate sulfonate donor 3'-phosphoadcnosine- S'-phosphosulfate (PAPS) to an acceptor compound, usually possessing a hydroxy! group, to form a sulfate ester. Typically, f ³⁵ S)PAPS is employed in the SULT assay. The assay is often terminated by chloroform extraction and the sulfated products in the aqueous phase are resolved by thin layer chromatography (TLC) on silica gel TLC plates and detected by autoradiography. However, to define the sites of sulfate conjugation, an LC- S/MS approach is usually required. Nonradioactive reactions can be run in parallel with reactions containing ^3S S |PAPS and monitored by TLC. The non-radioactive reaction* are extracted with chloroform and the water phase loaded onto a Sep-Pak cartridge, washed with water and eluted with MeOH. The elnates can be concentrated by evaporation under a stream of Nj. Identification of the sulfated compounds is by HPLC-mass spectroscopy using, for example, a Sciex API-4000 Triple Qiiadrnpole mass spectrometer with two Perkin Elmer series 200 micropumps (Perkin Elmer Life and Analytical Sciences, Boston, MA) and a Synergi Fusion (100 x 2 mm I ) analytical C- 1 column with a C-18 guard column (Phenomenex).

In an analogous manner, die glucuronidation reaction can be monitored as the transfer of the glucuronosyl group from UDP[U- ^l4 C] glucuronic acid to the target substrate. HPLC on a polar amino-eyano bonded phase colum is employed to separate radioactive ghieuronides from unmetabo!ked UDP[U- ^,4 C]glucuronic acid (Coughlrie et al, 1 86).

The immunoassays described herein can be utilized to detect activity of DOT and SULT or any other enzymes that alter a hydroxycholesterol such that it no longer binds to die antibodies described herein. For example, the extracerebral double conjugation of 24-OH cholesterol to generate (24S)~24-hydroxycholesterol 3-suifafe, 24~glucuronide can be analyzed by

immunoassay using the immunoassays of the present invention in combination with, e,g., immunoassays selective lor sulfation, or alternatively in combination with techniques allowing the selective separation of the sulfated form of the analyte from the nonsul fated form. For example, approaches that allow the selective precipitation of the sulfated form of the analyte, as described in US. Patent Application 12/806,950, filed August 24, 2010, would facilitate the analysis. Such an approach eliminates the need for the time-consuming analysis of UGT and SULT activity now utilized, as described above. The potential for high throughput analysis as can be employed with the immunoassays described herein, as well as the high sensitivity of the immunoassays, provides opportunities to utilize these immunoassays to monitor phase II drug metabolism assays and screen drugs and new chemical entities for their capacity to modulate phase II enzyme activity in native or recombinant fractions.

Thus, the present invention is also directed to a method of detecting an enzyme or enzymes utilized in phase 11 drug metabolism in a sample, where the enzyme or enzymes are capable of altering 22-hydroxycholcsteroI, 24S-hydr xycholesterol, 25-hydroxycholesterol, 26- hydroxycholesterol or 27-hydroxycholesterol. The method comprises combining the sample with 22-hydroxycholesterol, 24S-hydroxychoIesterol, 25-hydroxyehoIesteroI, 26- hydroxycholesterol or 27-hydroxycholesterol for a time sufficient for the enzyme or enzymes to modify the 22-hydroxycholeslerol, 24S-hydroxycholesterol, 25-hydmxycholesterol, 26- hydroxychoiesterol or 27-hydroxycholesierol, then assaying the sample using any of the above- described immunoassays, in these methods, a reduction in the amount of the 22- hydroxycholesterol, 24S-hydroxyeholesterol, 25-hydroxycholesterol, 26-hydroxyeholesterol or 27-hydroxyeholesterol detected by the assay indicates that, the sample comprises the enzyme or enzymes.

In some embodiments of these methods, the enzyme is a cytosolie sulfotransferase, a U P-glucuronosyliran«ierase, or a combination thereof. In certain specific embodiments, the enzyme or enzymes are capable of altering 24S-hydroxycho1esterol.

These methods can also be utilized to detenmne whether a compound is eapable of modulating the enzyme or enzymes, or reducing their expression (tire latter case where the sample includes cells that produce the enzyme or enzymes). In these embodiments, the compound is added to the sample with the enzyme or enzymes. If the compound modulates the enzyme or enzymes, the difference in the amount of the 22-hydroxycholesterol, 24S- hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27«hydroxycholesterol detected by the immunoassay when the compound is included vs. when the compound is excluded indicates that the compound modulates the enzyme. For example, if the compound inhibited or reduced the expression of the enzyme or enzymes, less of the hydroxychoiesterol would be altered, and the immunoassay would show more of the hydroxychoiesterol present than when the compound was not included with the enzyme or enzymes. Conversely, if the compound enhanced the activity, or increased expression of the enzyme or enzymes, more of the hydroxychoiesterol would be altered, and the immunoassay would show less of the

hydroxychoiesterol present than when the compound was not included with the enzyme or enzymes.

In a specific embodiment, inhibitor screening for UGT and SUL can be accomplished using a competiti ve binding assay format. A decrease in the formation of 24-OH cholesterol metabolite compared to the vehicle control is used to calculate an C50 value of the test agent. For example, a UOT isozyme inhibition assay can be performed as follows: The UOT substrate, 24-OH cholesterol, is incubated with a cDNA-expressed human UGT isoform, such as UGTIAI, as well as with UDP-glucuronic acid (UDPGA), and a range of lest compound concentrations (typicall 0.4 - ΙΟΟμ ) for 30 mint at 37°C. Optionally, a microsomal preparation can also be incorporated to enhance enzyme activity. Due to the luminal location of the UGT's within the endoplasmic reticulum of microsomal preparations, the passage of the water soluble cefaclor UDPGA to the active site can be challenging. In order to circumvent this latency phenomenon, the pore forming agent, alamethicin, can be included to improve access to the active site. At the end of the incubation period, the formation of the metabolite, 24-OH cholesterol 3-glueuronide is monitored by immunoassay, using antibodies of the present invention, at each of the test compound concentrations. A decrease in the formation of the metabolite compered to vehicle control is used to calculate an IC50 value of the inhibitor (test compound concentration which produces 50% inhibition of UG'FIAI ). The selective UGT1A1 inhibitor, silybin, can be used as the positive control inhibitor in the described assay.

These methods can be further utilized to quantify the enzyme or enzymes, e.g., by comparing the reduction in the amount of the 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesierol, 26-hydroxycholesterol or 27-hydroxycholesterol detected by the assay in the sample with the reduction in the amount of 22-hydroxycholesterol, 24S-hydroxychole«terol, 25-hydmxycholesterol, 26-hydroxycholesterol or 27-bydroxyeholesterol present when treated by a standard amount of the enzyme or enzymes.

The above-described immunoassays can also be utilized in a method to detect an enzyme that synthesizes 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hycuOxycholesterol, 26- hydroxycholesterol or 27-hydroxycholesterol in a sample. The method comprises combining the sample with cholesterol for a time sufficient for the enzyme to modify the cholesterol, then assaying the sample using any of the above-described immunoassays. In these methods, an increase in the amount of 22-hydroxycholesterol, 24S-hydroxycholesterol, 25- hydroxychoJesterol, 26-hydroxycholesterol or 27-hydroxycholesterol detected by the assay indicates that the sample comprises the enzyme. In some embodiments of these methods, the enzyme is CYP46 and the assay detects 24S-hydroxycholesterol.

In certain, embodiments of these methods, the enzyme in the sample is quantified by comparing the increase in the amount 22-hydroxycholesterol, 24S-hydroxycholesterol, 25- hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol detected by the assay in the sample with the increase in the amount of 22-hydroxycholesterol, 24S-hydroxycholesterol, 25- hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol present when cholesterol is treated by a standard amount of the enzyme.

Since reductions in plasma 24MC levels are associated with certain diseases, in particular multiple sclerosis and Huntington's disease (see Background above), the immunoassays described herein can be used to monitor the progression and efficacy of treatment for those diseases.

Thus, in some embodiments, a method of evaluating progression of multiple sclerosis in a patient is provided. The method comprises measuring 24S-hydroxycholesterol in blood plasma or serum of the patient over time by the above-described assay method, in this method, progression of multiple sclerosis is indicated by decreasing levels of 24S-hydroxyeholesterol in the blood plasma or serum over time.

Also provided is a method of determining whether a treatment for multiple sclerosis in a patient is effective is also provided. Tire method comprises measuring 24S-hydroxyeholesterol in blood plasma or serum of the patient over time by the above-described assay method. I n this method, the treatment is effective if the 24S-hydroxycholesterol levels do not decrease over time.

A method of evaluating progression of Huntington's disease in a patient is also provided. The method comprises measuring 24S-hydroxyeholesterol in blood plasma or serum of the patient over time by the above-described assay method, in this method, progression of

Huntington's disease is indicated by decreasing levels of 24S-hydroxycholesteroI in the blood plasma or scrum over time.

Additionally provided is a method of determining whether a treatment for Huntington's disease in a patient is effective is additionally provided. The method comprises measuring 24S- hydroxycholesterol in blood plasma or serum of the patient over time by the above-described assay method. In this method, the treatment is effective if the 24S-hydroxycholesterol levels do not decrease over time.

Preferred embodiments are described in the following example. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples. firaUfflrffr flrcWffftfrHI tlftWffirft 24S iYdroxvc p.estgyq. m tt m m liil y detection of same

Since 24S-hydroxychoiesterol (24HC) is a hapten that is too small to illicit an antibody immune response by itself, it must be covalently coupled to a carrier protein, which, when injected in a vertebrate, presents 24HC to the vertebrate's immune system as an epitope on the protein, .such that antibodies are generated to the 24HC epitope. However, 24HC does not have a moiety to which it could be conveniently conjugated to a carrier protein. Therefore., the 3-0- succinoyl derivative of 24HC was prepared as follows:

Preparation of 3-0-Succinoyl-24S-hydroxycholesterol

To 24HC (2lmg [0.052 mrnolj) in 3 mL dry pyridine was added succinic anhydride (l mg (0.10 mmol]). The mixture was heated under Ar at 80° for 3 days. The reaction was then diluted with ethyl acetate and washed with 1 M I1CI and brine. Flash chromatography (2% MeOH/CH2G2) yielded 12 mg (46%) of product. This scheme is illustrated below:

An activated ester of 3-0-8uccinoyl-24S-hydroxycholcsierol, 3-0-succinoyl-24S- hydroxyeholesterol N-hydroxysuccimmide ester, was then prepared as follows:

Preparation of 3-0~Soccinoyl-24(SV-h droxycholestero} N-hydroxysuccinimide ester

To 3-0-Succinoyl-24(S)-hydroxycholesterol (9,2mg 10.018 mmoTJ) and N- hydroxysuccinimide (NHS) (2.3g [20mmo1|) in 2 ml. dr IMF was added l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (3.9mg (O.020mmol]). 'The reaction mixture was stirred overnight at room temperature- It was then diluted with diethyl ether and washed with water and brine. Flash chromatography (100% H2CI2) gave 4.7mg (43%) of the desired product. This scheme is illustrated below:

The 3-0-Sueeinoyl-24S-hydroxycholesterol NHS ester was then conjugated to the carrier protein keyhole limpet hemocyanin (KLH) as follows:

Preparation of3-0»Succ¾noyl«24S*hydroxycholesterol»KIJ-.

1. Ten mg of KLH lyophilized in 0.1 M sodium phosphate, 0.15 M NaCl; pH 7.2 (Pierce product # 77600) was resuspended in 1 ml, water.

2. Four rng of the 3-0-suecinoyl-24S-hydroxytな>lesterol NHS ester was resuspended in 100 μΐ of dimethyl sulfoxide.

3. The 3-0-succ½oyi-24S-hydroxycholesterol NHS ester solution was added dropwise to the KLH solution 10 μΐ. at a time. After 70 μΙ.< was added, a precipitate was observed and 0.5 ml. of H2O was added to clear the solution before addin the rema inder of the NHS ester.

4. The solution was gently mixed on a tilting platform mixer tor 1 hour at room temperature and then transferred to a dialysis bag (1 kD pore size). The solution was diarysed for 4.5 hours at room temperature against 1 liter 0.1 M sodium phosphate, 0.15 M NaC!; pH 7.2. The dialysis buffer was changed and dialysis continued overnight. The dialysis buffer was changed one more time and dialysis was completed after an additional 6.5 hours.

5. 'Hie final solution was brought to 10 ml, .final volume and aliquoted.

This 3-0-Succinoyl-24S-hydroxycholesterol- LH conjugate was used as an immunogen to elicit antibodies in rabbits by the following protocol: immunization protocol

1. Female New Zealand White rabbits were immunized intradermally with 500 μg of 24S-hydroxycholesterol - KLH conjugate in PBS. The Immunogen with complete adjuvant was prepared by mixing 0.5 mL of I mg mL hydroxycholesterol - KLI I conjugate with 0.5 mL Freund's complete adjuvant and emulsifying by passage through glass syringes.

2. Prebleeds were collected 1 day prior to the first immunization.

3. Rabbits were injected I mL of immunogen intradermally over 10 sites on day 1.

4. Immunogen with incomplete adjuvant was prepared by mixing 0.5 mL of 1 mg mL 24S-hydroxycholesterol - KLH conjugate with 0.5 mL Preund's incomplete adjuvant and emulsifying by passage through glass syringes.

5. Rabbits were injected with 1 mL of this immunogen intradermally over 10 sites on day 21 , 42 and 63.

6. Rabbits were bled on Day 74 and Day 77.

7. Rabbits were injected with 1 ml- of immunogen with incomplete adjuvant (described in 4. above) intradermally over 10 sites on day 84.

8. Rabbits were bled on Day 95.

1 1. Rabbits were injected with 1 mL of immunogen with incomplete adjuvant intradermally over 10 sites on day 105.

12. Rabbits were bled on Day 1 1 .

14, Rabbits were injected with 1 ml., of immunogen with incomplete adjuvant intradermally over 10 sites on day 126.

15. Rabbits were bled on Day 136 and 138.

I some cases, the antisera was screened in an ELISA using 96 well mierotiter plates coated with a 3-0-succinoyl-24S-hydroxycholesterol linked to bovine serum albumin (BSA), prepared by conjugating the 3-0-succtnoyl-24S-hydroxychoIesterol HS ester described above to BSA by the method described above with KLH. This screening protocol is as follows:

Antisera screening assay using 24S*hvdroxycholestcrol - BSA

1. 100 uL of 24(S)l:.ydroxycholesterol - BSA conjugate was coated to plate at a concentration of I pg/mL in lOm sodium phosphate buffer pH 8.0 overnight at 4° C. Plates were blocked for 1 hour in lOmM sodium phosphate, 150 mM sodium chloride, 1% BSA butter. 2. Rabbit anfisera was serially diluted in 100 mM phosphate, 150 mM NaCl, pM 7.2 assay buffer and 100 μΐ.. of each dilution was transferred to individual wells on a 96 well plate. The plate was incubated at room temperature for 1 h with orbital shaking.

3. The plates were washed 4 times with 300 pL of 50 mM Tris, 100 mM NaCl, 0.05% Tween-20 butter.

4. Cioat anti-rabbit "horseradish peroxidase (H P) conjugate was diluted to 200 ng/m.L and 100 μΐ, was added to each well and incubated for 1 hour at room temperature.

5. The plate was washed 4 times with 300 μΐ, of 50 mM Tris, 100 mM NaCl, 0.05% Tween-20 butter to remove excess H !* conjugate. 100 μ£. of 1MB substrate solution was added to each well. The plates were incubated at room temperature for 30 min. An BRP- catalyzed reaction generates a blue color in the solution.

6. After color development, 50 uL of I HCl stop solution is added to each well to stop the substrate reaction. The HCl solution converts the blue color to yellow. The resulting yellow color was read at 450 nra. The intensity of yellow color (read as absorbance at 450 nm) is directly proportional to the amount of a«ti-24S-hydroxycholestetx l antibody in the in the sample.

In other cases, the antisera were screened using an assay that employs a biotinylated 3-0- succinoyl-24(S)-hydroxycholesterol _> which was prepared as follows:

Preparation of biotinylated 3-0-succinoyl-24S-hvdroxycholesteroi

To 3-0-Succinoyl-24S-hydroxychoJesterol N-hydroxysucemimide ester (4.7mg

[?.8μηκ>}}) in ImL dry CJfcCfe was atlded biotinyl-3, , 9-trioxaundecanediamine (3.3mg | ^' 7.8μηιοΙ)). After stirring overnight at room temperature, the mixture was subjected to flash chromatography (1 % MeOH/CHaCb) to yield 4.4mg (62%) of product. This scheme is illustrated below:

The screening assay using the biotinylated 3-0~succinoyl«24S-hydroxychoIesteroi reagent was as follows:

Antisera screening assay using biotinylated 24I1C

1. A microliter plate ( well) was coated with goat anti-rabbit IgG antibody. Rabbit antiserum to 24HC (100 μΐ), serially diluted in 1 0 m phosphate, 150 mM NaC!, pH 7.2 assay buffer, was added to 100 μϊ.. biotinylated 24HC diluted in the same buffer before adding to the microliter plate wells coated with a goat anti-rabbit IgG antibody. The plate was incubated at room temperature for 1 hour with orbital shaking. During this incubation the antibody binds the biotinylated 24S~hydroxycholesterol and becomes bound to the goat anti-Rabbit IgG coated on the plate surface.

2. The plate is washed 4 times with 300 uL of SO mM. Tris, 100 mM NaCl, 0.05% Tween-20 buffer. After washing only bound 24S-hydroxycholesteroI or bound biotinylated 24S- hydroxycholesterol remains.

3. Slrepuividin conjugated to horseradish peroxidase was diluted to working

concentration in 100 mM phosphate, 150 mM NaCl, pH 7.2 assay buffer and 200 μΐ was added to each well to allow detection of the bound biotinylated 24S-hydroxycholesteroL The plate was incubated at room temperature for 30 minutes. 4. The plate was washed 4 times with 300 μΐ., of 50 mM Tris, 100 mM NaCl, 0.05% Tween-20 buffer to remove excess MRP conjugate. TMB substrate solution (200 μϊ.) is added to each well The plates were incubated at room temperature for 30 min.

5. After color development, 50 uL of 1 N HCl stop solution is added to each well to stop the substrate reaction. The resulting yellow color was read at 450 nm. The intensity of yellow color is directly proportional to the amount of anti-24S-hydroxycholest.erol antibody in the in the sample.

The following protocol was used to detect varying amounts of 24MC to create a standard curve for 24HC:

24HC Enzyme Immunoassay Protocol

1. Known concentrations of 241 IC were prepared in KM) mM phosphate, 150 mM NaCl, 1% BSA, pH 7.2 assay buffer containing biotinylated 241 IS (described above) and added to wells of a % well microtitcr plate that were coated with a goat anti-rabbit IgG antibody. Rabbit polyclonal antibody to 24HC was diluted to a limiting concentration in 100 mM Tris, 150 mM NaCl, 1% BSA buffer and 300 μΐ was added to each well. The plate was incubated at room temperature for 1 hour with orbital shaking. During this incubation the anti-241 IC antibody binds to the goat anti-rabbit IgO antibody on the plate. Also, the 24I1C standard competes with the biotinylated 241 IC for anti-241 IC antibody binding sites. Thus, with more 24HC standard, less biotinylated 24HC binds to the antibody binding sites on the plate.

2. Following this incubation, the plate was washed 4 times with 300 μΐ. of 50 mM Tris, 00 mM NaCl, 0.05% Tween-20 buffer. After washing, bound 24HC or bound biotinylated 24I1C is bound to the binding sites on the antt-24HC antibodies.

3. Sireptavidin conjugated to HRP (200 \L), diluted in 100 mM phosphate, 150 mM NaCl, 1% BSA, pH 7.2 assay buffer, was then added to each well to allow detection of the biotinylated 24HC. The plate was then incubated at room temperature for 30 minutes.

4. The plate was then washed 4 times with 300 μΐ.. of 50 mM Tris, 100 mM NaCl, 0.05% Tween-20 buffer to remove excess streptavidin-HRP conjugate. TMB substrate solution (200 μΐ,) is added to each well. The plates were incubated at room temperature for 30 min.

5. After color development, 50 μΐ. of I HCl stop solution is added to each well to stop the substrate reaction. The resulting yellow color was read at 450 nm. The resulting yellow color was read at 450 nm. The intensity of yellow color is inversely proportional to the concentration of 24HC that was added to the well. A typical standard curve for this assay is shown in FIG, 1 , The multiple regression line shown therein has an " of 0.096. The detectable range lor 24HC for this assay is as shown is 1.56 ··· lOOng/mL. The total assay time is 2 h.

Cross-reactivity of the antisera tested above (Day 77 bleed, rabbit #72) to the following structurally similar molecules was determined:

Results of thai cross-reactivity study are shown in Table 2:

This study shows that the cross-reactivity to these structurally similar compounds is minimal.

Cross-reactivities of antisera taken from another rabbit at Day 77 (rabbit #71), subjected to the same immunization protocol, as well as repeat of the rabbit #72 antisera, were also determined. The results are shown in Table 3.

As with the assay described in Table 2, the cross-reactivities of structurally simi lar compounds wit these two antisera were minimal. The small differences in cross-reactivities described in Table 1 and the "Bleed #72" data in Table 3 represent variation i the assay.

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in view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

AH references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by ihe authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accurac and pertinence of the ci ed references.