BACKGROUND OF THE INVENTION

Sterols, steroids and steroid-like compounds share a common structure and biosynthetic pathway. The distinctive feature of these compounds is a four-membered polycyclic ring structure which in nature is synthesized from triterpenoids, and initially arises from the condensation of 2,3-oxido-squalene by the enzyme lanosterol-cyclase (EC 5.4.99.7) in the biosynthesis of cholesterol. Sterols such as cholesterol are the precursors to steroids and steroid-like compounds and undergo enzymatic oxidative metabolism and methyl group removal to fcrm specific families of steroids.

The polycyclic ring structure common to all sterols, steroids, and steroid-like compounds may be represented by the following chemical structure:

The rings may have various substituents, particularly methyl groups, hydroxyl groups, oxocarbonyl groups, ether groups, and a ino groups. For the most part, the steroids of interest will have at least one, usually 1 to 6, more usually 1 to 4 oxygen functionalities such as alcohols, ether, ester, or keto. The steroids will

usually have from 18 to 27 carbon atoms, or as a glycoside derivative up to 50 carbon atoms.

The steroids find use as male and female sex hormones, adrenocortical hormones, gestogens, bile acids, cardiotonic glycosides, as well as saponins and sapogenins. The sex hormones may be divided into two groups: the male steroid hormones, or androgens; and the female steroid hormones, or estrogens. Illustrative androgens include testosterone, androsterone, isoandrosterone, etiocholanolone, and methyl testosterone. Illustrative estrogens include β- estradiol, estrone, estriol, 2-hydroxyestrone, 2- methoxyestrone, equilenin, and α-ethinyl-estradiol. Illustrative adrenocortical steroids (glucocorticoids and mineral corticoids) include 17- hydroxydiox corticosterone, deoxycorticosterone, cortisone, corticosterone, 11-dehydrocorticosterone, cortisol, aldosterone, and prednisolone. Illustrative gestogens include progesterone, pregnenolone, allopregnane-3α:20α-diol, and allopregnan-3α-ol-20-one.

While substitution of the 4-membered ring structure with different functional groups such as hydroxyls, ketones, acids, esters, and hydrocarbon chains alters the biological properties of the steroid or sterol-like compounds, their physical and chemical properties remains defined by the 4-membered ring. For example, derivatives containing this structure are lipid-like, hydrophobic, and sparingly soluble in aqueous solutions.

The physical-chemical properties engendered by this common 4-membered sterol nucleus, are also responsible for the distinct biological activity and function of these compounds. Since these compounds are of limited solubility in physiological fluids, specific mechanisms and carrier proteins have evolved to facilitate transport

and control the biological activity of the compounds. For example, cholesterol is transported in association with several serum proteins, apolipoprotein A and apolipoprotein B. In humans, cortisol is bound to two naturally occurring serum proteins known as

Corticosteroid Binding Globulin (CBG) or transcortin. The sex hormone steroids, androgens, progestins, and estrogens circulate in a bound form with Sex Hormone Binding Globulin (SHBG) . These compounds will also bind non-specifically, but very weakly, to serum albumins.

The concentration of carrier proteins determines the biological activity of the transported compound or hormone by controlling the amount of "free" or biologically available compound or hormone. Only the "free" or bioavailable fraction can interact with cellular receptors to exert the compound's or hormonal effect. The bioavailable fraction is usually only a few percent of the total hormone concentration, but in certain natural conditions such as pregnancy, exposure to some drugs, or in certain pathological states, the bioavailable fraction can be drastically altered.

Investigators have recognized the clinical significance of the bioavailable fraction of circulating steroid hormones and have measured levels of the sex hormone binding globulin SHBG, or the ratio of total hormone to binding protein to determine a "free hormone index" ([hormone]/[SHBG]) . Immunodiagnostic assays have also been recently developed to indirectly measure the free steroid hormone levels.

With respect to indirect immunological methods now commonly in use, U.S. Patent No. 4,366,143 to Midgley et al. discloses a method to determine the free portion of a ligand in biological fluids by mixing a sample of the fluid with a lableled derivative of the ligand and a

specific binder for the ligand, incubating the mixture, and determining the portion of the labeled derivative bound to the specific binder. Also, U.S. Patent No. 4,311,690 to Buehler et al. discloses a test set and method for the determination of free hormone, wherein the sample is incubated with a specific antibody to the hormone and a distinguishable analogue of the hormone, both of which are separated from the sample by a semipermeable membrane capable of excluding passage of natural protein, but which allows passage of hormone and analogue. The amount of analogue present either in association with the antibody or in the reaction system is indicative of the level of free hormone in the sample. Several other European patent applications disclose methods for measuring free ligands in biological fluids by competitive binding of analyte with a labeled analogue or analogue: enzyme conjugate. Further examples include European Applications EP 218309 A2, April 15, 1987 (A.S. El Shami) ; and EP 8980 Al, Sept 28, 1983 ( J.C. Charlton, J.E. Midgley, and T.A. Wilkins) .

In actual practice, however, the indirect methods based upon competition in the presence of a specific binder or antibody result in only relative estimates of free ligand levels because the specific binder or antibody disturbs the equilibrium between the free and protein-bound ligand, particularly between the free and weakly bound albumin-bound fraction. These indirect estimates of the amount of bioavailable hormone, moreover, have not provided significant improvement over measurement of the total hormone concentration. For example, investigators compared the diagnostic value of information given by total testosterone (I) , free testosterone by indirect competitive radioligand binding (II) , free testosterone index (testosterone/SHBG) (III) , and albumin-bound testosterone (ABT) by a direct precipitation method (IV) in a group of hirsute and normal women (Loric et al.,

Clin. Ch - », 34.: 1826-1829 (1988)). Values for I, II, and III v;ere all significantly (P<0.01) higher in the hirsute group, but there was overlap in values between the groups.

By contrast, the direct measurement of bioavailable testosterone as albumin-bound testosterone found a more significant (P<0.001) difference between groups with no overlap. This finding led the authors to suggest that IV is the preferred discriminator, and could be used to diagnose hi sutism with a high predictive value.

Previous indirect methods generally are also unable to recognize o ¹ account for the two distinct pools of bioavailablw steroid, that is, non-protein-bound steroid (%NPB steroid) and ali;min-bound steroid (%AB steroid) . Methods to determine the albumin-ioound fraction of steroids by specific chemical precipitation methods have been described previously. Relevant literature includes: Rosner, J. Clin. Endocrinol. Metab. , 34: 983-988 (1972) ; and, O'Connor et al., J. Steroid Biochem.. 4.: 331-339 (1973) .

With respect to determining %NPB steroid, numerous methods have been used to separate bound and free steroids, including equilibrium dialysis (Kley et al. , Acta Endocrinol., 85: 209-219 (1977)); flow dialysis (Moll et al., Biochem. Med.. 18: 344-352 (1977)); zonal chromatography (DeMoor et al., J. Clin Invest.. 41: 816- 821 (1962)); steady state gel filtration (Greenstein et al.. Steroids. 30i 331-341 (1977)); and ultrafiltration through a dialysis membrane (Plager et al., J. Clin. Invest.. 43: 1066-1072 (1964)). Although qualitatively similar results may be obtained by several of these methods, each has one or more drawbacks such as large sample requirement, and variable increases in sample volume during the procedure. Moreover, alterations in

the composition and concentration of low molecular weight constituents relative to serum are inherent in most of the above methods. Such changes would alter important low affinity interactions beween the binding proteins and ligand being studied. As will be discussed below, these methods are also inherently limited in their accuracy and reproducibility due to one significant property of steroids, that is, their inherent tendency to adsorb to nearly any surface including glass, plastics, and natural and synthetic fibers.

A relatively new method which overcomes many of the previous limitations is the determination of free steroid in undiluted serum by centrifugal ultrafiltration- dialysis (Hammond et al. , J. Biol. Chem. , 255: 5023-5026 (1981)). For example, this method requires less than 0.5 ml of serum, no dilution or change in sample composition occurs, and all surfaces in contact with steroid were specially treated glass or were used in such a manner that any binding of steroid could be taken into account. This "home-made" device, however, is extremely difficult to construct and use.

Since the introduction of this method, several commercial centrifugal ultrafiltration devices have become available, devices which emulate the centrifugal ultrafiltration device of Hammond et al. described above. These devices potentially provide many of the features necessary to overcome previous limitations to accurate, reproducible determination of the fraction of free ligand in serum. These commercial devices, however, are composed of plastics such as polystyrene, polypropylene, polyethylene, and styrene-acrylonitrile. Unfortunately, these devices are limited by the fact that all steroid molecules will bind to these plastics to an extent sufficient to seriously compromise their use for accurately determining the levels of free steroids.

SUMMARY OF THE INVENTION

The present invention relates to a test kit for the determination of analytes in biological fluids. More particularly, this invention concerns a test kit for the determination of analytes in biological fluids, wherein said analytes are selected from steroids and sterol-like compounds and are disposed in a solution containing at least one protein, said test kit comprising the following compo; mts:

A. a generally cylindrical filter support housing having a filter means disposed at one end thereof, and an opening at the opposite end thereof;

B. a receptacle for said filter support housing with releasable closure means adapted to engage said filter support housing at said opening;

C. a receptacle with releasabl-t closure means for tne precipitation of said protein; and

D. a plurality of disposable pipette tips; wherein the surfaces of said components are non- adherent to said steroids, said sterol-like compounds and said protein.

Accordingly, it is a principal object of the present invention to provide a useful test kit for the analysis of steroids and sterol-like compounds that avoids the disadvantages and inaccuracies of the prior art methods.

It is a further object of the invention to provide a test kit as aforesaid that has surfaces that are non-adherent to said steroids and sterol-like compounds whereby the binding of these compounds is inhibited and highly accurate analyses may be achieved.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description which proceeds with

reference to the following illustrative drawing.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE is a graph presenting the results of the performance of an assay of blood serum samples for the purpose of measuring %NPBE and %ABE, wherein the test kit and corresponding assay are prepared and performed in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

It has been found that a test kit providing reproducible, accurate results for the determination of analytes in biological fluids, which analytes comprise steroids and sterol-like compounds disposed in solution containing at least one protein, can be produced by rendering the surfaces of the components of the test kit non-adherent by the application thereto or inclusion thereof, of a lubricant that is non-reactive with said analytes, said protein and any of the reagents as may be used with said test kit.

Any loss of steroid to a receptacle surface is likely to introduce a significant underestimate of non-protein- bound estradiol. Proteins in physiological fluids also bind to plastics, and since steroids also bind to some of these proteins, the steroids may be lost due to binding of the protein to the device. Therefore, to measure the bioavailable fraction of a steroid accurately all surfaces that come into contact with the sample must be non-adherent for steroids and proteins. This would include any devices used to transfer the sample and experimental fractions or products of said sample.

Typical plastic components used in the test kits of the present invention are of various shapes and sizes, and

include reception cups, pipettes, cuvettes, test tubes, and the like.

The non-adherent surface characteristics can be imparted to the surfaces of the kit components in a variety of ways.

For example, silicones (polydimethylsiloxanes) are well known for their ability to lubricate and render non- adherent to a variety of compounds. Although such treatment is not generally thought to be effective for plastic surfaces, the efficiency in rendering plastic surfaces non-adherent for steroid and protein molecules was investigated in accordance herewith.

A variety of silicone and silane coatings were evaluated as to their ability to prevent binding of ³ H-estradiol and ³ H-estradiol in protein solutions. All silicone and silane coatings were effective in reducing the binding of estradiol to plastics, but some were more effective than others. A preferred treatment was coating of the plastic surfaces, for example a polypropylene receptacle, with dichlorooctamethylsilane in heptane. Such treatment rendered polypropylene pipette tips non-adherent to ³ H- estradiol and protein.

Illustrative procedures and results are set forth in greater detail in Table I, below. Specifically, polypropylene disposable pipette tips were treated with dichlorooctamethylsilane. Aliquots of 50 microliters of PBS containing ³ H-estradiol were drawn up into the pipette tips and completely ejected. The tips were washed 4 times with 100 microliters of PBS, and then subjected to scintillation counting. The fraction bound was determined from the ratio of counts tritium bound to total counts aliquoted X 100. This method underestimated the actual fraction of estradiol bound. The results are

as follows.

TABLE I ESTRADIOL BINDING TO PIPETTE TIPS: EFFECT OF SILANIZATION

VENDOR FRACTION BOUND (%) Untreated Treated

Sigma Chemical Company 1.22

Cole-Parmer 2.63 0.16

Elkay 0.99 0.06

Eppendorf 0.70 0.06

Thus, silicone coatings were found to be effective in reducing steroid hormone adsorption to components of the test kit.

A further method for providing a non-adherent surface is by compounding of the silicone into the plastic resin before molding. During the thermoplastic resin molding process, silicone leaches to the surface of the molded part forming a very thin silicone film. A preferred compounding formula is to include greater than 200 parts per million (ppm) , and preferably 1000-1500 ppm, Dow

Corning 200® silicone fluid mold release agent prior to molding.

A still further method for providing a non-adherent surface is by gas plasma treatment of the surfaces of the components. Adhesion of a specific molecule to a surface depends upon the surface chemistry. A relatively new technology known as cold gas plasma surface treatment provides an efficient, economical and versatile method of modifying surface chemistry. Through plasma processing, it is possible to re-engineer the surface chemistry and corresponding properties of any polymer surface.

Plasma3is a partially ionized gas containing ions, electrons, and various neutral species at many different levels of excitement. Free electrons gain energy from an imposed high energy electric field for example, as may be imposed by radio frequency radiation, wherein the transfer of energy results in formation of free radicals, ions, and atoms. These particles interact with solid surfaces placed in the plasma leading to drastic modifications of the molecular structure and properties of the surfaces. Competing molecular reactions alter the surface of a polymer simultaneously, the extent of each depending upon the surface chemistry and plasma process variables.

The most dramatic and widely used effect of plasma treatment is the surface modification of polymers to create chemical groups on the surface capable of interacting with adhesives. See, Hook, T.J. , et al., J. Mater. Res. , 2 : 132 (1987); Everhardt, P.S., et al., Anal. Chem. _r 53: 665 (1981); Yasuda, H. , et al. J. Polv. Sci.. 15: 991 (1977) . The inherently low surface energy of untreated polymers hinders the wetting and interaction with adhesive systems. Typically, plasma treatment is used to add polar functional groups which increase the surface energy of polymers.

One of the most common plasma processes is treatment in a cold gas oxygen plasma. Within an oxygen plasma exists positive and negative oxygen ions, charged molecular oxygen, neutral oxygen radicals, ozone, ionized ozone, and free electrons. All of these species react with the polymer surface leading to addition of oxygen atoms and oxygen-containing functional groups to the surface. This treatment increases surface energy making the surface more wettable, or hydrophilic. When nitrogen fluoride (NF ₃ ) is added to a process cold gas containing oxygen, the efficiency of the plasma is greatly increased. The

resultant plasma yields excited forms of 0, OF, CF ₃ , CO, and F which react with the hydrocarbon polymer chain to produce HF. Addition of a noble gas such as argon (Ar) to the process plasma helps create free radicals on the surface of the polymer. Gas in a plasma may undergo polymerization as by a free radical initiation process. A gas may polymerize and adhere to a dissimilar material in the plasma to deposit a thin surface film. Polymerization of CF ₄ gas in plasma creates surfaces similar to Teflon® on polymers such as polypropylene. A wide range of polymer surfaces have been modified by this method including, polyethylene, polycarbonate, polystyrene, poly(tetrafluoroethylene) , and poly(tetrafluoroethylene-co-hexafluoropropylene) (Hansen et al. J. Pol. Sci.. B4: 203 (1966) ; Schohorn et al. ^~ L_ APPI. Polvm. Sci.. 11: 1461 (1967) ; Westerdahl et al, J_z_ Coll. Interface Sci.. 47: 610 (1974)). The effects of surface treatments are usually characterized in terms of wettability, lap shear strength, autoadhesion, and surface chemistry. However, heretofore, no studies have been done on the effect of plasma treatments of surfaces on steroid or steroid-like binding to the surface.

The efficacy of gas plasma treatment in modifying the steroid-binding properties of polypropylene receptacles is illustrated in Table II, below. In this instance, the components were subjected to plasma treatments performed at 200 milliTorr pressure and 2000 watts of radiofrequency (RF) power. Aliquots of 200 microliters of PBS containing ³ H-estradiol were incubated in pretreated receptacles for 10 minutes at room temperature. The receptacles were washed 4 times with the same volume of PBS and subjected to scintillation counting. The fraction bound (%) was determined from the the ratio of counts tritium bound to total counts aliquoted X 100. The results are presented below.

TABLE II ESTRADIOLBINDING TOPOLYPROPYLENERECEPTACLES: EFFECT OF

GAS PLASMA PRETREATMENT

PLASMA TREATMENT FRACTION BOUND (%)

None 0.46 3% NF ₃ , 3% Ar, 94% O ₂ (NAO) 0.05 - 0.10

100% O ₂ 0.10

100% CF ₄ 2.10

Treatment with a pure oxygen cold gas plasma created a surface with reduced steroid binding properties. Making the plasma treatment more aggressive by the addition of nitrogen fluoride (NAO treatment) resulted ^* .n an even less adherent surface. By contrast, treatment of the polypropylene receptacles with a methane tetrafluόride (CF ₄ ) cold gas plasma led to a surface with greatly increased affinity for steroids. These experiments s; : ort the conclusion that high surface energy or hyάrophiliαity is one important surface property required to inhibit bindir.. of steroids to surfaces. Combination of different plae-t-. treatments, surface temperatures, and polymer compositions thus allows one to custom tailor a surface.to prevent the binding of any steroid or protein that is the subject of analysis.

The practical importance of the above treatments to render the ultrafiltration device non-adherent to steroid in the determination of non-protein-bound steroid, is demonstrated in Table III. In this experiment, the pc. cent non-protein-bound estradiol (%NPBE) was measured in :;hree equivalent ultrafiltration devices rendered nor-

adherent for steroid by the above methods, with the exception that a differently treated ultrafiltrate receptacle was used in each of the three measurements: untreated, silanized, and NAO plasma-treated. Any loss of ³ H-estradiol due to binding to the inner surface of the receptacle would result in reduced recovery of the tracer and a relatively reduced %NPBE. In support of this hypothesis, measurements with receptacles previously shown most effective reducing estradiol binding, that is, silanization and plasma treatment (Tables I and II) , resulted in the higher values for %NPBE in serum (Table III) .

TABLEIII

BIOAVAILABLEESTRADIOLDETERMINATIONBYULTRAFILTRATION (%NPBE):

EFFECTOFPRETREATMENTOFRECEPTACLE

TREATMENT BIOAVAILABLEESTRADIOLFRACTION(%NPBE)

None 0.90

Silanization I2

Gas Plasma (NAO) 1.25

The test kit of the present invention thus finds utility in the testing of a wide variety of analytes including steroids and sterol-like compounds. These test kits are not limited to ultrafiltration devices, although ultrafiltration-type assays are known and described for a wide variety of steroids and sterol-like compounds including estradiol, testosterone, cortisol, and combinations of estradiol and testosterone. Other types of test kits which rely upon a differing assay may likewise be prepared to serve as kits in accordance with the present invention, by rendering their surfaces non- adherent, and thereby achieving a corresponding increase in reliability, accuracy and ease of use.

The present invention will be better understood from a consideration of the following illustrative example.

EXAMPLE 1

A kit is assembled consisting of the following:

Ultrafiltration Device

The ultrafiltration device consists of a filter unit and a 1.5 ml centrifuge tube which holds and seals the filter unit. The filter unit holds an ultrafiltration membrane with a pore size that under centrifugal force prevents the passage of greater than 99% of human serum proteins. The ultrafiltration membrane contains stabilizers and preservatives that must be removed before use. The unique ultrafilter unit and microcentrifuge when used in combination are designed to minimize the binding of ³ H- estradiol and protein to all device surfaces. Twenty-two filter units are provided in each kit.

Stability: The filter units are stable indefinitely when stored at room temperature. The membrane in the filter unit, however, is fragile and any touching of the membrane surface may cause damage such that increased amounts of tracer estradiol will pass through giving spuriously high results for %NPBE. Removal of liquid from the ultrafilter unit should be done by inversion of the unit and vigorous shaking.

Pipette Tips

The estradiol molecule has a strong binding affinity for most surfaces including plastics. Proteins also bind to plastics. Therefore, the kit provides specially prepared pipette tips. These tips minimize, if not eliminate, the binding of free and protein-bound estradiol to tip surfaces, thereby ensuring a high degree of accuracy and reproducibility in measurement of bioavailable estradiol.

A box of 96 pipette tips are included in each kit.

Microcentrifuqe Tubes

The 1.5 ml microfuge tubes provided with the kit have been fabricated so as to largely eliminate binding of estradiol and proteins to their surface. The tubes are provided with caps that seal the top of the ultrafiltration unit during determination of %NPBE. This design is included to assure the integrity of the filtration unit seal, and to thereby avoid possible radioactive hazard.

Forty-five microcentrifugation tubes are provided with each kit. Of these, two are intended for use in purification of ³ H-estradiol, 20 are for use with ultrafiltration units in assay of %NPBE, and 20 are for use in determining % ABE. Three extras are provided. The tubes have surfaces rendered non-adherent preferably by compounding of the silicone, such as silicone fluid mold release agent, into the resin prior to molding, while the pipette tips are preferably treated by gas plasma processing, both as previously discussed.

To utilize the kit, in addition to the reagents and devices provided, the operator must have available: a) water bath or heating block maintained at 37°C; b) a centrifuge able to hold 1.5 ml microcentrifuge tubes also maintained at 37°C; and, c) a liquid scintillation counter for ³ H counting, preferably with quench correction, along with scintillation vials, cocktail, etc.

Only serum samples that have been allowed to equilibrate to room temperature should be used. If the serum sample contains precipitated material or fibrin, it should be centrifuged to clarify before use. Extremely viscous serum may indicate incomplete clotting or denaturation of

serum proteins. If high viscosity persists upon reclotting and clot removal, it may not be possible to use that sample. All serum samples should be handled as though biohazardous. Likewise, all devices and sera containing ³ H-estradiol should be treated as radioactive waste and disposed of in appropriate containers by approved procedures.

Assay Procedure:

1. Place 1 filter unit into one of the 1.50 ml microcentrifuge tubes provided with the kit. Add 0.40 ml of PBS to the reservoir of the filter unit and centrifuge at 2,000 x g for 10 minutes. Washing may be done at room temperature or 37°C.

2. Remove filter device from centrifuge, decant PBS from centrifuge tube, and add 0.30 ml of PBS to filter unit. To this 0.30 ml of PBS, add 0.03 ml of Estradiol affinity solution and mix.

3. Add 0.012 ml of ³ H-estradiol tracer solution from the comb-V-vial to the PBS-Estradiol affinity solution in the filter device. Let sit at room temperature for 30 minutes.

4. Centrifuge filter device from Step 3. above for 15 minutes at 2,000 x g at 20-37°C.

5. Carefully discard the radioactive liquid in the centrifuge tube, and add 0.35 ml of PBS provided with the kit and repeat Step 4. above. Repeat this step two more times, i.e., add another 0.35 ml and spin.

6. After the final wash step, check to see that nearly all liquid is gone from the filter reservoir (hold-up volume is 0.01 ml). Add 0.05 ml of the estradiol elution

solution to the filter unit. Centrifuge for 10 minutes at 2,000 x g. The ultrafiltrate contains the purified estradiol.

7. Cap the centrifuge tube with the purified estradiol and store at 2-8°C.

Determination of % NPBE

1. Assemble one filter device for each serum sample.

2. Wash filter

3. Add 0.001 ml (1 microliter) of purified ³ H-estradiol (3-4 x 10 ^s dpm) to each filter unit, being careful not to place aliquot onto the surface of the ultrafilter.

4. Add 0.40 ml of each serum sample to filter unit reservoir containing the radiolabeled estradiol. Seal filter unit with cap of the microcentrifuge tube and mix by gentle vortexing.

5. Incubate filter devices in water bath or heating block at 37°C for 1 hour.

6. Centrifuge for 20 minutes at 2 _f 000 x g in a temperature controlled centrifuge at 37°C.

7. Remove filter devices from centrifuge, and let cool for 10 minutes at room temperature. Cooling will increase accuracy of pipetting.

8. Remove duplicate 0.03 ml aliquots of the ultrafiltrate (bottom) from each filter device into duplicate appropriately labeled liquid scintillation vials. Use only the pipette tips provided with the kit. Submerge pipette tip into ultrafiltrate, eject a bit of

air and release to draw up liquid. Depress to eject liquid into scintillation vial. Change tip for each aliquot.

9. Gently mix concentrated serum in filter devices by vortexing and aliquot duplicate 0.03 ml into duplicate scintillation vials. Follow pipetting procedures recommended in Step 8. above.

10. Add scintillation fluid to each vial (at least 3.00 ml) and count for at least 10 minutes (see "Calculation of-Results") .

Determination of % ABE

1. For each serum samp. , add 0.001 ml (1 microliter) of purified ³ H-estradiol tt. a 1.5 ml microcentrifuge tube.

2. Add 0.40 ml of serum sample to tube containing the radiolabeled estradiol, cap tube and mix by gentle vortexing.

3. Incubate for 1 hour at 37°C in a water bath or heating block.

4. Remove duplicate 0.05 ml aliquots of serum containing tracer estradiol into duplicate scintillation vials using pipette tips provided with the kit. Change tip for each aliquot, do not allow sample to cool.

5. Add to each sample tube 0.30 ml of SHBG Precipitation Solution which had been equilibrated to 37°C. Mix each tube gently by vortexing.

6. Centrifuge for 2 minutes at 37°C at 10,000 x g to pellet precipitate.

7. Remove duplicate 0.05 ml aliquots of the supernatants into duplicate liquid scintillation vials.

8. Count in liquid scintillation counter for at least 1 minute.

Calculation of Results

The %NPBE and %ABE are calculated from the average disintegrations per minute (dpm) of ³ H-estradiol in each fraction. For highest accuracy and reproducibility, it is recommended that samples be counted for a time sufficient to give an accuracy of at least ± 5%. Some samples from the %NPBE ultrafiltrate may need to be counted longer than 10 minutes to obtain this accuracy.

%NPBE = dpm ³ H-E2 fserum ultrafiltrate) X 100 (Eqn 1) dpm ³ H-E2 (serum in ultrafilter)

%ABE = dpm ³ H-E2 fserum supernatant- X 2 X 100 - %NPBE (Eqn 2) dpm ³ H-E2 (serum)

These calculations provide the investigator with the bioavailable estradiol fraction that is "free" (%NPBE) , or associated with albumin (%ABE) . The concentration of estradiol ([E2]) in each of these fractions is the product of the total serum estradiol concentration times the fraction.

Therefore;

[E2] "free" = % NPBE X [E2] total, and (Eqn 3)

[E2] albumin bound =- % ABE X [E2] total. (Eqn 4)

For example, if % NPBE were found to be 1.35%, and % ABE

were 42%, and total estradiol was 100 pg/ml, then [E2] "free" = 1.35 pg/ml, and [E2] albumin bound = 42 pg/ml. The bioavailable estradiol concentration would be 43.35 pg/ml.

The bioavailable estradiol fractions %NPBE and %ABE are, however, independent biomarkers which are of clinical significance independent of the total circulating estradiol concentration.

EXAMPLE 2

A kit was assembled and employed in an assay, both as described in Example 1, above. Accordingly, 15 blood serum samples were provided that had been taken at random from women subjects who had been examined by an oncologist for signs of breast cancer. The samples were subjected to ultrafiltration for the purpose of measuring %NPBE and %ABE. The results are set forth in the FIGURE which represents a correlation between the two parameters measured.

Referring to the FIGURE, it can be seen that the data obtained accurately and consistently reflect an accurate measurement of the noted parameters, and validate the operability and utility of the present invention. The. data are favorably comparable with extant data derived from like measurements that have been previously obtained with the prior art kits and methods (See for example, Hammond et al., supra.) _f while the availability, and the speed and reliability of performance of the present method are significantly improved.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all

respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.