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WO1993019374A1 - Kit d'essai ameliore permettant de determiner des steroides et des composes apparentes - Google Patents

Kit d'essai ameliore permettant de determiner des steroides et des composes apparentes Download PDF

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Publication number
WO1993019374A1
WO1993019374A1 PCT/US1993/002377 US9302377W WO9319374A1 WO 1993019374 A1 WO1993019374 A1 WO 1993019374A1 US 9302377 W US9302377 W US 9302377W WO 9319374 A1 WO9319374 A1 WO 9319374A1
Authority
WO
WIPO (PCT)
Prior art keywords
test kit
steroids
estradiol
protein
containing composition
Prior art date
Application number
PCT/US1993/002377
Other languages
English (en)
Inventor
Thomas L. Klug
Original Assignee
Immuna Care Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immuna Care Corporation filed Critical Immuna Care Corporation
Publication of WO1993019374A1 publication Critical patent/WO1993019374A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • 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.
  • 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.
  • 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 ⁇ - estradiol, estrone, estriol, 2-hydroxyestrone, 2- methoxyestrone, equilenin, and ⁇ -ethinyl-estradiol.
  • Illustrative adrenocortical steroids include 17- hydroxydiox corticosterone, deoxycorticosterone, cortisone, corticosterone, 11-dehydrocorticosterone, cortisol, aldosterone, and prednisolone.
  • Illustrative gestogens include progesterone, pregnenolone, allopregnane-3 ⁇ :20 ⁇ -diol, and allopregnan-3 ⁇ -ol-20-one.
  • CBG Corticosteroid Binding Globulin
  • SHBG Sex Hormone Binding Globulin
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • silicones polydimethylsiloxanes
  • silicones 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.
  • 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.
  • 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
  • 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.
  • Plasma3 is 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.
  • One of the most common plasma processes is treatment in a cold gas oxygen plasma.
  • 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.
  • 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.
  • a noble gas such as argon (Ar)
  • Ar argon
  • 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..
  • test kit of the present invention thus finds utility in the testing of a wide variety of analytes including steroids and sterol-like compounds.
  • 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.
  • a kit is assembled consisting of the following:
  • 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.
  • the filter units are stable indefinitely when stored at room temperature.
  • the membrane in the filter unit 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.
  • 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.
  • 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.
  • kits 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.
  • the operator 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.
  • 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)
  • estradiol fraction that is "free” (%NPBE) , or associated with albumin (%ABE) .
  • concentration of estradiol ([E2]) in each of these fractions is the product of the total serum estradiol concentration times the fraction.
  • bioavailable estradiol fractions %NPBE and %ABE are, however, independent biomarkers which are of clinical significance independent of the total circulating estradiol concentration.
  • 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.
  • 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.

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Abstract

Kit d'essai amélioré permettant de déterminer des analytes présents dans des fluides biologiques, tels que des stéroïdes et des composés du type stérol qu'on place dans une solution contenant au moins une protéine. Ce kit d'essai permet d'obtenir une plus grande précision étant donné que les surfaces des éléments qui le composent ont été conçues pour ne pas adhérer aux stéroïdes, aux composés du type stérol et aux protéines.
PCT/US1993/002377 1992-03-16 1993-03-16 Kit d'essai ameliore permettant de determiner des steroides et des composes apparentes WO1993019374A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US85157392A 1992-03-16 1992-03-16
US07/851,573 1992-03-16

Publications (1)

Publication Number Publication Date
WO1993019374A1 true WO1993019374A1 (fr) 1993-09-30

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PCT/US1993/002377 WO1993019374A1 (fr) 1992-03-16 1993-03-16 Kit d'essai ameliore permettant de determiner des steroides et des composes apparentes

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AU (1) AU3809993A (fr)
WO (1) WO1993019374A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117783545A (zh) * 2023-12-28 2024-03-29 深圳市新产业生物医学工程股份有限公司 经封闭剂封闭的软质材料、容器、液态校准品及检测试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0354799A1 (fr) * 1988-08-11 1990-02-14 Lundbeck Diagnostics A/S Procédé et moyen pour diagnostic d'allergie
EP0395137A2 (fr) * 1989-04-21 1990-10-31 ENIRICERCHE S.p.A. Capteur comprenant un antigène lié chimiquement à un dispositif à semi-conducteur
EP0420053A1 (fr) * 1989-09-26 1991-04-03 W.R. Grace & Co.-Conn. Amélioration des systèmes de support pour des essais en phase solide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0354799A1 (fr) * 1988-08-11 1990-02-14 Lundbeck Diagnostics A/S Procédé et moyen pour diagnostic d'allergie
EP0395137A2 (fr) * 1989-04-21 1990-10-31 ENIRICERCHE S.p.A. Capteur comprenant un antigène lié chimiquement à un dispositif à semi-conducteur
EP0420053A1 (fr) * 1989-09-26 1991-04-03 W.R. Grace & Co.-Conn. Amélioration des systèmes de support pour des essais en phase solide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CHROMATOGRAPHY vol. 126, 1976, AMSTERDAM NL pages 161 - 169 MADANI ET AL. 'NEW METHOD FOR THE PREPARATION OF HIGHLY STABLE POLYSILOXANE-COATED GLASS OPEN-TUBULAR CAPILLARY COLUMNS AND APPLICATION TO THE ANALYSIS OF HORMONAL STEROIDS.' *
THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 255, no. 11, 10 June 1980, pages 5023 - 5026 G. L. HAMMOND ET AL. 'ESTIMATION OF THE PERCENTAGE OF FREE STEROID IN UNDILUTED SERUM BY CENTRIFUGAL ULTRAFILTRATION-DIALYSIS.' cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117783545A (zh) * 2023-12-28 2024-03-29 深圳市新产业生物医学工程股份有限公司 经封闭剂封闭的软质材料、容器、液态校准品及检测试剂盒

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