CA2028254A1 - Non-aqueous solvent specific binding protein assays - Google Patents

Abstract

NON-AQUEOUS SOLVENT SPECIFIC
BINDING PROTEIN ASSAYS

ABSTRACT OF THE DISCLOSURE
Novel specific protein binding assays are provided employing a solvent system comprising a solvent characterised as hydrophobic, essentially anhydrous and water immiscible. Particularly, lipophilic analytes are shown to be sensitively determined using antibodies in hydrocarbon media, optionally substantially saturated with aqueous buffer and/or comprising an anionic surfactant.

36.

Description

~ ~ 2 ~ HABI-003/ol us NON-AQUEOUS SOLV~T SP~CIYIC
BINDING PROTEIN ASSAYS

CROSS-REFERENCE TO ~ELATED APPLICATIONS
This application i5 a continuation-in-part of USSN 07/425,759 filed October 23, 1989, which disclosure is hereby incorporated by reference.

I NTRODUCT I ON
. ~ ' Technical Field The field of this invention is the det0rmination of the presence of an analyte involving the presence of a specific binding protein, particularly in water-immiscible solvents.
~ . -., -Backqround The use of specific receptors or the detection of such receptors has found extensive exploitation, particularly in the medical field. In the medical field, one has been interested in a wide ;
variety of drugs and naturally occurring compounds and physiological fluids. These compounds are for the most part water soluble at the concentrations of - 25 interest. The assays are frequently based on the ; specific rscognition by a protein of a proteinaceous or non-proteinaceous analyte, where the binding of the protein receptor and its complementary ligand normally occurs in an aqueous medium.
~ In many situations, there may be an intelrest~
in using a non-aqueous medium. In some situations, one wishes to extract a hydrophobic compound from a specimen such as soil, fat, or the like, where it would be desirable to retain the extracted compound in a non-aqueous medium. In chemical processing, where organic solvent~ are employed, there will be many 202i3840 101990 1 .

situation~ where one wishes to monitor the pre~ence of minor impurities in the reaction mixture. The need to transfer the sample into an aqueous medium may preclude any measurement.
There is, therefore, substantial intere4t in being able to develop assays which provide for specific recognition of analytes while allowing for the use of non-aqueous solvents.

Relevant Literature Durfor et al., J.A.C.S. (1988), 110:8714-8716, describe antibody catalysis in reverse micelles. ~
Zaks and Xlibanov, J. Biol. Chem. (1988), 263:3194- ~ -3201, describe enzymatic catalysis in non-aqueous solvents. See also Chemical and Engineering News, July 2, 1984, page 23. Zaks and Klibanov, Science (1984), 224:1229-1231, describe enzymatic catalysis in organic media at 100~C. Russell and Klibanov, J.
Biol. Chem. (1988), 263:11624-11626, describe inhibitor-induced enzyme activation in organic solvents. Kazand~ian et al., BiotechnolooY and . Bioènaineerinq (1986), 23:417-421, describe enzymatic analyses in organic solvents. Kazand~ian and -~
Klibanov, J.A.C.S. (1985~, 107:5448-5450, describe regioselective oxidation of phenols catalyzed by ~:~ polyphenol oxidase in chlorofonm. Klibanov, Enzymes That Work In Organic Solvents, ChemtQch (June 1986), 354-359, discus~es enzymatic catalysis in organic ~`; solvents. Zaks and Rlibanov, J.A.C.S. (1986) 108:2767-2768, compare substrate specificity of enzymes in organic solvents in relation to water.
Kli~anov/ TIBS (1989) 14:141-144, describes enzymatic ~ ~','r' catalysis in anhydrous organic solvents. Russell et al., Biochem. and BioPhvs. Res. Comm. ~1989), 158:80 85, describe antibody-antigen binding in hydrophilic organic solvents.
.~,~ , , , `~; 101990 2.

2~2~
SUMMARY OF THE INVENTION -:
Specific binding protein a~says are provided for detecting the presence of a lipophilic analyte in a sample using a solvent system comprising an essentially anhydrous, hydrophobic, water immiscible solvent. The process employs a proteinaceous receptor wherein the lipophilic analyte and the receptor are members of a specific binding pair which bind to form a complex. The method comprises the steps of combining the sample, the -solvent system and a reagent system for providing a detectable signal, together with any additional reagents for detecting complex formation between the analyte and the receptor, and detecting the presence of the signal, where a decrease in the amount of signal detected in the sample as compared to a control containing no analyte is indicative of the presence of said analyte. Optionally, a small amount of aqueous ~ `
buffer and/or surfactant may be added to the solvent system.

DESCRIPTION OF SPECIFIC EMBODIMENTS
Novel specific binding protein assays are provided employing a solvent system comprising an essentially anhydrous water immiscible organic solvent, particularly hydrocarbons or halohydro-carbon, a~ the assay medium. The sample, containing an analyte which is one member of a specific binding pair may be pretreated as desired, and dispersed in the assay medium. The assay medium may then be ;~
comb~ned with'the complementary member of the specific binding pair, generally a proteinaceous receptor, and the mixture allowed to incubate for reaction to occur between the specific binding protein and its complementary ligand. For heterogenous assay~, the as~ay medium may be separated from the solid support, ~;~ the solid support washed and the distribution of the 101990 3.

~ Q2 ~ 2 ~3fl label determined by determining the amount of label bound to the support or the amount of label in the supernatant, optionally combined with the washes. A
decrease in the amount of bound label detected in the test sample as compared to a control sample containing no ligand is indicative of the presence of ligand in the test sample. -~
The use of hydrophobic solvents offers the advantage that ligands not readily assayed in aqueous media may be readily assayed. The solvent system may optionally contain a small amount of aqueous buffer and/or a surfactant. The addition of surfactant to the solvent system offers the advantage that the -amount of non-specific binding, or signal to noise ratio, generally is lower when surfactant is used.
The omission of surfactant offers the advantage of a more direct method, and finds particular use in an automated system using for example a biosensor system which automatically blanks out the non-specific binding.
The sample may be any industrial or ~ biological material in which an analyte i~ present, ; particularly where the analyte is a hydrophobic compound. ~iological samples may be animal or plant tissue, physiological fluid, feces, bile, fat or ~~
plasma samples. Industrial samples may include soil sample~ and industrial organic chemicals whether they be final products, process intermediates or wastes from the pharmaceutical, pesticide and like industries. In many plant and animal tissues, the presence of various organic compounds may be of interest. ~For example, detection of aflatoxin in ~ peanuts, pesticides in animal fat, or the like may be ;~ of interest. In soils, particularly soils associated with industry, such as chemical processing, synthesis of organic compound~, particularly pesticides, the ;~` electronic industry, or the like, various organic ~ ~ compounds may seep into the soil. The determination ,: , 20243840 ~-101990 4. ~ -.~ . ~ .
~ .-. . .

of the presence of these compound5 can be important in determining whether waste storage is leaking, whether there has been violation of rules and regulations concerning disposal of compounds, S presence of hazardous materials in chemical waste drums, Quality Control of bulk organic chemicals and process intermediates, or the like. In addition, there are a number of lipid soluble compounds in ~
physiological fluids or tissues which are of interest. ~-These include endogenous compounds, toxic compounds, therapeutics and illicit drugs.
With many of these samples, it may be necessary or desirable to extract lipid soluble material into an organic solvent. Thus, the extract will provide the sample to be used. For extraction, the same solvent as used as the assay medium is not required, so long as the extracting solvent is substantially miscible with the assay medium in the amounts uRed. Thus, various solvents may be used, which have higher hydrophilicity than the solvents used in the sub~ect assays. In some instances, the compound of interest may be dispersed in an aqueous medium due to the presence of solubilizing compound~
~; such as proteins or detergents. In this instance, so long as the sample is small and can be dispersed in the hydrophobic assay medium without separation, the sample may be used directly.
In carrying out the assay, the sample suspected of containing the analyte will be combined with the assay medium. The assay medium has a major portion an organic solvent, a liquid at the ~-temperature of the assay, preferably combined with not more than about 2% usually not more than about 0.2%, by volume of an aqueous buffer and up to about 5 mM of a surfactant, e.g., an anionic, non-ionic or - ~--cationic, particularly anionic. Larger amounts of ~;~ water, up to 5% ~w/w) may be used. The surfactant while not essential for obtaining ~pe~ific binding as 20243840 --~ -~

.

~ & ~
compared to non-specific binding is particularly desirable.
The organic solvent i8 characterized as being hydrophobic, essentially anlydrous, and water immiscible. By hydrophobic is intended that the solvent will usually dissolve less than 7.0% (w/w) more usually less than S~ (w/w) of water. 9y essentially anhydrous is intended annhydrous grade solvents which depending upon the solvent, generally contain <0.1% (w/w) water, more usually <0.05% (w/w) waters may contain ~0.005~ (w/w) water. By water immiscible is intended solvents that cannot be uniformly mixed or blended with water, i.e. two phases are formed upon mixing with water. For the most part, the solvents will be hydrocarbons or halohydrocarbons, aliphatic, aromatic and alicyclic, where the halohydrocarbons have halogen of atomic numbers 9 or 17, although 0-2 heteroatoms, e.g., 0, N or S, may be `~
present where the carbon/heteroatom ratio is at least about 4:1. Desirably the solvents are aliphatic hydrocarbons of from 5 to lB carbon atoms, particularly of from 6 to 16 carbon atoms, particularly straight chain. The halohydrocarbons will generally be of from about 1 to 6 carbon atoms, more usually from 1 to 4 carbon atoms, generally ~ ~-containing at least one halogen and up to perhalo.
Illustrative solvents include hexane, heptane, octane, decane, dodecane, pentadecane, hexadecane, 2,6-dimethyloctane, toluene, benzene, the xylenes, ~ -cumene, cyclooctane, chloroform, dichloromethane, carbon tetrachloride, etc. The solvents may also contain heterogroups such as nitro, for example !
nitrobenzene and heteroatoms such as halogens so long -~
as the characteristics of the solvent are maintained.
The assay medium will normally contain not more than about 5% (w/w), usually not more than about 2~, preferably not more than about 0.2% by volume of a ~; buffer solution, at a pH from about 5 to 10, more 101990 6.
~ : ~, ;.

usually 6 to 9. For the most part, the solventq which dissolve less than about 0.2~ ~w~w) of the buffer medium will be saturated with the buffer medium, i.e. the buffer will be present at the solubility limit. Generally, the buffer will be present in from about 10 to 200 mM, usually 50 to 150 mM, and various buffers may be used, such as phosphate, phosphate buffered saline, carbonate, Tris, MOPS, HEPES, or the like. Of particular interest, are inorganic buffers. Salts may be present, e.g., NaCl, other than the buffer, generally ranging in an amount from about 0 to 1 . 0% (w/v) -The third ingredient of the medium may be a surfactant, particularly an anionic surfactant, which may be aromatic or aliphatic, particularly aliphatic, normally a salt of an organic acid or ester, including carboxylates, sulfonates, sulfates, phosphonates, ~;
cationic surfactants include CTAB, while nonionic surfactants include polyoxyethylene glycol ethers and esters, or combinations thereof. Other functionalities may be present in the surfactant, particularly oxygen containing functionalities, such ~ ~
as ethers, esters, carbonyls, hydroxyl, or the like. ~ ~-Of particular interest is the diester of sulfosuccinate where the alkyl groups may be of 6 to 12 carbon atoms, particularly 8 carbon atoms. The concentration of the surfactant will vary depending upon the particular surfactant, generally not -~
exceeding about 5 mM, preferably being from about 0.02 to 3 mM, more preferably from about 0.1 to 2 mM. The ~ -surfactant may be addqd~to the solvent either in the presence or absence of buffer, preferably in the presence of buffer. The surfactant may serve to minimize non-~pecific binding.
The dilution of the sample into the assay medium will depend upon the concentration of ~he --~
analyte in the sample, the nature of the medium 101990 7.
.~ .

~ 2~2~L~
containing the sample, and the like. Usually, the sample will be le~s than about 50 volume ~ of the assay medium, usually less than about 20 volume % of the assay medium and may be as small as 1~ or less of the assay medium.
In carrying out the subject invention, various assay protocols may be used, particularly heterogeneous protocols. By heterogeneous is intended that bound and free are separated. At least one of the components of the assay system may be bound to a solid support. ~enerally, the component bound is one member of a specific binding pair (~receptor~) capable of binding specifically to an analyte or ligand ~ ~ ;
(second member of the specific binding pair). The receptor generally will be proteinaceous, and will usually be an antibody or fragment thereof, including the various isotypes, e.g., murine IsGl, IgG2a~ IgG2b~
IgG3, IgM, IgA, IgD and IgE, fraqment~ such as F(ab')2, Fab', Fv, etc. Following combination of the ~:
sample with the assay medium a~ described above, the solvent medium may then be combined with the immo~ilized receptor and the mixture allowed to incubate for reaction to occur between the receptor and the ligand. For heterogeneous assays, the solvent medium may be separated from the solid support, the solid support washed and the distribution of a label obtained by determining the amount of label bound to the support or the amount of label in the supernatant, optionally combined with the washes.
The protocols involve a reagent system comprising a labeled compound which provides for detection of~comple,x formation between the ligand and the receptor. The term label is intended to mean any compound which allows, either directly or indirectly, the detection of the presence of a member of a specific binding pair, particularly as to the presence or absence of complex formation between the liqand and it~ receptor. Variou~ label~ which find use include : , , 101990 8.
:

`3~-~t~3`3 radioisotopeæ, fluorescers, chemiluminescers, enzymes, including PEG-modified enzymes, beads, including beads containing a chromophore, graphite, metallic beads, etc. The label need not be directly bound to the member of the specific binding pair forming the complex, but rather may be bound indirectly, such as using a second specific binding pair. For example, biotin may be present as the label on the first specific binding pair, which may then be bound to avidin or streptavidin, which is labeled with a label capable of providing a detectable signal. Fluorescers include fluorescein, rhodamine, umbelliferone, BBD (7-benzylamino-4-nitrobenzoxadiazole), and the like.
Radioisotopes include 14c, 32p, 125I, 3H, and the ; ~;
like. For the most part, the labels are well known and do not require further exemplification here.
The reagent system will be comprised of the labeled conjugate and such other reagents as are necessary for detection of the label. For enzymes, this will usually include substrates. For chemiluminescers, other reactants may be required to produce the chemiluminescence. In other ~ituations, the reagent system may involve antibodies to -~
fluorescers, quencher conjugated antifluorescer, and ~-the like. The components of the reagent system may be combined concurrently or consecutively, depending on -~
the nature of the reagent system.
For heterogeneous assays, the receptor will normally be bound to a solid support. Solid supports may include rods, beads, membranes, vessels, for example, microtiter wells, or the like. Various matçrials may be involved, such as glass, nylon, Mylar, controlled pore glass, or other support which will not be adversely affected by the assay medium.
Immobilization of the receptor may be achieved in a ~
` variety of conventional ways, usually employing ~ -covalent binding in accordance with conventional techniques. The glass products can be readily 10199~ 9.

activated by heating the glass at elevated temperature, generally in the range of about 400 to 600C. After cooling, the glass i5 functionalized by reaction with an appropriately functionalized silane.
Controlled pore glass needs no activation prior to silane functionalization. Thus, halodialkoxy or trialkoxycarboxyalkyl or -aminoalkyl silanes may be employed. Functionalized supports may be readily conjugated to the receptor using various reagents, such as glutaraldehyde, maleimidobenzoic acid, where the protein has a mercapto group, or where sugars are ~-present on the protein, these may be oxidized to dialdehydes for linkage under reducing conditions to an amine, to form a methyleneamine or imine. After reaction of the specific binding protein to the surface, the surface may then be washed with an aqueous medium, particularly one containing inert ~-globulin. After incubation with ~-globulin containing solution, the surface may then be exhaustively washed with an aqueous medium, particularly phosphate buffered saline. ~
The particular order of addition will vary ;~-depending upon the nature of the protocol. In ~ ;i heterogeneous assays, usually the sample containing the ligand analyte and the receptor will be combined either concurrently with the labeled ligand analog or -~ prior to the addition of the labeled ligand analog.
For homogenous assays, a similar order of addltion may be employed, although one may be preferred over the ^
other. Depending upon the nature of the assay, either ~; a rate or equilibrium determination may be made, using one qr more~determinations. Generally, the time far the determination will be at least about 30 sec and may be 24 hr or more, usually at least about 2 min and not more than about 12 hr. Rate determination~ will v~ry, generally beginning at least about 1 min after combining with the labeled ligand analog and usually requiring at least 1 min between readings, more lOl9gO 10.
.~ ~
- . ' ~ , ~2~
usually at least about 2 min and not more than about 6 hr.
The temperature for the reaction may vary from about -20 to 40C, more usually from about 0 to 22C. For the most part, the receptors will be naturally occurring receptors or antibodies, particularly monoclonal antibodies, althouqh antisera may find application. Various naturally occurring receptors include thyroxine binding protein, lipo-lC proteins, surface membrane proteins, etc.
The analytes of interest may be varied widely, including pesticides, such as insecticides, herbicides, nematocides, fungicides, pollutants, toxins, such as aflatoxin, physiologically active compounds, and such other compounds as may be hydrophobic and of interest. For the most part, the ~ ~;
ligands will be haptenic and less than about S kD, usually less than about 2 kD. ~ -Depending upon the nature of the label, various techniques may be employed for detection of `~
the signal. Equipment is generally available, such as ~; scintillation counters for radioisotopes, spectrophotometers for dyes, fluorimeters for ~`
fluorescence and chemiluminescence, and the like.
The following example~ are offered by way of illustration and not by way of limitation.

. EXPERIMENTAL
.: : , .
Abbreviations ~ PBS-T = PBS + 0.05~ (v/v) Tween-20 ; NRIgG = normàl rabbit IgG
BSA = Bovine serum albumin PBS = 0.01 M Sodium phosphate, 0.15 M NaCl (pH 7.0) PBS/BSA = PBS ~ 1% BSA

~ ~ *

~ 101990 11.
::~

~2~$l~ ~
OPP = Octane containing 1 M l-propanol and 0.1% PBS (v/v) CPG = Controlled pore glass Materials Antigen specific sera were purchased from the suppliers listed in Table 1. They were stored at 4C until use.
Purified normal rabbit IgG (Pierce) Protein G columns (Genex, GammaBindY-G) ~;~
Rabbit IgG standards (Sigma~
3H]Estradiol (Amersham), 94 Ci/mmol [3H]Progesterone (Amersham), 85 Ci/mmol ~3H]Digitoxin (New England Nuclear), 15.8 Ci/mmol Ecolume liquid scintillation cocktail (ICN) CPG glass beads, 20 mesh, pore dia 1273 , ;~
pore dist ~ 8.4%, CPG Inc. Fairfield NJ lot # lOOCC
; 20 O9DOl ~; Nylon 6J6 rods (Aldrich Chemical Co.), 3x2 mm - ;
Pyrex~ rods - 5x3 mm Pyrex~ beads - (Baxter) 3 mm dia.
Karl Fischer reaqent - (Fisher Scientific) -Solvents - (Aldrich Chemical Co.), anhydrous `
~`~ grade AOT (Sigma) - dioctyl sulfosuccinate sodium ~ salt :~; 30 1. AntibodY PreParation and QualitY Control a. PreParation of purified rabbit IaG fractions from whole sera ` ~ Protein G'chromatogr;aphy was used to isolate~
the IgG fraction of rabbit sera. Protein ~ columns ~` ~ containing 1.0 ml recombinant protein G covalently immobilized on Sepharose~ 4B were used to isolate the IgG fraction from.rabbit sera. Protein G
chromatography was performed as followss Serum ~
, ~ - ~:.

` 20243840 ~101990 12.
:

., i .

samples were clarified by centrifugation for 15 min (16,000 x g) and diluted 1:1 in PBS loading buffer.
Protein G columns were equilibrated with 10 ml PBS and one milliliter of diluted serum was then loaded onto the column. Unbound protein was washed from the ~-column with PBS at a flow rate of 1 ml/min, using a peristaltic pump. Eluted protein was monitored by UV
absorbance at 280 nm. When A280 had returned to ~ -baseline values, bound IgG was eluted with 2 ml of 0.5 M ammonium acetate (pH 3.0). IgG fractions were ~-collected into tubes containing equal volumes of 1.5 M
Tris-HCl (pH 8.8) in order to allow rapid neutralization of the elution buffer pH. Fractions containing IgG were combined and dialyzed extensively against PBS. Immunoglobulin concentration was estimated by UV spectroscopy at 278 nm using an extinction coefficient of 1.4 mg/ml.
Purity of rabbit IgG fractions was assessed by SDS-PAGE (Laemmli, Nature (1970), 277:680) and ; 20 visualized by the silver staining method (Hukeshoven and Dernick ElectroPhoresis (1985), 6:103). Protein G
chromatography resulted in purification estimated to be > 90~ when compared to rabbit IgG standards.
~ Protein G purified IgG fractions were ; 25 checked for antigen binding capacity by a -precipitation RIA, as described below. Binding capacity of the IgG fraction~ was retained following chromatography.

b. Precipitation RIA method:
0.36 ml of antibody preparation serially dil~ted in PBS~+ 1% BSA and 0.04 ml radiolabeled !
antigen (O.l~Ci) were combined and mixed, then incubated for 1 hr at 37CC in a water bath. Then 0.1 ml of a O.lM EDTA, (ethylenediamine tetraacetic acid) in water solution pH 7.0 and 0.2 ml of a 2~ (v~v) normal rabbit serum in PBS ~ 1~ BSA ~olution were -~ added to each sample and mixed. Then 0.1 ml of an .
202438~0 ' 101990 13.

optimally diluted goat anti-rabbit IgG and 0.5 ml of a 6~ (w/v) polyethylene glycol 6000 in PBS + 1~ BSA
solution was added to each sample, mixed, and incubated for 5 min at ambient room temperature.
Each sample was then centrifuged at 15,000 x 9 for 15 min at 4C. The pelleted material was washed by centrifugation 3 times with 0.5 ml PBS ~ 1% BSA and the precipitate solubilized with 0.3 ml of 0.1N NaOH.
0.15 ml of the dissolved pellet was then added to 3 ml scintillation cocktail and the radioactivity quantified using standard liquid scintillation countin~ techniques.

c. Immobilization Four different types of support materials ~ -were evaluated for the immobilization of the antibodies: Controlled pore glass (CPG) beads, nylon ;~
rods, and activated Pyrex~ glass rods and Pyrex qlass beads. The CPG and Pyrex~ beads (or rods) were cleaned by treatment with 5~ nitric acid at 100C for ~ -1 hr followed by extensive rinsing with distilled water and acetone. The glass surface of the Pyrex beads and rods was activated by heating at 500C for 5 hr~followed by cooling to room temperature in a desiccator (Hamaguchi et al., J. Biochem. (1976), 80:895-898). No activation was necessary for the CPG -~
beads. ~he beads (or rods) were then immersed in a 2 solution of 3-aminopropyltriethoxysilane in acetone and allowed to stand at 45C for 24 hr. After cooling to room temperature, the aminated glass beads (or rods) were washed repeatedly with acetone and stored in a desiccator until used. Nylon 6~6 rods were activated by incubation in 3.5 M HC1 for 24 hr (Hendry and Herman J. Immunol. Methods (1980), 35:285-296).
The nylon rods were then washed with distilled water, washed for 1 min in 0.1 M carbonate buffer (pH 9.5), wa~hed with PBS and stored in a de~iccator at 0C ;-until needed.

~;~ 20243840 101990 14.
' Antibodies were immobilized on the supports using Robinson~s modification (Robinson et al., Biochem. BioPhYs. ACTA, (1971), 242:659-661) of the general m0thod of Weetall (Meth. Enzymol. (1976), 44:134-147). Thirty-six aminated Pyrex~ bead~ (or rods)l 36 nylon rods or 500 mg of aminated CPG beads were treated with 8% aqueous glutaraldehyde for 30 min at room temperature. The beads (or rod~) were thoroughly washed with distilled water and suspended in 2 ml of PBS containing 720 ~g of purified IgG and allowed to stand at 4C for 24 hr. The beads were .
then washed with PBS and resuspended in 2 ml of PBS
containing 720 ~g of normal rabbit IgG. After standing at room temperature for 2 hr, the beads were exhaustively washed with PBS. The liquid was decanted ~;
off and the beads were frozen and lyophilized.
Experiments to determine the optimal solid phase were conducted in parallel with organic phase antigen-binding assays. Initial assays employed IgG
immobilized on CPG. However, the use of CPG beads in preliminary experiments proved to be technically cumbersome. Not only did each sample require accurate weighing, but full recovery of the beads following sample washing was difficult. In an effort to find a more reliable solid phase support, nylon rods and Pyrex glass rods were evaluated. The Pyrex~
rod~ proved to be most suitablo, however, there was too great a variation in size of the available rods.
The Pyrex~ beads were readily available in quantity and proved to be just as suitable as the Pyrex~ rods.
While the size distribution of these beads was not as narrow as preferred~, they were~more uniform than the rods. Since size variations of the beads could increase the variability of the assays, we sorted the beads as much as possible.

.. :, 101990 15.

2 & 2 ~ ~d ~3 lC
- d. Storaqe Immobilized antibodies were stored in a vacuum desiccator over Drierite~ at 4C until use. -~
They were allowed to equilibrate to room temperature before the vacuum was released.

, 2. Solvents a. Purity Organic solvents were anhydrous grade, certified to contain <0.005~ H20. The water content of the solvents was checked by Karl Fischer titration and found to be below the level of detection.

Example 1 :
Solid Phase Antiqen-bindinq AssaYs Determination of the immobilized antibodY bindinq activity bY aqueous RIA
Controlled pore glass (CPG): Five milligrams of beads with immobilized anti-estradiol (lot #554-C) or immobili~ed BSA were added to duplicate 1.5 ml Eppendorf tubes. Beads were incubated in 0.4 ml PBS/BSA containing 0.1 ~Ci [3H]estradiol for 1 hr at room temperature, with shaking at 200 rpm. Following incubation, samples ware washed three times by centrifugation with 1.5 ml PBS/BSA. ~eads were transferred to scintillation vials and bound radioactivity was determined by liquid scintillation counting in 3 ml Ecolume cocktail.
An~iestradiol (lot #554-C) immobilized on controlled pore glass beads was shown to bind approximately 2500 cpm vs 500 cpm for the immobilized BSA background. -~
Glass and nylon rods: One glass or nylon ~-rod with immobilized anti-estradiol or immobilized BSA ~
was added to duplicate 7 ml glass scintillation vials. ~ f`
Samples were incubated in 0.~ ml PBS/BSA contalning , 101990 16. -: , :

0.1 ~Ci [3H]estradiol for 1 hr at room temperature, without agitation. Rods were washed three times with 4 ml PBS/BSA. Bound radioactivity was determined by liquid scintillation counting in Ecolume cocktail.
Aqueou~ phase testing of IgG immobilized on nylon rods showed less specific signal and higher ~;~
background signal than did glass rods used under the same conditions, and therefore, were not selected for further evaluation.
In general, the activity of the immobilized IgG fractions paralleled the activity of the raw sera.
Thus, in PBS, two lots of anti-estradiol (NEIA lot #554-C and 462-C) were shown to have high tracer binding capacity, while anti-digitoxin antibodies (Wein lot #04737) and anti-progesterone antibodies (NEIA lot #262-C) were found to be less active.
Example 2 Organic Phase Antigen-binding Assays General procedure Except as otherwise noted, antigen binding assays were performed according to the following protocol. Either 10 mg of CPG beads or one glass rod or one glass bead with immobilized antibody was added to each 7 ml glass scintillation vial. Each sample then received 1 ml of the specified solvent and 10 ~1 ; 30 (0.1 ~Ci) of 13Hl-tracer, dissolved in toluene.
Samples were incubated at the specified temperature for the indicated time and then washed three times with 2 ml incubation solvent. Bound radioactivity was ~
dqtermined in 3 ml of E~colume liquid scintillation ~-3S cocktail. The aqueous phase binding capacity of ~ immobilized IgG was determined concurrently with all ;~ organic phase experlments. Methods for these aqueous phase determinations were identical to those for solvent, except PBS or PBS~BSA was substituted for orqanic solvent incubation media, and tr~tiated 20~43840 101990 17.

~: .

2 ~ ~
tracers were diluted in PBS instead of toluene.
Statistical analysis was performed on a MacIntosh -computer using the Statview statistics program.
Initial screenin~
Preliminary organic phase experiments utilizing rabbit IgG fractions immobilized on CPG were performed in octane containing lM propanol and 0.1~
PBS (OPP). Tritiated tracer was bound by immobilized anti-estradiol more than to BSA background controls in a preliminary experiment (see Table 1, Exp. l). ~ :
Effect of increasinq amounts of AOT on antiqen bindina A 50 mM (AOT) in hexane solution was prepared. Dilutions were prepared from this stock solution with anhydrous hexane to give final AOT
concentrations of 10 mM, 2mM, 0.2mM, and 0.02 mM.
Anti-estradiol and anti-digitoxin were tested at each concentration. One milliliter of hexane containing specified amounts of AOT was added to each sample, followed by 10 ~l of l3H]-tracer (0.1 ~Ci/sample) in anhydrous toluene (Table 23.
~; It is noteworthy that the background binding of digitoxin tracer was substantially reduced in the `~ ~ presence of AOT. More than 20,000 cpm were bound by ; ~ anti-digitoxin IgG when compared to normal rabbit IgG
(NRIgG) background, at 0.02 m~ and 0.2 mM AOT. A
differential binding of this magnitude after only two hours suggests that significant binding could have been detected with a shorter period of incubation.
Based on these results~ 0.2 mM AOT was selected for use in future experiments.
The results presented in Tables 2 and 3 `~ suggest that precise optimization of incubation conditions may be necessary with each antigen/solvent/
~`~ antibody combination. Antigen binding by ;~ :antiestradiol IgG was greatly enhan~ed by an 18 hr., -~ 101990 18.
.

~!' ':' ;, ., ' ',; ' ' i ; ; ; ;

~3~
- 4C incubation, when compared to a 2 hr, room temperature incubation.- However, this was not the case for anti-digitoxin IgG. Following the 18 hr, 4C
incubation, labeled digitoxin tracer was bound to NRIgG background beads and anti-digitoxin IgG
comparably. The result presented in Table 2 shows that after a 2 hr, room temperature incubation, substantial amounts of tracer were bound in excess of background. Anti-progesterone IgG preparations did not seem to bind labeled tracer beyond background under any of these conditions.
Effect of time and temperature on antiqen bindinq in hexane/AOT
Using a preferred combination of estradiol, antibody, and solvent, the time course of tracer binding was studied at room temperature and at 4C.
Antigen-binding assays were performed in PBS-saturated hexane containing 0.2 mM AOT. Samples were incubated for specified time periods at ~C or at room temperature (22-25C), then washed three times with 2 ml of ice cold hexane/AOT at room temperature hexane/AOT followed by measurement of bound label by scintillation counting.
Unlike the previous observations (Table 2), significant binding of labeled tracer was not detected at any time point when the incubation was performed at room temperature. However, clear antibody bindLng activity wa~ seen when the incubation was performed at 4C. Equilibrium binding was achieved following 4 to 8 hr of incubation.

Specific inhibition of tracer bindinq by unlabeled antiqen in hexane/AOT
Antigen-binding assays were performed in P8S
saturated hexane with 0.2 mM AOT, prepared as described above. Estradiol inhibitor was prepared as a saturated solution in PBS saturated toluene, then lOlg90 19. , ~ .
: ~ .

2 ~ 4 diluted to the indicated concentrations in hex~ne/AOT.
Progesterone was dissolved in hexane/AOT. A toluene vehicle control was included as the zero inhibitor sample (final added toluene concentration = 0.1 (v/v)). Radiolabeled tracer, [3H)Estradiol (0.1 ~Ci/sample), was then added in 10 ~1 of in anhydrous toluene.
The following Table indicates the results.

.. .'' '''' ,;~ ' .

~ .
.~; .

. ' ~;' 101990 20.
~ .

~ 32~ ~
., ~ ~ X
~ , ~
~ ,t ~ ~ ~.
o o ~
1-- N ~D
~D
r~
O CO

~ D) ~ a, P ~ ~ g ~n g u~ ~ ~ 3 O
Z I I O C7' O u~ Q U~ ~
t ~ .
X X C) 11 ~ ~ N
-C 1- ~ O- O ~
~ D) ~ ~t o ~ o ~ t~ n , - ~
~ ~ ~ U~ ~ ~
~ o 1 -O O D~ ~0 ~ ~
O~ Ul O) tO C~ C~ ~ ~. 3 ~3 0 0 ~ :1.
O O ~ ~ ~ ~
~n r- X X
1~ 1 1 1 1 511 ~ O ~3 ~t ~
l~ a. P ~ -:
oo O O' ~ :~ t' 11 ~- 0 r; ~_ . ~ ~ u~
g ~
~ ~ ' n n CD CD ,- O o ~
~. ~. ~ ~ O ~ ~ . o l~ ~ ~ ~_ ~ . ~' ~ : ':
O O N ~-- ~ tr ~
O 11 ~ ~ C~ ,. '' ~ o o a.~ r-~. .) n CL ~ . .
r- r- ~ ~ ~ 11 ~ O

~ ,_ 11 11 11 ~t t~
- b~ ~ ~ Cr~ ~n O
o It ~ ~: .
~D ~ ' ~ ~D
ut v) o ' ~ ' '' ` It'' ' It ~' CD ' ':
Q, Q, ~ô o ;:
O O o ~` ~ r r- ~n ;;
~ ~ o 0~

. .:
.. : : ~
::: .: :

.
' :

~ ~ 2 ~
. , Z U~
,t~ ~ ~ Z ~ Z ~ ~
,--~ 11 11 ~ ,-~ a 1'~
o o ~ I G~ ~ G ~
, ~ ~_ ~ ~ , ~ , ~ ~ .
~o .P ~ W U~ ,,- ~ U~ tq ., ~ W , ~ ~ } ~ ~ o ~
o c~ ~ . . g ~ .
O . ~ 0~ ~n rt r~
1-- 11 ~t ~ O
X P~ 3 w n ~ ~-~- ~ o ol Q. 11 : ' -~ C~' ~t N O ~1 a~ _ D1 D1 a~ N ~ ~0 ~ O 3 Dl ~ :
n ~ ~ ~ ~ x u- IV U~
~ ~ 1- 0 00 o ~ ~ , . , tl ~ O ~ . ~
u~ w ~ I_ a~ o O~ ~P ~IW ~ O O
Dl w ,P W ~O 3 ~3 ~ ~
,.~ 3 O C~ 0 ~3 .. : .

3 ~D n ~
~ w ~ ~ ~ ~3 1 . ~ .:
3ul ,_ a~ ~ ~ ID O
D ~ e~ o ~w ~ w vl j~ n tr w co ~I ~ (D

~ vw~o O 3 1 ~
. ~ . , o .t ,~
U~
~ ~ Z ~ ~n "~ ! . i ' ~ t~ ~. ce~, O

`` ' "'::

' ? ~ . `:

u~ o ~
~t U~
O O U~ O
~ - ~ ~ rt D ~ 1_. ~ ~ll C
- ~ U~
o c~
o . ~ ) . ~ ~
~ ~ ~ ~ ~-Q ~ G~ O, -O-I ~ ~ ~-O ~X ~ ~ ~
r ~ o o~ o ~ c~ ~ O
Q ~ ~n o o o~ w o Q
CO ~ W :~ ~
~t O ~ ~ U~ O
~ g ~ ~ ~
~ h ~ ~ ~o g W ~ ~D O ~ ~ O~ _l O ~ ~
n ~ , ~ :~ U7 : :
~:~ ~ It ~ ~:
_ ~ : o Q. ~ `, ' N ~ ~ rt ~ . 1~.
w ~ vl W a~ o~ ~ O
1~ Dl ~t I_ ~ ~

~: t~ ~ .P ~ ~ I ~Z ~1 w ,`
,~ ~ ~)~ O u~ ~n ~ ~ 0 O
~ ~ ~ w ~ o w P ~ ~D
,~ ~ C~
a. ~ o ' ~ `;
~,, ~ ~ ~ ~ ~ ~
-- ~ O W U- ~ ~ r~
W ~ ~ I U~ 1-'-X ~ I
~1 ~0 ~, ` O ~ 1~. `
~ o ~ ~ ~ ;:
t~ ~ ~
~D ~O w ~ 1 o ~ ~ ~ ;
I ~ W ~ ~ O Ul C~ ! X

~ ~D . ~-'.`~.~ t:1' ~ ` ' ~
~ ,~ ~ cn ~ o ~- ~ C~ .
!~ ~ ~ ~ P ~ t ~
~''` 11 W W ~ ~ ~ ~ ~ O i-~- 1 .
i`.'"~ O ~ ~O W ~ O O ~ X I _ :~ :
P ~ ~ ~ ~ `~
W
U~ , .`. ~ ' '``' ~ ~r~ ?~

~ ~ ~ z: cl~
c~ ~ ~ z ~
F
o o ~ n . - ~.
ow ~ ~ ~ , o ~ . ~ ~ ~ ~
1~. CO ~ ~D
O ~D~ ~
o o , ol o~ , , o~w .
. .
.; , ~o o o ,.
or~ ~
o ~ o o ~ ~ ~ ~ -~a o W tD
~w ~ ~t ~ .
oo~ ~ o tL ~ 3 It ;
~ E ~ ~ g ~ ~ `~
+ w ~ ~ ~ ~D
w~ o~ ~ o ~ ~
:~ ~ `~:
. ~ . 11 "~ , : :~ ~ ~ ~n ~ ~-~`: ~o, o o ,+ ,+ ,+ .
~ ~ , ,. ~ , ~ o ~W ~ ,.. .

:' '~ ' ''"','' ~ ~- .' ' "~
,, ;: ~ W '~ .',~,' ': '' CD O ~ '; ~ '~
1+ 1~ 1+ ~ ~: '' . Vl1- 1~1 :. ~'-. ~;

, ~:' ~ ' The antigen binding activity of anti-e~tradiol was significantly greater than for NRIgG
(p<0.0005). A detection limit of 200 pg/ml was achieved with both lots of anti-estradiol (lot #554-C, 0.01 ~p < 0.025; lot #462-C, 0.005 <p c 0.01~.
Specificity of the reaction was demonstrated by the lack of cross-reaction with 1 ~g/ml of progesterone (lot #554-C, 0.1 <p < 0.375; lot 462-C, p ~ 0.4).

Example 3 Comparison of Solvents for Or~anic Phase RIA

Duplicate samples of rabbit anti- ~-estradiol IgG (lot 554-C, New England Immunology Associates) covalently immobilized on 3 mm Pyrex~
glass beads (Aldrich) were incubated in 1 ml of the specified medium for 18 h. at 4 C with increasing amounts of [3H~Estradiol (Amersham, specific activity ;
110.5 Ci/mmol). Duplicate samples of antigen non- -~
specific rabbit IgG (Pierce) similarly immobilized were incubated in parallel, and served as controls for non-specific binding of [3H]Estradiol. A 0.1 ml sample o-f the supernatant, containing free, unbound ~ -[3H]Estradiol, was taken prior to washing of the solid phase. Following incubation, the solid-pha~e antibodies were washed three times with 2 ml of 4 C
incubation medium. All solvents were of anhydrous ; grade (Aldrich). Bound and free [ H]Estradiol was counted using standard liquid scintillation counting ~; techniques. Scatchard analysis of the data was performed usling thei"EBDA~ and "LIGAND" programs (Biosoft) on a 2.5 megabyte i~AM Macintosh SE computer (Apple).
` 35 . .

101990 25.

- ,.

Determination of Solvent ~ater Content Solvent water content was determined by Karl Fischer titrations performed by the dead-stop technique, using an Aquametry II apparatu~ (Lab Industries). Stabilized pyridine-free Karl Fi3cher reagent (KFR) was purchased from GFS Chemical (Powell, ;~
Ill.). Our limit of detection was determined by ~he minimum buret reading (0.05 ml), the concentration of KFR and the sample aliquot (2 ml). The commercial KFR
had a titer of 6.16 mg H20/ml KFR. This was diluted to a titer of 1.61 mg H20/ml KFR. Further dilutions gave irreproducible titration results. The limit of ~-detection was 0.08 mg H20 (approximately 0.002% (w/w) dependinq on the density of the solvent). A solution comprised of 15% methanol in methylene chloride (v/v) served as diluent. The water equivalence titer, F, of the KFR was determined prior to each experimental session (F = mg H2O/ml KFR). The titration endpoint -was established by the use of a conductivity meter.
Weighed samples containing unknown ~ amounts of water were added to the reaction vessel and ;~ titrated to the endpoint with KFR. Water content of the samples was calculated with the following formula:
.

% H2O (w/w) = ml ~FR x F x 0.1 wt. of sample (grams) .. ..
Scatchard AnalYsis of Antiaen - Antibody Bindina in Water Immiscible Solvents i, According to Rodbard (in Liaand ASSAY5 Langan and Clapp (eds) Massan Publishing, N.Y.
pp 45-101), the Scatchard plot is one of the most useful methods for the analysis of antigen-antibody binding. When an equilibrium between antigen and antibody is reached, there is a linear relationship between the bound to free ratio for the ligand and the concentration of bound ligand. The ~lope of thi3 line i8 -1 times the affinity constant ~Kaff)~ the : . .

101990 26.

- ~ .

intercept on the horizontal axis is the concentration of binding sites present (BmaX), and the intercept on the vertical axis is (Kaff x Bmax). In ~ystems containing a single homogeneous ligand which reacts with multiple classes of receptor, a nonlinear or -~concave Scatchard plot may be observed. Such i9 ~ ~ .
often the case with polyclonal antibodies since they contain multiple populations of antibodies which may vary both in their frequency and equilibrium constant.
Equilibrium-binding data of heterogenous receptor (e.g. antibody) systems may be evaluated with mathematical models which assume the presence of a single receptor site, two receptor sites, and so on.
We have performed Scatchard analysis of antigen-antibody binding in aqueous medium and in a series of nearly anhydrous organic solvents using one- and two-site models for this interaction. While neither of these models is expected to provide an exact measurement of the Kaff and BmaX~ since a polyclonal~ ~ -IgG represents a heterogeneous receptor population, reasonably accurate values for these parameters can be estimated.
The data presented in ~ables 5 and 6 illustrate that specific high affinity antigen~
antibody interaction does occur in nearly anhydrous ~- -organic media. In all cases, a one-site Scatchard plot model could be applied to the data ~Table 5). In several experiments, the data could also be interpreted with a two-site Scatchard plot model (Table 6).
In the ~'conventional" system, where the ~antibody-antigen binding takes place in aqueous PBS-T, the data were best expressed by a one-site model. The equilibrium constant obtained (2.33 x 10 ~ 0.24 x 109 M 1) can be compared to the systems employing nearly anhydrous organic media, organic - media saturated with PBS, pH 7, and organic media saturated wi~h PBS and supplemented with 0.2 mM AOT.

20243840 ;-101990 27.

In all cases, the water content of the organic media was less than 0.1~ (w/w), as determined by the Karl Fischer titration (Table 7). The method is described above.
In hexane, the affinity constant derived from the one-site model was comparable to that obtained in the aqueous system. However, when a 2-site model was applied to the data, a fraction of the -total antibody could be detected which had an increased affinity constant, when compared to that obtained in the aqueous system. It was not necessary to saturate hexane with P~S, or to add surfactant to the system in order to observe this effect. However, the addition of surfactant did lower non-specific binding of [3H]Estradiol to antigen non-specific IgG
controls, thus serving to enhance the "signal to noise" ratio of the system. This surprising result suggests that certain antibodies can be selected which will function well under nearly anhydrous conditions.
Antibodies which exhibit high affinity in organic media might be exploited to produce more sensitive immunoassay procedures, rapid immunoaffinity separations, and catalytic antibodies for use with lipophilic analytes such as estradiol or those analytes having intermediate solubility in hydrophobic solvents, such as digitoxin.
Significant binding with the estradiol/anti-estradiol system was al90 observed in nearly anhydrous toluene and carbon tetrachloride. In these solvents there was a modest reduction in the apparent affinity constants, when interpreted with one~site Scatchard models. ~owever, in those casçs where a two-site Scatchard model could be employed, populations of antibodies with affinity comparable to the aqueous system were detected. This observation -~ suggests that antibodies can be selected which will function in water immiscible aliphatic, alicyclic and 101990 28.
.

L
~ ' :
: . :~

~n ~ ~ o~
~ ~ U~ ~ ~ ~ U~ ~ ~ Y~ ~ o 1 ~:
Z o ~ . :' O ~ :
o o o o o o o o o o 0 ~ ~
~J 0 ~ K K K K K U K K K ~ ~:
U~ .C . ~ ~ U ~ ~ o ~-1 t.) ~ ~ ~ 0 ~1 ~ ~ o _i o o o o o o o o . :, .
a ~+,~ +l+,+,+,+,~,+l +l +l +l ~ X
O ~ ~ 0 ~. I ~. I I
. .,1 ~ OOOOOOO O O O :~:
O ~ ~ :. :' I
0 ~ a~ O 'D ~ <~ 'O 0 N 0 ::
U~ L~ O O 1~ 0 ~1 O O C~ ~ to 1~ a~
:~
o o a~ 0 0 0 ~o 0 ~ ~
O O ,~ ,~ ,0~ ,0~ ,0~ 0 0 0 ,C - ~
,~ U ~ O ~ O~ U~ ~ ~ ~ ~ 0 ~ : - :~ ' ~r~ ~ ~ ~ 0 ~1 1 ~ ~ 0 ~ _~ O ~ O O O O O O O
~ ~ ~ ~ +~lX ~i +l +l +l +l +l +l +l +l +l ~:
~: c ~- ~ ~ ~ a~ ~ ~ 0 :1 0 o. o '~: l ~ 0 O O O O O O O O O O
0 ~C
l ~ ~
~g ~ t 0 ~
~:~' I ~ O o ~rl ':
l ~ ~ ~ ~ ~ 0 ~ i ~! ~; : l ~ ~ I o o ~ o o ~1 o O ~ o ,a o ~
. ~ "
':: U~ ~J 0 0 0 0 00 0 0 1 . ~ .
.: ~ ~ ~ ~ Il> ~ O o~ a~ o ~ ~ I
~ ~ O ~ P~ 0 ~ C ; P~ ~ ~
' + ~ ~ ~''''''':' p ~o ~ Q' ~ o ~ a ~ ~0~
~ O ~ X X
U ~ ~ 10 ;
~ Iq ~ ~ .

; ~ ~ . ;- .
.~ .
~ ~ .

_ r 0 0 c ~ ~.1 0 ul ~ n ~ o ,a~ ~ ~
Z~a ~ :, ~ O O l .c ~ ~ ~ x c ~ c ~ c ~. c U _ _I ~O ~O ~ rl O ~ ~ O
0 ~ ~ O
u ~ o +l +l +l ~ +l ~ +I ~11 U~ ~ O ~ J
.
C' lt +~ ~ rl ~ ~ ~ ~ O
I ~ V JJ JJ ~ ~J
VJ KC -- ~4 K X O X O O O X O
0 O $-o~ ~1 ~0 CO Z 0Z Z Z 0 z 3 ~ . . . . .
~--~ r~
O 0 ~ CO ~ t 0 o o o c o ~ a ~ o 0 v~ ~ o ~ u~ ~ ~ .3 .3 .~ ~ ~
v~ 0 ~ ~ O 0` ~ ~ ~` 6 F~ Fi ~ 6 tl~ V~t ~'~ C I~ t 0, ~ O ~t _I 0C O + l + l + l J + l ~ I v ' : ':
~V ~ ~ ~ ~ r- ~ ~ t ~ lt ~ a~ o,t .. ..
C ~- --~ ~ O o o ~ o ~ ~ ~o ~ : - ':,. .
<: t _1 0 ~ X X X ~ J : ,`,:
J o'a 30 ~ ~`J z ~ z z z ,.1 Z .,:
O ~ 0~ ~ "'" ~.:.
0 ~ ~ ~1 ~1 ~
O
_ 3t ~ 0 ~ tr) ~ ~ v~ -l ~ 3 0 ~ .e ~ ~ ~
u _~ 0 ~ a~ 6 ~1 ~i G 13 ul 6 .1 +1 ~ ~ .~ o ~ , ' U ~ +l +l +l +l - ~ +l ~
l v~ 0c e ~ 1 ~ ot ~lt at 0,t ~ ~
E~ o ~ 1 ~ . .
" _ ~, o o o .~ o . .- ~ o .- : ..
_~ O r10 0 0 _~ O
l ~ ~ l O Z Z Z ~
l o ~ oo Cl~ ~ o o ~ ~
., ~ P.
o ~ _ . 0~1 ~ ~ ~ ~o ~o o~ o. ~
I ~ ~ ~ 3 c 3 ~ ~ ~ 3 c ~ ~ +~ .~ ~ 0 ~

~ ., ~ ~ ~ . .
I V~ 0C 8 o+lo+l +, .. ~, ~ ...... .. +, ~ ~ :
3t ~6 ~1 ~ ~ ~ lI,t 0~ 3t 11~ It O~ ~J
~rl ~ ~ ~1 ~ O O O 'O O 'O 'O ~ O ~ ~:
_~ ." 0 0 ~ ~" ~ o ~j ! o o O ~ ~ V
o ~ o~ o~ ~o o ~ z ~ z z z o~ z o e~ ~ x ~ ~ ~ ~
~` ~ ~ E~ O O ~ O al o~ O O ~o O
~ ~ c c ~ c ~ ~ c c ~ c :
~ ~ o ~-~ ~ p~
d O ut 111 0 m CO O ~ al I
~ a v~. c~c~ , . ~
P~ d ~ ~ ~ ~ ~ 'O ~ ~ ~ N :
c- a o, ~t ~ c c c c ~ --~ c ~ ~ d ~
J !~ ~ C ~ D a O ~ ~ O ~ ~ O ~ ~ ~ C
~ t~ ~ V~ O

~ .
lJ

~ater content of solvent sYstem3 emPloYed in orqanic Phase immunochemistry exPeri~nents.
__ . _ __ PBS
Saturated PBS with 0.2 mM
Trial Sample Anhydrous Saturated AOT -[~ H2O (w/w mean + standard deviation) .. --- :
1 Hexane 0.0038 + 0O0152 N.D.
0.0002 ~:
2 Hexane N.D. 0.0118 + 0.008 .
0.002 3 Hexane 0.0093 + 0.0133 + 0.0110 +
0.0002 0.0018 0.0020 ~
1 Toluene 0.0049 0.0605 + 0.0515 + ~ :
0.004 0.0029 2 Toluene 0.01C5 + 0.0663 0.0622 0.0013 0.0008 -~
~: 3 Toluene 0.0094 + 0.0507 + 0.0478 +
~; 0.0004 0.0041 0.0023 ~ ~

4 Toluene 0.0095 + 0.0721 + 0.0478 + : ::
~ 0.0004 ~ 0.0017 ~ 0.0023 ~ ~-;~ 1 Carbon 0.0080 + 0.0133 1 0.0130 +
Tetrachloride 0.0004 0.0013 0.0005 ~ :
2 Car~on 0.0069 + 0.0116 + 0.0144 + :.:
Tetrachloride 0.0006 0.0013 0.0005 : 3 Carbon 0.0081 + 0.0113 + 0.0107 + `-Tetrachloride 0.0004 0.0001 0.0001 ','`' ~:` ; '~, `:

! ' ` :

~ 31 :~: :

aromatic hydrocarbons, and chlorinated solvents, with binding activity comparable to that obtained in aqueous media.
It is evident from the above results, that accurate assays may be carried out with a wide variety of analytes in hydrophobic media.
Therefore, the sub~ect methodology offers many advantages in those situations where an aqueous medium -is undesirable or not available. The subject invention finds particular application with hydrophobic or lipophilic analytes, in systems -involving organic solvent media, and the like.
All publications and patent applications cited in this specification are herein lS incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in ~ome detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appendQd claims.

"';
: , ~ :, ~; ~

, ~ .
. ~ .

101990 32.

Claims (10)

1. A method for detecting the presence of a ligand analyte, consisting of a lipophilic compound in a sample, employing a proteinaceous receptor wherein said lipophilic compound and said receptor are members of a specific binding pair which bind to form a complex, said method comprising:
combining to form an assay medium, said sample, a solvent system comprising a solvent characterised as hydrophobic, essentially anhydrous and water immiscible and a reagent system for providing a detectable signal, where said reagent system comprises a labeled conjugate and a receptor, and any additional reagents for detecting complex formation between said ligand and said receptor;
removing any unbound labelled conjugate, with the proviso that said assay medium may be separated from any complexes which are formed prior to addition of any remaining members of said reagent system; and detecting the presence of said signal, where a decrease in the amount of signal detected in said sample as compared to a control containing no analyte is indicative of the presence of said analyte.

2. The method according to Claim 1, wherein said solvent system further comprises:
at least one of not more than about 5% (w/w) of an aqueous buffered solution at a pH in the range of about 5 to 10 and up to about 5 mM of a surfactant.

3. The method according to Claim 2, wherein said solvent is an aliphatic or aromatic hydrocarbon or halohydrocarbon and said solvent is saturated with said aqueous buffered solution.

33.

4. The method according to Claim 1, wherein said receptor is bound to a solid support.

5. A method for detecting the presence of a ligand analyte, consisting of a lipophilic compound in a sample, employing a proteinacous receptor bound to a solid support, wherein said lipophilic compound and said receptor are members of a specific binding pair which bind to form a complex, said method comprising:
combining to form an assay medium, said sample and a solvent system comprising a solvent characterised as hydrophobic, essentially anhydrous and water immiscible, where said solvent is an aromatic hydrocarbon, an aliphatic hydrocarbon or a halohydrocarbon, not more than about 5% (w/w) of an aqueous buffered solution at a pH in the range of about 5 to 10, and up to about 5 mM of an anionic surfactant, and a reagent system for providing a detectable signal, where said reagent system comprises a labeled conjugate and said receptor;
separating said assay medium from any complexes which have formed;
adding any additional reagents for detecting complex formation between said compound and said receptor to said support or said assay medium;
and detecting the presence of said signal, where a decrease in the amount of signal detected in said sample as compared to a control containing no analyte is indicative of the presence of said analyte.

6. The method according to Claim 1 or 5, wherein said receptor is an antibody or binding fragment thereof.

34.

7. The method according to Claim 5, wherein said aliphatic hydrocarbon is a straight chain hydrocarbon of from 6 to 16 carbon atoms and said detergent is a sulfo salt.

8. The method according to Claim 1 or Claim 5, wherein said label of said labeled conjugate is a radioisotope, a fluorescer or a chemiluminescer.

9. The method according to Claim 2 or Claim 5, wherein said aqueous buffered solution is present in not more than about 2% (w/w) and said surfactant is an anionic surfactant present at from about 0.01 to 1mM.

10. A kit for use in the detection of an analyte comprising:
a solvent system comprising a solvent characterised as hydrophobic, essentially anhydrous and water immiscible, not more than about 5% of an aqueous buffered solution at a pH in the range of about 5 to 10, and up to about 5 mM of a surfactant for inhibiting non-specific binding; and a reagent system comprising a labeled conjugate, wherein said labeled conjugate is cross-reactive with said analyte.

35.