CA1140463A - Solvent extraction for separation in immunoassay - Google Patents
Solvent extraction for separation in immunoassayInfo
- Publication number
- CA1140463A CA1140463A CA000346390A CA346390A CA1140463A CA 1140463 A CA1140463 A CA 1140463A CA 000346390 A CA000346390 A CA 000346390A CA 346390 A CA346390 A CA 346390A CA 1140463 A CA1140463 A CA 1140463A
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- CA
- Canada
- Prior art keywords
- free
- ligand
- bound
- fraction
- organic solvent
- Prior art date
- Legal status (The legal status 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 status listed.)
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/815—Test for named compound or class of compounds
- Y10S436/816—Alkaloids, amphetamines, and barbiturates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/815—Test for named compound or class of compounds
- Y10S436/817—Steroids or hormones
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/824—Immunological separation techniques
Abstract
ABSTRACT OF THE INVENTION
The present invention relates to a simple and accurate method to be applied in immunoassay technique for separating the free ligand from the antibody-bound ligand.
The method involves the use of a solvent extraction operation which freezes the equilibrium between the free- and bound-fraction. The solvent to be utilized should be slightly water miscible or completely water immiscible, having the properties of marked selective extraction power towards the antigen. The method is applicable in a liquid-liquid system, a solid-liquid system and solid-solid system.
The method is useful for radioimmunoassay, free radical assay or metallo-immunoassay, being most versatile having also the advantage that as a result of the free ligand being extracted into the solvent, the determination of the free and/or bound fraction compares very favourably with the known prior are methods in immunoassays.
The present invention relates to a simple and accurate method to be applied in immunoassay technique for separating the free ligand from the antibody-bound ligand.
The method involves the use of a solvent extraction operation which freezes the equilibrium between the free- and bound-fraction. The solvent to be utilized should be slightly water miscible or completely water immiscible, having the properties of marked selective extraction power towards the antigen. The method is applicable in a liquid-liquid system, a solid-liquid system and solid-solid system.
The method is useful for radioimmunoassay, free radical assay or metallo-immunoassay, being most versatile having also the advantage that as a result of the free ligand being extracted into the solvent, the determination of the free and/or bound fraction compares very favourably with the known prior are methods in immunoassays.
Description
The present invention relates to immunoassay technique. klore specifically the invention relates to a simple, accurate and efficient operation to be applied in the specific binding assay techniques for separating the free ligand from the antibody-bound ligand.
As known, specific binding assays are based on the principle of monitoring specific binding reactions in which the extent of binding is a function of the unknown ligand present, by means of a labelled component. Among the known methods the following five specific binding assay techniques can be mentioned: Radioimmunoassay (RIA), metalloimm-unoassay (~IA), free radical assay technique (FRAT), hemaglutination inhibition (HI) and enzyme multiplied immunoassay technique (~lIT).
In the first t~Yo techniques, tlle mixture comprising the unlabelled ligand, labelled ligand and antibody is allowed to reach an equilibrium and the antibody-bound ligand is separated from the free ligand.
lS In the radioimmunoassay, the ligand is labelled with a radioactive isotope, whereas in the metalloimmunoassay the ligand is labelled with a metal-containing reagent which contains also a suitable functional group by means of which one can attach the metal reagent to the hapten desired to be assayed. A full description of the latter is gi~en in the U.K.
Patent Number 1.550.540.
The opeTation of separating the free fraction from the bound one, is of great iD~oortance and its accuracy determines the sensitivity and precision o~ the entire specific binding assay technique. In selecting and assessing a separation operation, it is useful to consider the criteria that should be fulfilled in order to obtain the desired resu~.
The following major requïrements of an ideal separation can be mentioned:
~a~ A complete separation between the bound and free fraction s~ould occur ~ith a ~Jide margin for error in the conditions used for separatinn.
~lis implies that the method should not interfere with -the prir,lary binding reaction.
. 2 -~, , :~14q;)~63 ~b) The operation should be simple7 quic~ and inexpensive, utilizing known reagents and available equipment.
(c) The operation should not be affected by the ligancl utilized, since otherwise difficulties in standardization will be encountered. A number of separation operations can be found in the prior art and a brief description thereof is heTeinafter presented:
- Adsorption methods. Usually the free fraction is adsorbed; the adsorbents are commonly used for small peptides, steroid and thyroid hormones. The adsorption is determined by many factors, including the Telative surface area of adsorbent, size and charge of antigen, nature and concentration of the competing proteins, temperature, ionic strength and pH. It is essential that the conditions for the use of the adsorption met~lods are optimized experimentally for eacl~ antigen.
~xamples of col~only encountered adsorbents are charcoal and silicates.
~le major problém is that always charcoal contains some unactivated material which has a high non-speci:Eic avidity for a wide range of substances, so that the bound fraction as well ~he free one may be adsorbed. In addition, charcoal may remove the bound antigen, particularly when a low-avidity binding agent is employed. The drawbac~
of silicates is that t~ne adsorption is dependent on the protein present, so that the optimal relative proportions of adsorbent and serum must be determined experimentally for each antigen.
- ~ractional precipitation with neutral salts or organic solvents which leaves in solution the free fraction. The forces that determine this effect are largely electrostatic and can be in1uenced by ~any factors such as temperature, p~-l, protein concentTation and dielectric constant of the medium. Optimal conditions must be determined ex~er1~.entally, with attention to the parameters of: pH, buffer, protein and salt concentrations. The applicability of these simple precipitations, depends mainly on the properties of the free antigen.
_ 3 -A~nonium sulfate has been successfully employed in assays of plasma and urine extracts for many small peptides. Ethanol is perhaps more versa-tile, being suitable for a range of peptides. Polyethylene glycol has proved satisfactory for assays of insulin and may be useful for preci-pitation of peptide hormones which do not bind to standard adsorbents.
All the methods described in this para suffer from the disadvantage that they require at least one centrifugation step.
- Gel filtration. Since bound complexes are considerably larger in their molecular volume than the free antigens, they can be clearly separated by using an appropriate grade of a gel such as *Sephadex. The gel has been used in two ways for phase separation; the incubate can be passed through a column under proper conditions such that the bound moiety is eluted, leaving the free moiety within the gel. A second method widely employed in competitive protein-binding technique is that of gel equili-bration. The gel is present throughout the incubation and can be pre-dispensed in equilibrium with the tracer binder solution. The free moiety within the pores o~ the gel is thus in equilibrium with that without. When the test sample is added to the gel suspension, equili-brium is rapidly reestablished, since the binding of tracer to gel is rev~rsible. After allowing the gel particles to settle, a sample o~
the supernatant, which includes the bound fraction, can be removed and counted.
- Paper chromatoelectrophoresis. A suitable paper is selected which ad-sorbs the free hapten at its site of application, while the bound frac-tion move from the origin under the influence of an electric current andbuffer flow caused by evaporation. The disadvantage of this method is that it is too complex, time consuming and expensive to be used as a routine and also does not lend itself easily to automation. Also papers and antigens vary in their affinity characteristics which makes repro-ducibility difficult.
* Trade Mark ~4~)463 In view of the drawbacl.cs of the above prior art methods it appears tllat it is a long felt need for an i~proved method of separating the free fraction from the bound fraction in the specific binding assay technique. The invention ~herefore consists in!an improvement in the specific binding assay method for assaying a medium for a ligand involving the steps of :
(a) contacting said mediu~ with reagent means which includes a labelled constitu0nt comprising a conjugate of a labelling substance and a binding col~ponent and which forms, with said ligand to be determined, a binding Teaction system producing a bound-fraction and a free-fraction of said labelled constituent, the quantity of labelling substance resulting in said bound-fraction being a function of the amount of said ligand present in said liquid medi~j (b~ separating said bound-fraction rom said free-fraction; and lS ~c) determining the quantity of labelling substance in either the bound-fraction or the free-fraction, the improvement consisting in that the separation of said bound-fraction from said f~ee-fraction lS carried out by a solvent extraction operation usm g an organic solvent whicl~ is water immisclble having the property of freezing the equilibrium between the free-fractlon and bound-fraction.
The method is applicable for any immunoassay technique (e.g. radioimmu-noassay, free radical assay and metalloimmunoassay as described in the U.K. Patent No. 1,550.5~0), which requires a separating opeTation between the bound and free f~acti~n. ~ven though the general principles of solvent extraction have been well ~no~ for a long time, the use of solvent e~traction for this separation has not been used in im~unoassay techniques. The major reason for this lies in the expected interference of the solvent with the i~unological reaction, either through damage caused to the specific binding protein by the solvent or through drastic ch~nges caused by the solvent in the antibody-antigen equilib~ium ~:~4~63 reaction at the time of separating the bound and free fractions by the solvent extraction operation. It is therefore not surprising that the extensive literature on immunoassay techniques does noi report any successful example of applying solvent extraction techniques for separating bound and free frac~ions in rout me immunoassay techniques. The use of a scintillation liquid containing toluene has been suggested to extract tritium-labelled aldosterone by repeated operations for subsequent measurement in the scintillation counter, but the cumbersome procedure described (J.P.Jowett et.al, Clinical Science and ~lolecular Medicine, 1973 pages 607-623) did not indicate any possibility of general applicability. It was therefore surprising and unexpected, when it was found according to the present invention that by the use of appropriate solvent and conditions, a general method could be devised for seyarating the bound and free ligands in immunoassays without adversely affecting either the binding ability of the bindin2 protein or the binding protein-ligand equi~brium. Moreover the method accordlng to the present invention can be of wide and general application to radioiminunoassay systems as well as to other immunoassays using non-isotopic labelling agents for mar};ing the haptens to be analyzed. One of the advantages which characteri~e the method according to the present invention is its versatility and the variety of configurations of the liquid-liquid system which can be applied for achieving t~e desired separation of free from bound ligand. In addition to the common liquid-liquid system, it can be envisaged various configurations in which the liquid-solvent and/or the aqueous mediu~ of the immunological reaction, are immobilized in a solid matrix. Thus the method wouid be applicable in a liquid-liquid system, a solid-liquid system and even a solid-solid system.
In a liquid-liquid system, the in~mUnological reaction woul~ be allowed to proceed in the usual manner in tile aqueous medium. Follo~Ying attainment of equilibrium in the reaction, an appropriate amount of a suitable solvent 6~3 is added, and after a thorough r,lixing, the t\~o phases are allol.ed to separate, the free ligand being trans~erred into the extracting solvent.
If a ~-radiating Tadioisotope has been used for hapten-labelling, separation into zones takes place spontaneously,f the bound and/or the free fraction being determined without actual separation of the aqueous and solvent phases into dir~erent vessels. This is an important advantage by itself, particularly for labelled radioactive elements since it decreases the number of operational steps. If deemed desirable to separate the aqueous and solvent phases, this can be done by any usual procedures, including the use of membrane systems.
Another configuration for separating the free from bound fraction is a solid-liquid system, in which the liquid is held in a solid matrix.
After the immunological reaction is carried out in the usual manner in an aqueous solution and the required incubation period for attainment of equilibrium is allowed, an appropriate so:lvent is introduced and brought into intimate contact with the aqueous phase. After a thorough mixing, the two phases ~aqueous and solvent~ are allo~ed to separate, and an appropriate hydrophobic but solvent~adsorbent material in the form of a strip, or other suitable form, is introduced into the reaction ~ixture.
Adsorption of the solvent onto tlle solid material will carry with it the free ligand. The bound ligand in the aqueous phase and/or the free ligand adsorbed with the solvent onto the solid phase, can be measured by th~ appropriate anal~rtical method chosen according to the type of labelling agent used for marking~the hapten.
In a variation of the above configuration, the aqueous phase of the immunological reaction after the necessary incubation period, is passed through a porous cellJ such as for instance through a pDrOuS spiral tubing surrounded by the solvent. By choosing the appropriate length of spiral tubing and the suitable solvent, on passage from one end of the tubing to the other end, all the free ligand ~ill be extracted into the solvent 6~
leaving the aqueous phase to carry only the bound ligand. ~ile free ligand extracted into the solvent cell and/or the bound ligand carried into the aqueous phase can be determined by a suitable analytical method chosen according to the labelling agent used to marl~ tpe hapten. The configuration just described is eminently sui~able for automation of the whole procedure.
Another configuration is the use of solid-solid separation systems which provides additional versatility and great simplicity in supplying the present invention of solvent extraction for separation of bound and free ligands in in~n~nological reactions. In this case the both liquids ~solvent and immunological system) are held in two solid matrixes which may be different,or the same. For example a strip of suital~le hydrophilic material is impregnated with the aqueous medium of the immunological reaction and the binding and ligands are adsorbed into this medi~ and allowed to incubate till equilibrium. A second strip of suitable hydrophobic material and impregnated witll the appropriate solvent is brought into intimate contact with the f:irst llydrophilic strlp. Owing ~o the distribution of the rea~ents over the very large surface area of the two strips, the extraction of the free li~and from the hydro-philic strips onto the solvent in the hydrophobic strip will be ext,remely rapid and complete. Se~aration of the two strips will then allow for datermining the labeiled hapten in both the bound and free ~actio~s.~lis configuration is also readily ammeanable to automation without Tequiring complicated auxilliary equipment.
Another simple configuration of the solid-solid extraction system -for use witll our invention consists in the preparation of two intert-wining strips one made of hydrophilic and the other of hydrophobic material. The aquèous i~imunological reagents are added to the hydrophilic portion of the twinned-strip. After 'the necessary incuoation ?eriod, the extracting solvent is added to the hydrophilic portioll. Tr.is solvent will then r3pidly migrate onto the hydrophobic portion.
During this process of migration the solvent will exert its extraction operation thus conveying with it the free iigand onto *he hydrophobic portion. Separation of the two intertwining strips will then allow for analysis of the bound and/or the free labelled ligand.
The method is also useful in enzyme immunoassay and particularly suitable in the case ~hen the labelling agent is a fluor~ent label, the measurements being carried out with a suitable spectrometer.
l~hen the labelling agent in the assay is a gamma-emitting radioisotope such as IodiPe - 125 and the upper phase is an organic solvent, the aqueous phase, after the solvent extraction will contain the anLibody -antigen complex. Accordin~ly the lower phase will be determined by the gamma counter.
The method can also bc used in the various kits available on the market for immunoassay, as appear from the ex2eriments described in some of the ~xamples.
The method according to the present invention is also applicable in the separation of free antigen from bound antigen-antibody complex in ~he case of proteins3 cells and other high molecular weight compounds if one uses two water soluble but mutuall~ incompatible polymers to induce unmixing. This generates two aqueous phases bet~een which various species may distribu~e. Such a phenomenon has been described in the literature (P.A.Albertson et.al, Nature, 184, 1465,1959; G.Johanson et.al., ~ur~J.Biochem., 33, 379, 1973). Incompatible pairs of polymers are numerous ~see for example A.Dobry e~.al., J.Polym.Sci.2, 90, 1947).
The method according to the present invention is teclmically simple, expeditious and inexpensive and should be considered as an ideal method in immunoassay technique. In order to enphasize-the long felt need in the art for the teclmique described above, it ~ould be worthwhile to quOte from a wcll known specialist text-boo}~ "Principles of co~3eti~ive 3G protein-binding assays" (~`~.D.Odell and ~,.II.Dauglladay, ~ditors), g i3 J.B.Lippincott Co., Philadelphia and Toronto, 1971, Chapter XI, page 303:
"The fact that so many dif~erent separation tec]miques have been proposed is indication of some dissatisfaction with existing methods".
It appears that the technique accordlng to the present invention S comes nearest to the requirements of the ideal method than any of the existing prior art methods.
The method is useful in the specific binding assay method, for the detection of minute concentrations of chemical substances in biological fluids. From the known nomenclature of these chemical substances the following groups can be envisaged-for their analysis:
Alkaloids, such as: morphine, codeine, dihydrocodeine, heroin, oxymorphone~
metopon, pholcodine, etc.
Barbiturates, such as: Veronal, luminal, seconal, phenobarbital, barbital, etc.
Steroids, estrogens such as: ~-estradiol, estrone, estriol, 17~ - ethyiny estradiol etc., and~rogens, progestogens;
adrenocortical hormones, etc.
-Cannabinoids and their matabolites.
Vitamins~ such as: Carothene, riboflavine, thiamine, niacin, ascorbic acid, tocopherol, phytyl - 1,4,- naphtoquinone, etc.
Amino acids and polypeptides Sugars, including saccharides and polysaccharides.
Tranquilizers, suc}- as: meprobamate, valium, oxazepam, phenotiazines, etc.
In addition to the above haptens other miscellaneous compounds suc'n as cocaine, prostaglandin, antibiotics such as penicillin, chloromycetin, actinomycetin and nucleic acids and nucleotides; insecticides, f~ngicides, bacteriocides and nematocides such as malatilion, carbamates, etc. can also be assayecl with the method according to the present invention. In general, antigens, ~aptens and their antibodies, hormones, vitamins, drugs, 3~ metabolites and tlleir receptors and binding materials may be determined using the present method.
.
Among the particular constituents to be determined the following can be mentioned:
Compensated T4; T3 uptake;
Cortisol; Insulin;
Digoxin; Triiodothyronine, Polate; Thyroxine ~Total T4);
h G H; T S H;
The organic solvents useful for the present invention must fulfil the following requirements:
~a) It should be slightly water miscible or completely water immiscible.
~b) It must possess a marked selective extraction power towards the antigen.
~c) Does not have any specific intereference with any constituent of the system.
Organic solvents to extract antigen as such are very numerous and most of them are unsuitable for the present invention. Thus all organic solvents that have complete or very marked miscibility with water cannot be used e.g. e*hanol, acetone etc. However the present literature is fortunately abundant with a large number of organic solvents which possess indeed the above criteria. Generally the organic solvents will be selected from hydrocarbons9 halohydrocarbons, oxygen derivatives of hydrocarbons, nitrogen derivatives of hydrocarbons, phosphorus - derivatives of hydrocarbons in which the hydrocarbon moiety can vary from C3 to C20. Typical examples of such organic solvents are:
methyl iso-butyl ketone7 chloroform, dichloromethane, carbontetrachloride, t-amyl alcohol, benzyl alcohol, ethyl acetate, tert-butyl methyl-ether, hexane, heptane, isooctane etc. ~e choice of the preferred solven~ for each system is made by carrying ou~ a simple experiment in each case as illustrated hereinafter.
- 11 . -~4~L63 In the follo~ing Table 1, there are illustrated the data of some of the useful solvents in respect to the criteria mentioned hereinbefore in an experiment to choose an adequate solvent for estriol. 25I estriol was selected as ligand tested for its extraction in the presence of 250 ng/ml unlabelled estriol and withou~ it.
TABLE 1 ~~
_ Solvent % Estriol extracted % solubility in Tracer onlv with 250 ng/ml water Methyl isobutyl ketone 91 94 ~0-94 1.9 ~thyl acetate 90-94 90-92 3.3 Toluene 85-91.5 90.5 0.05 Triethyl amine 86-91 86-90 1.5 Benzyl chloride 7~-80 76-78 0,05 Tert-amyl 78-82 80-S5 12.5% ~separated only alcohol after centrifugation~
Chloroform 52-53 53-54 l.01 Dichloromethane 45-47 52-51 2 ~arbon tetra- 35-38 38-40 0.08 chlor~ide -The method o~ separation according to the present invention will be hereinafter illustrated with estriol digoxin and T-4 taken as ligands, and the results ob~ained are compared with the known chromatographic column or precipitation methods.
EXAMPL~ 1 - Solvent extraction method for Digoxin RIA.
.
Materials and methods.
~ _ _ .
After the incubation time, the appropriate amount of solvent was added into each test tube ~polyproplyene), test tubes were capped and vortexed for 30 seconds (or all test tubes to~ether were shaken vigorously by inverting them for 2 minutes). All test tubes were left to stand for a few minùtes (5 - 10) and half of the amount of the solvent ~o~ the aqueous) solution was transferred to new test tubes for counting.
Experiments were also carried out in order to avoid the "physical"
separation ~transferring a definite volume after phase separation).
;3~
A copper shielded well liner was used to cover the upper phase (usually the solvent~ and each test tube was inserted into the ~amma-counter so that only the lower part of the test tube ~containing the aqueous phase with the bound fraction) could be counted. Digoxin kit components were reconstituted according to kit dirçctions for use.
Reaction mixture included 200 ~l antiserum (lot no. 9002), 400 ~l of 5I Digoxin derivative (lot no. 9007~, l00 ~l of standards (lot no.
9902) or control sera (DAD~ three level control sera) all kit components contained EDTA phosphate buffer (pH 7.4 + 0.l). Reaction was carried out for 30 minutes at room temperature and 2 ml of methyl isobutyl ketone were added to each test tube and vortexed for 30 seconds.
Separation was achieved within 5 - l0 minutes and the aqueous phase was counted on a GAh~CORD II using a shielded ~Yell liner (15 mm spacer).
Results and Conclusions I1le attached Figure l represents the standard curve obtainedl plotted as logit bound/total (B/T) versus log Digoxin concentration. Total delta % retention (slope between 0.54 and 4.6 ng/ml) is 50%. Control sera values ~Figure l~ indicates good correlation with commercially available kits for digoxin deteTmination.
Figure 2 TepreSentS a comparison of solvent extraction with chromatographic tubes method.
~XAh~L~ 2 - Solvent extraction method for T-4 ~IA. (T=thyroxine).
h~terials and methods.
~ T-4 lot no 0047 containing 3 mg!ml 8-anilino-naphthalene sulfonic acid (ANS) in Tris-h'laleate buffer pH 7.3 + 0.l (Tris 1.47 g, h1aleic Acid 0.62 g. Disodium ~DTA 0.22 g) in 150 ml of doubly-distilled water.
Antis~rum lot no. 0007 reconstituted ~Yith same buffer. T-4 standards prepared in buffer solution, reaction mixture included 200 pl of 30 reconstituted I-T-4.50 ~l standards or serum samples and 400 ~l of ~14t;~463 antiserum was incubated for 30 minutes at room temperature. Three control sera (TRI-Rac - lot no. RCS-16) level I~ level II and Level III
were also assayed with the standard curve.
After the incubation, 200 ~l of saturated solution of(~l4)2S04 and 1 ml of tert-amyl-alcohol werP added to each test tube, All test tubes were capped and rotated (upside domn) for two minutes. llle aqueous phase was counted on GA~r~ACORD II ~ tcounter) using a shielded well liner ~no physical separation was required).
Results and conclusions Results were calculated as % Bound over Bound Zero ~BIBo), and are represented in the attached graph (Figure 3), Control sera values were calculated and also are represented. These values are compared ~o the values obtained by the Ames T-4 kits (Tetralute and Seralute T-4 RIA).
Prom the results obtained, it can be seen that the slope between the 1 - 5 mcg/dl standard and the 24 mcg/dl standard is 43%.
The control sera values correlate well with the values obtained with the commercial kits. This in~lies that the system can distinguish between normal and hyyerthyroid, and hypothyroid values.
EXA~LE 3 - SOLVENT EXTRACTION metllod for Estriol RIA.
Materials and methods.
. .. ~
Buffer:
Estriol RIA system was carried out in ~DTA phosphate buffer (0.01 M, pH 7.4~.
Antisera:
.
Two different sources of antisera used:
(1) antiserum which was produced by rabbits injected with ~striol 6-0 carbox~ethyl BSA (R-6S, obtained from Ames Yissu~ ReseaTGh Laboratories used as crude antiser~).
~2) Antisera which were produced by rabbits injected with ~striol-3-OBSA (R-63, R-64~ R-65, produced at tlle Technion, Haifa, and used as purified Immunoglobulins) 36~o ammonium sulfate precipitation.
_ 14 ~4V~;3 Estrogen Tracers:
5I-Estriol produced at the Ames-Yissum Laboratories. H Estriol pur-chased from New England Nuclear (N.E.N.) H Estrone and H Estradiol also purchased from N.E.N.
Estrogen and Estrogen derivatives:
Estriol (Merck), Estradiol (Sigma), Estrone (Sigma). Estriol-3-0 Carboxymethyl (produced at Technion).
A. Standard Cu~ve Determination 100 ~1 of tracer in EDTA phosphate buffer (containing 0.2% polyvinyl alcohol, P.V.A.), 200 ~1 of Estriol standard (in the same buffer) and 100 ~1 of antiserum (using the appropriate dilution) were incubated for 60 - 90 minutes at room temperature.
After the incubation time, the Estriol bound was separated from the free using different separation methods and the radioactivity of the free fractions was determined. Standard curve was plotted as a free versus log Estriol concentration (ng/ml).
B. Separation Methods 1. *Sephadex Col~ns - Reaction Mixture (200 ~1) was applied on top a G-10 *~ephadex column and left to penetrate into the column~
Columns were then washed with 3 ml of E~TA phosphate buffer to remove the bound fraction.
As known, specific binding assays are based on the principle of monitoring specific binding reactions in which the extent of binding is a function of the unknown ligand present, by means of a labelled component. Among the known methods the following five specific binding assay techniques can be mentioned: Radioimmunoassay (RIA), metalloimm-unoassay (~IA), free radical assay technique (FRAT), hemaglutination inhibition (HI) and enzyme multiplied immunoassay technique (~lIT).
In the first t~Yo techniques, tlle mixture comprising the unlabelled ligand, labelled ligand and antibody is allowed to reach an equilibrium and the antibody-bound ligand is separated from the free ligand.
lS In the radioimmunoassay, the ligand is labelled with a radioactive isotope, whereas in the metalloimmunoassay the ligand is labelled with a metal-containing reagent which contains also a suitable functional group by means of which one can attach the metal reagent to the hapten desired to be assayed. A full description of the latter is gi~en in the U.K.
Patent Number 1.550.540.
The opeTation of separating the free fraction from the bound one, is of great iD~oortance and its accuracy determines the sensitivity and precision o~ the entire specific binding assay technique. In selecting and assessing a separation operation, it is useful to consider the criteria that should be fulfilled in order to obtain the desired resu~.
The following major requïrements of an ideal separation can be mentioned:
~a~ A complete separation between the bound and free fraction s~ould occur ~ith a ~Jide margin for error in the conditions used for separatinn.
~lis implies that the method should not interfere with -the prir,lary binding reaction.
. 2 -~, , :~14q;)~63 ~b) The operation should be simple7 quic~ and inexpensive, utilizing known reagents and available equipment.
(c) The operation should not be affected by the ligancl utilized, since otherwise difficulties in standardization will be encountered. A number of separation operations can be found in the prior art and a brief description thereof is heTeinafter presented:
- Adsorption methods. Usually the free fraction is adsorbed; the adsorbents are commonly used for small peptides, steroid and thyroid hormones. The adsorption is determined by many factors, including the Telative surface area of adsorbent, size and charge of antigen, nature and concentration of the competing proteins, temperature, ionic strength and pH. It is essential that the conditions for the use of the adsorption met~lods are optimized experimentally for eacl~ antigen.
~xamples of col~only encountered adsorbents are charcoal and silicates.
~le major problém is that always charcoal contains some unactivated material which has a high non-speci:Eic avidity for a wide range of substances, so that the bound fraction as well ~he free one may be adsorbed. In addition, charcoal may remove the bound antigen, particularly when a low-avidity binding agent is employed. The drawbac~
of silicates is that t~ne adsorption is dependent on the protein present, so that the optimal relative proportions of adsorbent and serum must be determined experimentally for each antigen.
- ~ractional precipitation with neutral salts or organic solvents which leaves in solution the free fraction. The forces that determine this effect are largely electrostatic and can be in1uenced by ~any factors such as temperature, p~-l, protein concentTation and dielectric constant of the medium. Optimal conditions must be determined ex~er1~.entally, with attention to the parameters of: pH, buffer, protein and salt concentrations. The applicability of these simple precipitations, depends mainly on the properties of the free antigen.
_ 3 -A~nonium sulfate has been successfully employed in assays of plasma and urine extracts for many small peptides. Ethanol is perhaps more versa-tile, being suitable for a range of peptides. Polyethylene glycol has proved satisfactory for assays of insulin and may be useful for preci-pitation of peptide hormones which do not bind to standard adsorbents.
All the methods described in this para suffer from the disadvantage that they require at least one centrifugation step.
- Gel filtration. Since bound complexes are considerably larger in their molecular volume than the free antigens, they can be clearly separated by using an appropriate grade of a gel such as *Sephadex. The gel has been used in two ways for phase separation; the incubate can be passed through a column under proper conditions such that the bound moiety is eluted, leaving the free moiety within the gel. A second method widely employed in competitive protein-binding technique is that of gel equili-bration. The gel is present throughout the incubation and can be pre-dispensed in equilibrium with the tracer binder solution. The free moiety within the pores o~ the gel is thus in equilibrium with that without. When the test sample is added to the gel suspension, equili-brium is rapidly reestablished, since the binding of tracer to gel is rev~rsible. After allowing the gel particles to settle, a sample o~
the supernatant, which includes the bound fraction, can be removed and counted.
- Paper chromatoelectrophoresis. A suitable paper is selected which ad-sorbs the free hapten at its site of application, while the bound frac-tion move from the origin under the influence of an electric current andbuffer flow caused by evaporation. The disadvantage of this method is that it is too complex, time consuming and expensive to be used as a routine and also does not lend itself easily to automation. Also papers and antigens vary in their affinity characteristics which makes repro-ducibility difficult.
* Trade Mark ~4~)463 In view of the drawbacl.cs of the above prior art methods it appears tllat it is a long felt need for an i~proved method of separating the free fraction from the bound fraction in the specific binding assay technique. The invention ~herefore consists in!an improvement in the specific binding assay method for assaying a medium for a ligand involving the steps of :
(a) contacting said mediu~ with reagent means which includes a labelled constitu0nt comprising a conjugate of a labelling substance and a binding col~ponent and which forms, with said ligand to be determined, a binding Teaction system producing a bound-fraction and a free-fraction of said labelled constituent, the quantity of labelling substance resulting in said bound-fraction being a function of the amount of said ligand present in said liquid medi~j (b~ separating said bound-fraction rom said free-fraction; and lS ~c) determining the quantity of labelling substance in either the bound-fraction or the free-fraction, the improvement consisting in that the separation of said bound-fraction from said f~ee-fraction lS carried out by a solvent extraction operation usm g an organic solvent whicl~ is water immisclble having the property of freezing the equilibrium between the free-fractlon and bound-fraction.
The method is applicable for any immunoassay technique (e.g. radioimmu-noassay, free radical assay and metalloimmunoassay as described in the U.K. Patent No. 1,550.5~0), which requires a separating opeTation between the bound and free f~acti~n. ~ven though the general principles of solvent extraction have been well ~no~ for a long time, the use of solvent e~traction for this separation has not been used in im~unoassay techniques. The major reason for this lies in the expected interference of the solvent with the i~unological reaction, either through damage caused to the specific binding protein by the solvent or through drastic ch~nges caused by the solvent in the antibody-antigen equilib~ium ~:~4~63 reaction at the time of separating the bound and free fractions by the solvent extraction operation. It is therefore not surprising that the extensive literature on immunoassay techniques does noi report any successful example of applying solvent extraction techniques for separating bound and free frac~ions in rout me immunoassay techniques. The use of a scintillation liquid containing toluene has been suggested to extract tritium-labelled aldosterone by repeated operations for subsequent measurement in the scintillation counter, but the cumbersome procedure described (J.P.Jowett et.al, Clinical Science and ~lolecular Medicine, 1973 pages 607-623) did not indicate any possibility of general applicability. It was therefore surprising and unexpected, when it was found according to the present invention that by the use of appropriate solvent and conditions, a general method could be devised for seyarating the bound and free ligands in immunoassays without adversely affecting either the binding ability of the bindin2 protein or the binding protein-ligand equi~brium. Moreover the method accordlng to the present invention can be of wide and general application to radioiminunoassay systems as well as to other immunoassays using non-isotopic labelling agents for mar};ing the haptens to be analyzed. One of the advantages which characteri~e the method according to the present invention is its versatility and the variety of configurations of the liquid-liquid system which can be applied for achieving t~e desired separation of free from bound ligand. In addition to the common liquid-liquid system, it can be envisaged various configurations in which the liquid-solvent and/or the aqueous mediu~ of the immunological reaction, are immobilized in a solid matrix. Thus the method wouid be applicable in a liquid-liquid system, a solid-liquid system and even a solid-solid system.
In a liquid-liquid system, the in~mUnological reaction woul~ be allowed to proceed in the usual manner in tile aqueous medium. Follo~Ying attainment of equilibrium in the reaction, an appropriate amount of a suitable solvent 6~3 is added, and after a thorough r,lixing, the t\~o phases are allol.ed to separate, the free ligand being trans~erred into the extracting solvent.
If a ~-radiating Tadioisotope has been used for hapten-labelling, separation into zones takes place spontaneously,f the bound and/or the free fraction being determined without actual separation of the aqueous and solvent phases into dir~erent vessels. This is an important advantage by itself, particularly for labelled radioactive elements since it decreases the number of operational steps. If deemed desirable to separate the aqueous and solvent phases, this can be done by any usual procedures, including the use of membrane systems.
Another configuration for separating the free from bound fraction is a solid-liquid system, in which the liquid is held in a solid matrix.
After the immunological reaction is carried out in the usual manner in an aqueous solution and the required incubation period for attainment of equilibrium is allowed, an appropriate so:lvent is introduced and brought into intimate contact with the aqueous phase. After a thorough mixing, the two phases ~aqueous and solvent~ are allo~ed to separate, and an appropriate hydrophobic but solvent~adsorbent material in the form of a strip, or other suitable form, is introduced into the reaction ~ixture.
Adsorption of the solvent onto tlle solid material will carry with it the free ligand. The bound ligand in the aqueous phase and/or the free ligand adsorbed with the solvent onto the solid phase, can be measured by th~ appropriate anal~rtical method chosen according to the type of labelling agent used for marking~the hapten.
In a variation of the above configuration, the aqueous phase of the immunological reaction after the necessary incubation period, is passed through a porous cellJ such as for instance through a pDrOuS spiral tubing surrounded by the solvent. By choosing the appropriate length of spiral tubing and the suitable solvent, on passage from one end of the tubing to the other end, all the free ligand ~ill be extracted into the solvent 6~
leaving the aqueous phase to carry only the bound ligand. ~ile free ligand extracted into the solvent cell and/or the bound ligand carried into the aqueous phase can be determined by a suitable analytical method chosen according to the labelling agent used to marl~ tpe hapten. The configuration just described is eminently sui~able for automation of the whole procedure.
Another configuration is the use of solid-solid separation systems which provides additional versatility and great simplicity in supplying the present invention of solvent extraction for separation of bound and free ligands in in~n~nological reactions. In this case the both liquids ~solvent and immunological system) are held in two solid matrixes which may be different,or the same. For example a strip of suital~le hydrophilic material is impregnated with the aqueous medium of the immunological reaction and the binding and ligands are adsorbed into this medi~ and allowed to incubate till equilibrium. A second strip of suitable hydrophobic material and impregnated witll the appropriate solvent is brought into intimate contact with the f:irst llydrophilic strlp. Owing ~o the distribution of the rea~ents over the very large surface area of the two strips, the extraction of the free li~and from the hydro-philic strips onto the solvent in the hydrophobic strip will be ext,remely rapid and complete. Se~aration of the two strips will then allow for datermining the labeiled hapten in both the bound and free ~actio~s.~lis configuration is also readily ammeanable to automation without Tequiring complicated auxilliary equipment.
Another simple configuration of the solid-solid extraction system -for use witll our invention consists in the preparation of two intert-wining strips one made of hydrophilic and the other of hydrophobic material. The aquèous i~imunological reagents are added to the hydrophilic portion of the twinned-strip. After 'the necessary incuoation ?eriod, the extracting solvent is added to the hydrophilic portioll. Tr.is solvent will then r3pidly migrate onto the hydrophobic portion.
During this process of migration the solvent will exert its extraction operation thus conveying with it the free iigand onto *he hydrophobic portion. Separation of the two intertwining strips will then allow for analysis of the bound and/or the free labelled ligand.
The method is also useful in enzyme immunoassay and particularly suitable in the case ~hen the labelling agent is a fluor~ent label, the measurements being carried out with a suitable spectrometer.
l~hen the labelling agent in the assay is a gamma-emitting radioisotope such as IodiPe - 125 and the upper phase is an organic solvent, the aqueous phase, after the solvent extraction will contain the anLibody -antigen complex. Accordin~ly the lower phase will be determined by the gamma counter.
The method can also bc used in the various kits available on the market for immunoassay, as appear from the ex2eriments described in some of the ~xamples.
The method according to the present invention is also applicable in the separation of free antigen from bound antigen-antibody complex in ~he case of proteins3 cells and other high molecular weight compounds if one uses two water soluble but mutuall~ incompatible polymers to induce unmixing. This generates two aqueous phases bet~een which various species may distribu~e. Such a phenomenon has been described in the literature (P.A.Albertson et.al, Nature, 184, 1465,1959; G.Johanson et.al., ~ur~J.Biochem., 33, 379, 1973). Incompatible pairs of polymers are numerous ~see for example A.Dobry e~.al., J.Polym.Sci.2, 90, 1947).
The method according to the present invention is teclmically simple, expeditious and inexpensive and should be considered as an ideal method in immunoassay technique. In order to enphasize-the long felt need in the art for the teclmique described above, it ~ould be worthwhile to quOte from a wcll known specialist text-boo}~ "Principles of co~3eti~ive 3G protein-binding assays" (~`~.D.Odell and ~,.II.Dauglladay, ~ditors), g i3 J.B.Lippincott Co., Philadelphia and Toronto, 1971, Chapter XI, page 303:
"The fact that so many dif~erent separation tec]miques have been proposed is indication of some dissatisfaction with existing methods".
It appears that the technique accordlng to the present invention S comes nearest to the requirements of the ideal method than any of the existing prior art methods.
The method is useful in the specific binding assay method, for the detection of minute concentrations of chemical substances in biological fluids. From the known nomenclature of these chemical substances the following groups can be envisaged-for their analysis:
Alkaloids, such as: morphine, codeine, dihydrocodeine, heroin, oxymorphone~
metopon, pholcodine, etc.
Barbiturates, such as: Veronal, luminal, seconal, phenobarbital, barbital, etc.
Steroids, estrogens such as: ~-estradiol, estrone, estriol, 17~ - ethyiny estradiol etc., and~rogens, progestogens;
adrenocortical hormones, etc.
-Cannabinoids and their matabolites.
Vitamins~ such as: Carothene, riboflavine, thiamine, niacin, ascorbic acid, tocopherol, phytyl - 1,4,- naphtoquinone, etc.
Amino acids and polypeptides Sugars, including saccharides and polysaccharides.
Tranquilizers, suc}- as: meprobamate, valium, oxazepam, phenotiazines, etc.
In addition to the above haptens other miscellaneous compounds suc'n as cocaine, prostaglandin, antibiotics such as penicillin, chloromycetin, actinomycetin and nucleic acids and nucleotides; insecticides, f~ngicides, bacteriocides and nematocides such as malatilion, carbamates, etc. can also be assayecl with the method according to the present invention. In general, antigens, ~aptens and their antibodies, hormones, vitamins, drugs, 3~ metabolites and tlleir receptors and binding materials may be determined using the present method.
.
Among the particular constituents to be determined the following can be mentioned:
Compensated T4; T3 uptake;
Cortisol; Insulin;
Digoxin; Triiodothyronine, Polate; Thyroxine ~Total T4);
h G H; T S H;
The organic solvents useful for the present invention must fulfil the following requirements:
~a) It should be slightly water miscible or completely water immiscible.
~b) It must possess a marked selective extraction power towards the antigen.
~c) Does not have any specific intereference with any constituent of the system.
Organic solvents to extract antigen as such are very numerous and most of them are unsuitable for the present invention. Thus all organic solvents that have complete or very marked miscibility with water cannot be used e.g. e*hanol, acetone etc. However the present literature is fortunately abundant with a large number of organic solvents which possess indeed the above criteria. Generally the organic solvents will be selected from hydrocarbons9 halohydrocarbons, oxygen derivatives of hydrocarbons, nitrogen derivatives of hydrocarbons, phosphorus - derivatives of hydrocarbons in which the hydrocarbon moiety can vary from C3 to C20. Typical examples of such organic solvents are:
methyl iso-butyl ketone7 chloroform, dichloromethane, carbontetrachloride, t-amyl alcohol, benzyl alcohol, ethyl acetate, tert-butyl methyl-ether, hexane, heptane, isooctane etc. ~e choice of the preferred solven~ for each system is made by carrying ou~ a simple experiment in each case as illustrated hereinafter.
- 11 . -~4~L63 In the follo~ing Table 1, there are illustrated the data of some of the useful solvents in respect to the criteria mentioned hereinbefore in an experiment to choose an adequate solvent for estriol. 25I estriol was selected as ligand tested for its extraction in the presence of 250 ng/ml unlabelled estriol and withou~ it.
TABLE 1 ~~
_ Solvent % Estriol extracted % solubility in Tracer onlv with 250 ng/ml water Methyl isobutyl ketone 91 94 ~0-94 1.9 ~thyl acetate 90-94 90-92 3.3 Toluene 85-91.5 90.5 0.05 Triethyl amine 86-91 86-90 1.5 Benzyl chloride 7~-80 76-78 0,05 Tert-amyl 78-82 80-S5 12.5% ~separated only alcohol after centrifugation~
Chloroform 52-53 53-54 l.01 Dichloromethane 45-47 52-51 2 ~arbon tetra- 35-38 38-40 0.08 chlor~ide -The method o~ separation according to the present invention will be hereinafter illustrated with estriol digoxin and T-4 taken as ligands, and the results ob~ained are compared with the known chromatographic column or precipitation methods.
EXAMPL~ 1 - Solvent extraction method for Digoxin RIA.
.
Materials and methods.
~ _ _ .
After the incubation time, the appropriate amount of solvent was added into each test tube ~polyproplyene), test tubes were capped and vortexed for 30 seconds (or all test tubes to~ether were shaken vigorously by inverting them for 2 minutes). All test tubes were left to stand for a few minùtes (5 - 10) and half of the amount of the solvent ~o~ the aqueous) solution was transferred to new test tubes for counting.
Experiments were also carried out in order to avoid the "physical"
separation ~transferring a definite volume after phase separation).
;3~
A copper shielded well liner was used to cover the upper phase (usually the solvent~ and each test tube was inserted into the ~amma-counter so that only the lower part of the test tube ~containing the aqueous phase with the bound fraction) could be counted. Digoxin kit components were reconstituted according to kit dirçctions for use.
Reaction mixture included 200 ~l antiserum (lot no. 9002), 400 ~l of 5I Digoxin derivative (lot no. 9007~, l00 ~l of standards (lot no.
9902) or control sera (DAD~ three level control sera) all kit components contained EDTA phosphate buffer (pH 7.4 + 0.l). Reaction was carried out for 30 minutes at room temperature and 2 ml of methyl isobutyl ketone were added to each test tube and vortexed for 30 seconds.
Separation was achieved within 5 - l0 minutes and the aqueous phase was counted on a GAh~CORD II using a shielded ~Yell liner (15 mm spacer).
Results and Conclusions I1le attached Figure l represents the standard curve obtainedl plotted as logit bound/total (B/T) versus log Digoxin concentration. Total delta % retention (slope between 0.54 and 4.6 ng/ml) is 50%. Control sera values ~Figure l~ indicates good correlation with commercially available kits for digoxin deteTmination.
Figure 2 TepreSentS a comparison of solvent extraction with chromatographic tubes method.
~XAh~L~ 2 - Solvent extraction method for T-4 ~IA. (T=thyroxine).
h~terials and methods.
~ T-4 lot no 0047 containing 3 mg!ml 8-anilino-naphthalene sulfonic acid (ANS) in Tris-h'laleate buffer pH 7.3 + 0.l (Tris 1.47 g, h1aleic Acid 0.62 g. Disodium ~DTA 0.22 g) in 150 ml of doubly-distilled water.
Antis~rum lot no. 0007 reconstituted ~Yith same buffer. T-4 standards prepared in buffer solution, reaction mixture included 200 pl of 30 reconstituted I-T-4.50 ~l standards or serum samples and 400 ~l of ~14t;~463 antiserum was incubated for 30 minutes at room temperature. Three control sera (TRI-Rac - lot no. RCS-16) level I~ level II and Level III
were also assayed with the standard curve.
After the incubation, 200 ~l of saturated solution of(~l4)2S04 and 1 ml of tert-amyl-alcohol werP added to each test tube, All test tubes were capped and rotated (upside domn) for two minutes. llle aqueous phase was counted on GA~r~ACORD II ~ tcounter) using a shielded well liner ~no physical separation was required).
Results and conclusions Results were calculated as % Bound over Bound Zero ~BIBo), and are represented in the attached graph (Figure 3), Control sera values were calculated and also are represented. These values are compared ~o the values obtained by the Ames T-4 kits (Tetralute and Seralute T-4 RIA).
Prom the results obtained, it can be seen that the slope between the 1 - 5 mcg/dl standard and the 24 mcg/dl standard is 43%.
The control sera values correlate well with the values obtained with the commercial kits. This in~lies that the system can distinguish between normal and hyyerthyroid, and hypothyroid values.
EXA~LE 3 - SOLVENT EXTRACTION metllod for Estriol RIA.
Materials and methods.
. .. ~
Buffer:
Estriol RIA system was carried out in ~DTA phosphate buffer (0.01 M, pH 7.4~.
Antisera:
.
Two different sources of antisera used:
(1) antiserum which was produced by rabbits injected with ~striol 6-0 carbox~ethyl BSA (R-6S, obtained from Ames Yissu~ ReseaTGh Laboratories used as crude antiser~).
~2) Antisera which were produced by rabbits injected with ~striol-3-OBSA (R-63, R-64~ R-65, produced at tlle Technion, Haifa, and used as purified Immunoglobulins) 36~o ammonium sulfate precipitation.
_ 14 ~4V~;3 Estrogen Tracers:
5I-Estriol produced at the Ames-Yissum Laboratories. H Estriol pur-chased from New England Nuclear (N.E.N.) H Estrone and H Estradiol also purchased from N.E.N.
Estrogen and Estrogen derivatives:
Estriol (Merck), Estradiol (Sigma), Estrone (Sigma). Estriol-3-0 Carboxymethyl (produced at Technion).
A. Standard Cu~ve Determination 100 ~1 of tracer in EDTA phosphate buffer (containing 0.2% polyvinyl alcohol, P.V.A.), 200 ~1 of Estriol standard (in the same buffer) and 100 ~1 of antiserum (using the appropriate dilution) were incubated for 60 - 90 minutes at room temperature.
After the incubation time, the Estriol bound was separated from the free using different separation methods and the radioactivity of the free fractions was determined. Standard curve was plotted as a free versus log Estriol concentration (ng/ml).
B. Separation Methods 1. *Sephadex Col~ns - Reaction Mixture (200 ~1) was applied on top a G-10 *~ephadex column and left to penetrate into the column~
Columns were then washed with 3 ml of E~TA phosphate buffer to remove the bound fraction.
2. Solvent Extraction After the incubation, 200 ~1 of the reaction mixture was extracted with 400 ~1 of solvent (see results) by vortexing 30 seconds (using polypropylene, plug~stoppered test tubes). After phase separation, 200 ~1 of the solvent were transferred to test tubes and counted.
Results and conclusions -The solvent extraction method results were calculated as ~ free over log estriol concentration and are represented in Figure 4. mese values are * Trade Mark 4~i3 compared with the results obtained in the separation of the bound and free fractions on Sephadex columns. As can be seen from this comyarison the solvent extraction method witll RIA can be used to determine estriol in biological fluids.
EXA~L~ 4 - Solvent extraction method for Di oxin RIA
~ g A similar experiment as in Lxample 1 was carried out with the commercially available Di~xin RIA l;it supplied by Becton DiclcinSOn.
All kit components were used unchanged except for the 125I-Digoxin tracer wllich was prepared specially for this experiment by Becton Dickinson research laboratory, so that the appropriate amount of radioactivity was contained in 0.1 ml portions instead o~ 1 ml portions as provided in the original kit.
DAD~ Control sera (I, II and IV) were reconstituted and assayed together with the kit, according to the kit instructions. After the prescribed incubation time, 1.0 ml of t-amyl alcohol was added to each tube, vortexed to extract the free digoxin and after phase separation, 150 pl of the aqueous phase l~ere transferred for radioactivity counting. The results obtained for the standard cu¢ve (expressed as % B/Bo) and those for the control sera (expressed as % B/Bo and mg Digoxin/ml) ~e summarized in Table 2 below:
Table 2.
Digoxin B/Bo DADA CONTROL SERA
(ng/ml) (%~ oB/~O ng/ml Digoxin Found ~xPected 0.5 74.0 Leve] I 61.1 0.72 0.71-1.06 1.0 49.0 1.5 35.0 Level II 32.3 1.78 1.55-2.17 2.0 27.5
Results and conclusions -The solvent extraction method results were calculated as ~ free over log estriol concentration and are represented in Figure 4. mese values are * Trade Mark 4~i3 compared with the results obtained in the separation of the bound and free fractions on Sephadex columns. As can be seen from this comyarison the solvent extraction method witll RIA can be used to determine estriol in biological fluids.
EXA~L~ 4 - Solvent extraction method for Di oxin RIA
~ g A similar experiment as in Lxample 1 was carried out with the commercially available Di~xin RIA l;it supplied by Becton DiclcinSOn.
All kit components were used unchanged except for the 125I-Digoxin tracer wllich was prepared specially for this experiment by Becton Dickinson research laboratory, so that the appropriate amount of radioactivity was contained in 0.1 ml portions instead o~ 1 ml portions as provided in the original kit.
DAD~ Control sera (I, II and IV) were reconstituted and assayed together with the kit, according to the kit instructions. After the prescribed incubation time, 1.0 ml of t-amyl alcohol was added to each tube, vortexed to extract the free digoxin and after phase separation, 150 pl of the aqueous phase l~ere transferred for radioactivity counting. The results obtained for the standard cu¢ve (expressed as % B/Bo) and those for the control sera (expressed as % B/Bo and mg Digoxin/ml) ~e summarized in Table 2 below:
Table 2.
Digoxin B/Bo DADA CONTROL SERA
(ng/ml) (%~ oB/~O ng/ml Digoxin Found ~xPected 0.5 74.0 Leve] I 61.1 0.72 0.71-1.06 1.0 49.0 1.5 35.0 Level II 32.3 1.78 1.55-2.17 2.0 27.5
3.0 20.0 Level III 20.2 2.90 2.64-3.49 5.0 12.0 ~xample 5 - Solvent_extraction method for RIA-~stradiol 17~.
A commercially available ( ~1) RIA kit for estradiol supplied by Isopac ~las tested by the solvent extraction method as follows:
All kit components were prepared according to kit direc~ions.
S One experir,lent (A) was carried out exactly according to the Xit protocol.
A second, parallel experiment (B) was performed according to kit protocal up to, and including, the incubation step. After incubation time in experiment B, 1.6 ml of solvent (a 1:1 mixture of tert-amyl alcohol and tert-butyl methyl ether, presaturated with assay buffer) were added to all tubes and vortexed for 20 seconds. After phase separation ~about 10 minutes) the upper solvent phase, containing the free estradiol, was transferred to scintillation vials to which 5 ml of scintillation liquid (Instagel II, Packard) were added and tlle radioactivity determined.
Table III below presents the results calculated as % B/Bo for the two parallel ex~eriments. ~len plotted on a logit-log graph paper according to kit directions, a linear plot is obtained for both experiments, but the results of experiment B tusing the solvent extraction procedure) exhlbit a steeper standard curve thus allowing for a more sensitive assay.
Table 3.
~stradiol % B/Bo % B/Bo standards ~xpt. A ~it) ~xpt. B (solvent extn.) pg/ml 12.5 96.0 98.0 25.0 S9:0 S9.0 50.0 ~5.0 76.0 100.0 59.0 40.0 200.0 40.0 23.0 400.0 21.0 14.0 .
.
_ 17 ~x~lple 6 - Solvent extraction method for RIA -_phenobarbitone.
~le components used for a phenobarbitone RIA were a (311) phenobarbitone tracer (N.~.N), a phenobarbitone antiserum R-66X (Technion) and tris-maleate assay buffer (pH 7.3, 0.078 ~ Fris, 0.035 M maleic acid) containing O.l % PVP ~polyvinyl pyrrolidone).
- ~nè tracer, lO0 pl ~approx. lO nono Ci/tube) was added to test-tubes followed by the addition of 50 ~l phenobarbitone standards and lO0 ~l of diluted antiseruln. All test-tubes were incubated overnight at 4 C.
After incubation, 0.5 ml of solvent (tert-butyl methyl ether, TB~ in one e~eriment ~nd, MIBK in a parallel ex~eriment) were added to each assay tube and vortexed for 20-30 seconds. After phase separation on standing, the aqueous phase was transferred to scintillation vials for radioactivity determination. Tne follo~ing Table 4, sumn~arizes mean values ~of duplicates) ex~ressed as ~0 E/Bo.
Table 4.
Phenobarbitone % B/Bo standards TB~ rlIBK
ng/ml (tert-but~l methyl ether) (methyl isobutyl Xetone) 96.5 73.0 79.0 63.0 52.0 45.0 30.0 30.0 lO0 23.0 19.0 ~xam~le 7 - Solvent extraction method for RIA-rr1C.
_ hn assay using the solvent extraction method was developed for tetrahydrocannabinol ~nlC) metabolites using a ~ H)-THC tracer (~nersham).
All assay components were prepared 1n tris-maleate buffer (pH 7.3) containing 0.2% PVP and 0.1% Triton X450. The ~ H)-THC trace~, lO0 jul, was mixed with 50 ~l of THC standards and lO0 ~l of diluted an~i-T~IC antiserum, R-41Va (prepared at the Tec1mion) and incubated 1~ -:
A commercially available ( ~1) RIA kit for estradiol supplied by Isopac ~las tested by the solvent extraction method as follows:
All kit components were prepared according to kit direc~ions.
S One experir,lent (A) was carried out exactly according to the Xit protocol.
A second, parallel experiment (B) was performed according to kit protocal up to, and including, the incubation step. After incubation time in experiment B, 1.6 ml of solvent (a 1:1 mixture of tert-amyl alcohol and tert-butyl methyl ether, presaturated with assay buffer) were added to all tubes and vortexed for 20 seconds. After phase separation ~about 10 minutes) the upper solvent phase, containing the free estradiol, was transferred to scintillation vials to which 5 ml of scintillation liquid (Instagel II, Packard) were added and tlle radioactivity determined.
Table III below presents the results calculated as % B/Bo for the two parallel ex~eriments. ~len plotted on a logit-log graph paper according to kit directions, a linear plot is obtained for both experiments, but the results of experiment B tusing the solvent extraction procedure) exhlbit a steeper standard curve thus allowing for a more sensitive assay.
Table 3.
~stradiol % B/Bo % B/Bo standards ~xpt. A ~it) ~xpt. B (solvent extn.) pg/ml 12.5 96.0 98.0 25.0 S9:0 S9.0 50.0 ~5.0 76.0 100.0 59.0 40.0 200.0 40.0 23.0 400.0 21.0 14.0 .
.
_ 17 ~x~lple 6 - Solvent extraction method for RIA -_phenobarbitone.
~le components used for a phenobarbitone RIA were a (311) phenobarbitone tracer (N.~.N), a phenobarbitone antiserum R-66X (Technion) and tris-maleate assay buffer (pH 7.3, 0.078 ~ Fris, 0.035 M maleic acid) containing O.l % PVP ~polyvinyl pyrrolidone).
- ~nè tracer, lO0 pl ~approx. lO nono Ci/tube) was added to test-tubes followed by the addition of 50 ~l phenobarbitone standards and lO0 ~l of diluted antiseruln. All test-tubes were incubated overnight at 4 C.
After incubation, 0.5 ml of solvent (tert-butyl methyl ether, TB~ in one e~eriment ~nd, MIBK in a parallel ex~eriment) were added to each assay tube and vortexed for 20-30 seconds. After phase separation on standing, the aqueous phase was transferred to scintillation vials for radioactivity determination. Tne follo~ing Table 4, sumn~arizes mean values ~of duplicates) ex~ressed as ~0 E/Bo.
Table 4.
Phenobarbitone % B/Bo standards TB~ rlIBK
ng/ml (tert-but~l methyl ether) (methyl isobutyl Xetone) 96.5 73.0 79.0 63.0 52.0 45.0 30.0 30.0 lO0 23.0 19.0 ~xam~le 7 - Solvent extraction method for RIA-rr1C.
_ hn assay using the solvent extraction method was developed for tetrahydrocannabinol ~nlC) metabolites using a ~ H)-THC tracer (~nersham).
All assay components were prepared 1n tris-maleate buffer (pH 7.3) containing 0.2% PVP and 0.1% Triton X450. The ~ H)-THC trace~, lO0 jul, was mixed with 50 ~l of THC standards and lO0 ~l of diluted an~i-T~IC antiserum, R-41Va (prepared at the Tec1mion) and incubated 1~ -:
4~3 overnight at 4C. Three experiments were carried out in parallel.
In one experiment the separation of the free and bound fractions was carried out by the dextran-coated charcoal method and the o~her two ex~eriments used the solvent extraction metllod, tert-amyl alcohol in one and hlIBX in the other. The solvent, 500 ~1, was added to the assay tubes and vortexed for 20-30 seconds. ~fter phase separation ~10-~0~-minutes) 150 ~1 of the aqueous phase were transferred to scintillation vials for radioactivity determination. The following Table 5 su~ arizes the results, expressed as % B/Bo for the three experiments.
Table S.
% B/Bo THC standards t-amyl alcohol hlIBK Charcoal ng/ml ____ ___ 1 90 9~ 9 2 ~6 34 4 77 6~ 86 .
1~ -
In one experiment the separation of the free and bound fractions was carried out by the dextran-coated charcoal method and the o~her two ex~eriments used the solvent extraction metllod, tert-amyl alcohol in one and hlIBX in the other. The solvent, 500 ~1, was added to the assay tubes and vortexed for 20-30 seconds. ~fter phase separation ~10-~0~-minutes) 150 ~1 of the aqueous phase were transferred to scintillation vials for radioactivity determination. The following Table 5 su~ arizes the results, expressed as % B/Bo for the three experiments.
Table S.
% B/Bo THC standards t-amyl alcohol hlIBK Charcoal ng/ml ____ ___ 1 90 9~ 9 2 ~6 34 4 77 6~ 86 .
1~ -
Claims (25)
1. An improvement in the specific binding assay method including immunoassay technique for assaying a medium for a ligand involving the steps of:
(a) contacting said medium with reagent means which includes a labelled constituent comprising a conjugate of a labelling substance and a binding component and which forms with said ligand to be determined, a binding reaction system producing a bound-fraction and a free-fraction of said labelled constituent, the quantity of labelling substance resulting in said bound-fraction being a function of the amount of said ligand pre-sent in said ligand medium;
(b) separating said bound-fraction from said free-fraction, and (c) determining the quantity of labelling substance in either the bound-fraction or the free-fraction;
the improvement consisting in that the separation of said bound-fraction from said free-fraction is carried out by a solvent extraction operation using an organic solvent to provide the formation of two phases, said organic solvent being slightly water miscible or completely water immis-cible, having the property of a marked selective extraction lower towards the antigen and possessing the characteristic of freezing the equilibrium between the free- and bound-fraction.
(a) contacting said medium with reagent means which includes a labelled constituent comprising a conjugate of a labelling substance and a binding component and which forms with said ligand to be determined, a binding reaction system producing a bound-fraction and a free-fraction of said labelled constituent, the quantity of labelling substance resulting in said bound-fraction being a function of the amount of said ligand pre-sent in said ligand medium;
(b) separating said bound-fraction from said free-fraction, and (c) determining the quantity of labelling substance in either the bound-fraction or the free-fraction;
the improvement consisting in that the separation of said bound-fraction from said free-fraction is carried out by a solvent extraction operation using an organic solvent to provide the formation of two phases, said organic solvent being slightly water miscible or completely water immis-cible, having the property of a marked selective extraction lower towards the antigen and possessing the characteristic of freezing the equilibrium between the free- and bound-fraction.
2. The method according to claim 1, wherein the immunoassay method is selected from radioimmunoassay, free radical assay, enzyme assay, fluo-rescence assay and metalloimmunoassay technique.
3. The method according to claim 1, wherein the separation is carried out on free antigen from bound antigen-antibody complex present in proteins, cells or high molecular weight compounds.
4. The method according to Claim 1, wherein the bound and/or the free-fraction is analysed without actual separation between the phases into different vessels.
5. The method according to Claim 1, incorporating various configurations of separation consisting of immobilizing the liquid in a solid matrix and thus applied to a solid-liquid system or solid-solid system.
6. The method according to Claim 5, wherein after equilibrium between the two liquid phases is attained, a hydrophobic, and solvent adsorbent solid material is introduced, the solvent containing the free ligand being adsorbed on said solid material.
7. The method according to Claim 1, wherein the organic solvent is selected from the group consisting of hydrocarbons, halohydrocarbons, oxygen derivatives of hydrocarbons, nitrogen derivatives of hydrocarbons and phosphorus derivatives of hydrocarbon in which the hydrocarbon moiety is between C3 and C20.
8. The method according to Claim 7, wherein said organic solvent is methyl iso butyl ketone.
9. The method according to Claim 7, wherein said organic solvent is chloroform.
10. The method according to Claim 7, wherein said organic solvent is dichloromethane.
11. The method according to Claim 7, wherein said organic solvent is carbonetrachloride.
12. The method according to Claim 7, wherein said organic solvent is t-amyl alcohol.
13. The method according to Claim 7, wherein said organic solvent is benzyl alcohol.
14. The method according to Claim 7, wherein said organic solvent is ethyl acetate.
15. The method according to Claim 7, wherein said organic solvent is hexane.
16. The method according to Claim 7, wherein said organic solvent is iso-octane.
17. The method according to Claim 7, wherein said organic solvent is tert-butyl methyl ether.
18. The method according to Claim 1, being carried out in an automatic manner.
19. The method according to Claim 1, wherein said ligand is selected from the group consisting of antigens, hormones, barbiturates, steroids, vitamins, tranquilizers, drugs, alkaloids.
20. The method according to Claim 19, wherein said ligand is Digoxin.
21. The method according to Claim 19, wherein said ligand is Estriol.
22. The method according to Claim 19, wherein said ligand is Thyroxine (T-4).
23. The method according to Claim 19, wherein said ligand is Estradiol 17 .beta..
24. The method according to Claim 19, wherein said ligand is Phenobarbitone.
25. The method according to Claim 19, wherein said ligand is Tetrahydrocannabinol.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL56839 | 1979-03-11 | ||
| IL56839A IL56839A0 (en) | 1979-03-11 | 1979-03-11 | Improvement in specific binding assay technique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1140463A true CA1140463A (en) | 1983-02-01 |
Family
ID=11050917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000346390A Expired CA1140463A (en) | 1979-03-11 | 1980-02-25 | Solvent extraction for separation in immunoassay |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US4645747A (en) |
| JP (1) | JPS55160855A (en) |
| CA (1) | CA1140463A (en) |
| DE (1) | DE3009154A1 (en) |
| FR (1) | FR2451582A1 (en) |
| GB (1) | GB2103790B (en) |
| IL (1) | IL56839A0 (en) |
| NL (1) | NL8004290A (en) |
| SE (1) | SE442785B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4832850A (en) * | 1980-07-21 | 1989-05-23 | Technion Research And Development Foundation, Ltd. | Method and device for mass transport and separation through selective barriers |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI841023A0 (en) * | 1984-03-14 | 1984-03-14 | Labsystems Oy | FOERFARANDE FOER UTFOERING AV IMMUNOBESTAEMNINGAR |
| US5073341A (en) * | 1985-08-21 | 1991-12-17 | Biotope, Inc. | Devices for conducting specific binding assays |
| DE3682502D1 (en) * | 1985-08-21 | 1991-12-19 | Biotope Inc | METHOD AND DEVICES FOR SEPARATING, MIXING AND DETERMINING COMPONENTS IN SPECIFIC BINDING TESTS. |
| US5501949A (en) * | 1985-12-10 | 1996-03-26 | Murex Diagnostics Corporation | Particle bound binding component immunoassay |
| SE8703733L (en) * | 1987-09-28 | 1989-03-29 | Ragnar Soedergaard | METHOD OF ANALYSIS FOR DETERMINING PERCENTAGE OF FREE SUBSTANCE IN BIOLOGICAL SCIENCES THROUGH EQUAL DISTRIBUTION IN OIL / WATER SYSTEM |
| WO1989005456A1 (en) * | 1987-12-01 | 1989-06-15 | Biotope, Inc. | Methods and devices for conducting assays |
| US5252492A (en) * | 1991-03-12 | 1993-10-12 | University Of Utah Research Foundation | Fluorescence assay for ligand or receptor utilizing size exclusion |
| DE4111527C2 (en) * | 1991-04-09 | 1994-07-07 | Peter Dr Miethe | Affinity chromatographic method with mobile organic phase |
| GB2312746B (en) * | 1996-04-24 | 2000-07-19 | Molecular Light Technology Lim | Detection of an analyte in a Water Immiscible Solvent |
| US5856194A (en) | 1996-09-19 | 1999-01-05 | Abbott Laboratories | Method for determination of item of interest in a sample |
| US8993243B2 (en) * | 2011-01-10 | 2015-03-31 | Wisconsin Alumni Research Foundation | Method for isolating weakly interacting molecules from a fluidic sample |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3929981A (en) * | 1974-02-27 | 1975-12-30 | Squibb & Sons Inc | Method for determining thyroid function |
| DE2410033C2 (en) * | 1974-03-02 | 1975-09-11 | Merck Patent Gmbh, 6100 Darmstadt | Isolation of substances in aqueous solutions that are soluble in lipophilic solvents |
| GB1566098A (en) * | 1975-11-14 | 1980-04-30 | Nat Res Dev | Separation of solid and liquid components of mixtures |
| IL51224A (en) * | 1976-11-19 | 1979-09-30 | Ames Yissum Ltd | Assay method for the quanititative determination of a hapten in aqueous solution |
| US4220450A (en) * | 1978-04-05 | 1980-09-02 | Syva Company | Chemically induced fluorescence immunoassay |
| SE7812237L (en) * | 1978-11-28 | 1980-05-29 | Pharmacia Diagnostics Ab | DETERMINATION OF CONCENTRATION OF SUBSTANCES WITH THE ABILITY OF BIOSPECIFICALLY BINDING MOLECULES OF BIOLOGICAL ORIGIN |
| US4301139A (en) * | 1979-06-21 | 1981-11-17 | Ames-Yissum Ltd. | Multilayer column chromatography specific binding assay method, test device and test kit |
| IL58943A (en) * | 1979-12-12 | 1983-05-15 | Cais Michael | Method and device for mass transfer operations in immunoassays and other applications |
| IL63363A (en) * | 1981-07-20 | 1986-08-31 | Cais Michael | Non-centrifugation method for immunoassay of materials |
-
1979
- 1979-03-11 IL IL56839A patent/IL56839A0/en not_active IP Right Cessation
-
1980
- 1980-02-25 CA CA000346390A patent/CA1140463A/en not_active Expired
- 1980-03-10 DE DE19803009154 patent/DE3009154A1/en not_active Withdrawn
- 1980-03-11 JP JP2984480A patent/JPS55160855A/en active Pending
- 1980-03-11 FR FR8005450A patent/FR2451582A1/en active Granted
- 1980-04-22 GB GB08013179A patent/GB2103790B/en not_active Expired
- 1980-07-25 NL NL8004290A patent/NL8004290A/en not_active Application Discontinuation
- 1980-08-11 SE SE8005658A patent/SE442785B/en not_active IP Right Cessation
-
1983
- 1983-02-28 US US06/470,334 patent/US4645747A/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4832850A (en) * | 1980-07-21 | 1989-05-23 | Technion Research And Development Foundation, Ltd. | Method and device for mass transport and separation through selective barriers |
| US4897193A (en) * | 1980-07-21 | 1990-01-30 | Technion Research And Development Foundation, Ltd. | Device for mass transport and separation through selective barriers |
Also Published As
| Publication number | Publication date |
|---|---|
| IL56839A0 (en) | 1979-05-31 |
| SE442785B (en) | 1986-01-27 |
| FR2451582A1 (en) | 1980-10-10 |
| GB2103790B (en) | 1984-12-12 |
| SE8005658L (en) | 1982-02-12 |
| DE3009154A1 (en) | 1980-09-25 |
| JPS55160855A (en) | 1980-12-15 |
| GB2103790A (en) | 1983-02-23 |
| NL8004290A (en) | 1982-02-16 |
| US4645747A (en) | 1987-02-24 |
| FR2451582B1 (en) | 1984-03-02 |
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