CA1102789A - Immunological determination - Google Patents
Immunological determinationInfo
- Publication number
- CA1102789A CA1102789A CA297,676A CA297676A CA1102789A CA 1102789 A CA1102789 A CA 1102789A CA 297676 A CA297676 A CA 297676A CA 1102789 A CA1102789 A CA 1102789A
- Authority
- CA
- Canada
- Prior art keywords
- enzyme
- active material
- immunologically active
- receptor
- activity
- 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.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D475/00—Heterocyclic compounds containing pteridine ring systems
- C07D475/06—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
- C07D475/08—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D489/00—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula:
- C07D489/02—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula: with oxygen atoms attached in positions 3 and 6, e.g. morphine, morphinone
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
A b s t r a c t The present invention relates to a reagent for the d??ermination of an immunologically active material, comprising the immunologically active material or a receptor which can specifically bind said immunologically active material, labelled with a substance capable of modifying the extent or the mode of activity of an enzyme.
Description
The present invention relates to novel reagents for the determination of immunologically active materials and to immunological methods using said reagents.
In the last years many analytical systems based on competitive protein binding analysis (also called saturation analysis) have been developed.
The terms 'saturation analysis' and 'competitive protein binding analysis', which are synonymous, refer to analytical systems used for the determination of immunologically active materials (ligands). The results of these determinations, `
in biological fluids, are emplo~ed in medical and veterinary diagnosis. The dia~nosis is dependent upon the level of the determined substance being normal or pathological. The analytical principle is e.g. based upon the competition between a ligand and a labelled ligand for a common specific binding agent (receptor), as illustrated in the following equation:-. , , _ , .
Label Label Lalel Ligand ~ Ligand + Receptor _ Ligand + Ligand + Ligand + Ligand Equation 1 Receptor Receptor (1) (2) (3) (4) .
Hen/ 7.2.1978 ~ .
The primary reaction is a combination of one molecule of ligand with one molecule of receptor to form a bimolecular ligand/receptor complex (1). A secondary reaction is caused by addition of labelled ligand which similarly combines with receptor to form a labelled ligand/receptor complex (2). In saturation analysis the concentrations of the receptor and of the labelled ligand are constant. The concentration of receptor is limited so that the labelled ligand is in excess relative to the receptor. Under these conditions the addition of ligand causes competition between ligand and labelled ligand for binding with receptor.
Hence an increase in ligand concentration lowers the amount of labelled ligand/receptor complex. The assay principle is based on the determination of the percentage of the total labelled ligand bound to the receptor.
This percentage is inversely proportional to the amount of ligand added to the reaction mixture from the specimen to be measured or from the standard used in the assay. The decrease in the concentration of labelled ligand/receptor complex or the increase in the concentration of labelled ligand in the reaction mixture can be used to determine the ligand concentration.
The sensitivity of saturation analysis depends upon the use of a receptor which has a very high affinity for the ligand and labelled ligand. Secondly, the sensitivity also depends upon the use of a label which can be detected at very low concentration.
The specificity of saturation analysis is dependent upon the capacity of the receptor to bind exclusively the ligand and labelled ligand in a complex mixture of different molecules.
Saturation analysis has been employed using many different techniques, the differences primarily being related to the type of label used.
Generally these techniques are classified by using the broad titles 'radioassay' or 'non-radioassay', depending upon whether or not a radioactive tracer is used as the label.
Radioassay has been utilised to a greater extent than non-radioassays.
Radioassay can be further classified as radioimmunoassay or radio-receptor .
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assay, depending upon the type of receptor employed in the assay. In radioimmunoassay an antibody is used which specifically binds the ligand and labelled ligand. Alternatively, radio-receptor analysis refers to the use of any other type of biological receptor which will similarly specifically bind the ligand and labelled ligand.
In all radioassay techniques it is essential to physically separate the bound fraction (1 and 2 of Equation 1) from the unbound fraction (3 and 4 of Equation 1) of the reaction mixture. An index of the ligand concentration can subsequently be obtained by counting these fractions in a radioactivity counter and comparing the counts obtained for the unknowns specimens with those obtained for appropriate standard ligand samples subjected to the same assay. Many different and diverse methods have been described for the separation of the bound and free fractions of the radioassay reaction mixture utilising techniques including gel fitration, absorption and ion exchange chrom~tography, fractional precipitation, solid phase or electrophoresis.
Subsequent to the development of radioassay techniques in saturation analysis, methods employiny non-radioactive labels have been developed.
Methods utilising enzymes as the label have been demonstrated. These methods can have the advantage that physical separation of the bound (1 + 2) and free (3 + 4) fractions of labelled - ligand is not necessary ;n the assay procedure.
When antibody binds ligand labelled with an enzyme, the activity of the enzyme is modified. The degree of modification of the enzyme activity is indicative of the concentration of the labelled ligand in the bound fraction and hence gives an index of the concentration of ligand in the reaction mixture.
The chemical structure of the ligand-enzyme complex is extremely difficult to assess and this is a major disadvantage of enzyme-~0 immunoassay. This is undoubtedly due to the great multiplicity of amino acid side chains which are available on the enzyme surface for ` - 5 ~ 7~
complexing with the ligand. This causes great difficulty in reproducing the ligand-enzyme in different preparations of the complex.
The general lack of control of the complexing reaction results in the attachment of many ligand molecules to one enzyme molecule, although it is likely that the binding of only a few of these ligand molecules by antibody is involved in the inhibition of enzyme activity. Hence not all antibody/labelled antigen interactions result in a modification of the enzyme activity, thereby lowering the sensitivity of the technique.
Protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the multiplicity of ligand molecules on the enzyme surface. Protein-protein interaction is further increased if the ligand is also a polypeptide.
Hence the enzyme molecule induces a localised micro-environment of high protein concentration. In such situations it has been demonstrated that protein precipitation occurs, thereby causing a loss of one of the main advantages of enzymeimmunoassay by necessitating separation of antibody bound and free fractions.
A modification of enzymeimmunoassay has been described which partially overcomes these problems in that the ligand is labelled with a detector molecule which is ~f low molecular weight. In ~ this assay antibody binding of labelled ligand sterically hinders binding of detector molecule by another antibody specific for the detector molecule. The degree of hindrance is determined by competition for binding of the free ligand-detector molecule and detector molecule labelled enzyme with detector-molecule antibody.
The degree of modification of enzyme activity, as determined in normal enzymeimmunoassay, is indicative of the concentration of free ligand-detector molecule which is likewise indicative of the concentration of ligand in the reaction mixture. The advantage of this technique is that a small molecule rather than an enzyme is attached to the ligand, thereby allowing the chemical structure of the labelled ligand to be determined and thus overcoming many of the disadvantages of enzymeimmunoassay described previously. However, -, . . , - ~ .
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this system only overcomes the disadvantages of the primary blnding reaction involving the ligand and labelled ligand and transfers them to the detector system for determining the degree of antibody bound and antibody free fractions of the labelled ligand.
The disadvantages of the prior art methods are overcome by the present invention according to which an enzyme modifier is used as the label.
More particularlv the present invention relates to a reaaent for the determination of an immunologically active material, comprising the immunologically active material or a receptor which can specifically bind said immunologically active material,labelled with a substance capable of modifying the extent or the mode of activitv of an enzvme.
Furthermore the present invention relates to a method for the determination of an immunologically active material in a sample wherein the sample is contacted with a receptor which can specifically bind the immunologically active material, the immunologically active material labelled with a substance capable of modifyina the activity of an enzyme, an enz~me and an enzyme substrate, and wherein the extent or the mode of the resultina en-- zy~e activit~v is measured and ccmpared with that obtained with a standard.
Furthermore the present invention relates to a method for the determination of an immunologically active material in a sample wherein the sample is contacted with a receptor which can specifically bind the immunologically active material and which is labelled with a substance capable of modifyins the activity of an enzyme, with the immunologically active material in insolubilized form, and with an enzyme and an enzyme sllhstrate, and wherein the extent or the mode of the enzyme activitv, after the separation of the solid phase, is measured and compared with that obtained with a standard.
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The terms 'immunologically active material' or 'ligand' within this specification refer to any immunologically active substance or part thereof capable of being immunologically determined e.g. by using saturation analysis techniques. The essential requirement is that there be a receptor which will specifically bind the ligand. When the receptor is an antibody the ligand will be haptenic or ahtigenic such that specific antibody can be formed. A ligand is referred to as a hapten when it will only elicit antibody formation when attached to a compound having antigenic properties. Alternatively, a ligand is referred to as an antigen when it will elicit antibody formation without chemical modification. The ligand may vary widely in molecular weight ranging from approximately 100 - 1,000,000.
This range of molecular weight is not limiting in the assay providing receptor is available which will specifically bind the-ligand.
The ligand may be either polymeric or non-polymeric in structure. When polymeric, the ligand will usually be biological in origin and be classified as a nucleic acid polysaccharide and/or polypeptide. Alternatively, when the ligand is non-polymeric, it will generally have a molecular weight of less than 2,000 and may have a wide variety of structures, functionalities and physiological properties.
Ligands of ~articular i~portance which mav be used in the ~resent invention are amines, aminoacids, peptides, proteins, lipoproteins, gl~copr~teins, ~ sterols, steroids, lipoids, nucleic acids, mono- and polvsaccharides, alka-loids, vitamins, druas, narcotics, antibiotics, metabolités, pesticides, toxins industrial pollutants, flavourinq agents, hormDnes, enzymes, coenzymes, cellul~r or extracellular ccmponents of tissues and isolated antibodies from humans or animals. The assay is,however,not limited to only these li~ands.
1 Components of immediate potential for analysis with the system are hepatitis B-surface antigen; ferritin;tumour antigens like CEA; a-feto protein; rheumatoid factor; C-reactive proteins;
imnunoglobulin classes IgC-, IgM or IgP.;myoglobin, th~roi~ hormones includina T3 and T4, insulin; steroid horniones including testosterone or estra-diol; drugs of abuse including narcotic analgesics, like morphine;
barbiturates; stimulants like amphetamine; drugs for the treat-ment of epilepsy including diphenyl hydantoin and phenobarbjtal;
cardiac glycocytes like digoxin; vitamines like vitamine Bl?~nd folic acid. Furthermore, antibodies which have immediate po-tential for analysis with the system are those associated with infection in syphilis, gonorrhoea, brucellosis, rubella and rheumatism.
The term 'labelled immunologically active material' or 'labelled receptor' in this specification refers to a ligand, analog of a ligand or part ther~of or to a receptor which is labelled with an enzyme modifier. One, more than one, and generally less than lOO molecules of label, can be attached to a ligand or receptor molecule. Similarly, one, more than one and usually less than 5 ligand or receptor molecules can be attached to a label molecule. The attachment of extra label molecules to a ligand or receptor molecule generally increases the sensitivity of the assay provided the extra labels do not affect binding.
Attachment of a modifier molecule to a ligand or receptor molecule involves the formation of intermolecular bonds which in most cases, but not necessarily, are covalent in nature. Attachment may be accomplished in some cases in the presence of a coupling agent by the insertion of a linking group between the label and the ligand or receptor.
The mcdifier molecule can be attached directly to the liqand or receptor molecule. However it may be desirable to insert chemical bridges of various length between the mcdifier rolecule and the ligand or receptor molecules depending on the speciic assay - 9 - ~
1 en~isa~ed. In some cases it may even be advanta~eous to attach themodifier molecule and the ligand or receptor molecules sepa-rately to the same carrier molecule e.~. a macromolecule like a polypeptide or a polysacch~ride.
The term'receptor'in this specification refers to any substance which can specifically bind ligand and labelled ligand or part thereof. Generally the receptor used in the assay is a specific antibody to the ligand formed in the blood of vertebrates following injection of appropriate hapten or antigen.
Alternatively, receptors which occur naturally can also be used in the assay. This latter group includes,but is not limited to, proteins, nucleic acids and cellular membranes. Such receptors have been used in radioassay techniques for thyroxine, insulin, angiotensin and various steroid hormones.
In case the ligand is an antibody the receptor may be the antigen utilized for inducing said antibody in a host animal.
In an another embodiment the receptor can be an antibodv against the antibody to be determined.
It is not possible to ascertain with certainty the mode of receptor -action which, on binding of the labelled ligand, reduces interaction between enzyme and modifier. The most likely explanation is that the affinity of the enzyme for the modifier is reduced as a result of a change in size and net charge of the receptor/ligand-modifier complex compared to that of the ligand-modifier alone.
The tërm'modifier'within this specification refers to any substance which is capable of interacting with an enzyme such that the extent or the mode of enzyme activity is modified.
This modification may result in an inhibition, activation or a change in specificity or any other property of the enzyme which is detectable either directly or indirectly by a change in enzyme activitv or in the mode of the activity e.g. a chanae in reaction conditions like co-factor requirements or pH-opti-mum, in kinetic properties, or in activation energy. The :
1 modifier can range in size from a small molecule to a macromolecule, and its interaction w;th an enzyme molecule can be either reversible or irreversible depending whether t~e inter-molecular association is ionic or covalent in nature.
Sensitivity of the assay is dependent amon~ others upon the affinity of the receptor for the ligand and the ability of the modifier to cause a change in the extent or the mode of enzyme activity. Preferably, modification of the extent or the mode of enzyme activity is achieved with a minimal con-centration of modifier. The closer this concentration is to that of the enzyme on a molecular basis, the ~reater will be the sensitivity of the assay.
Preferably, the modifier will be an enzyme inhibitor which, on interaction with an enzyme, will inhibit its activitv. The mode of action of the inhibitor may be competitive, non-competitive, uncompetitive,allosteroic or a combination of two or more of these modes. Preferably the inhibitor should ha~e an inhibition z~ constant (concentration of inhibitor necessary for 50% inhibition of the enzyme system) below lO ~ moles/l, dependin~ on the assay performed. ~ost preferably the inhibition constant is between lO 5 and lO 5.
Any enzyme ~an be used in the assay provided there exists a modifier which will specifically modify the enzyme activity in the manner described above Enzymes of choice are stable, easily obtained at low cost and have a high turnover number and a simply performed assay system. Preferably the turnover number (molecules of product formed per molecule of enzyme in one minute) is superior to lO0 dependinq on the specific test performed. Most preferably, the turnover number is as hi~h as possible and at least 200.
Enzyme modifier systems which are particularly suitable for the present invention are dihydrofolate reductase/
methotrexate, dihydrofolate reductase/4-aminopterin, .
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8~3 1 dihydrofolate reductase/other specific inhibitors of this enzyme;
~-glucoronidase/4-deoxy-5-amino glucarlc acid and its derivatives;
biotin containing enzymes/avidin like carboxylase/avidin;
chymotrypsin/TPCK CH2-Ph ~M3 ~ H 0 ~-cystathionase/propargylalycine; alanine racemase/tri-fluoroalanine; trvptophanase/trifluoroalanine; tryptophan svnthetase/trifluoroalanine; ~-cystathionase/trifluoro-alanine; pyruvate-glutamate transaminase/trifluoro-alanine; lactic acid oxidase/2-hydroxy-3-butynoic acid;
monoamine oxidase/N,N-dimethyl nropargyl amine; and diamine oxidase/~2N-CH2-C_CCH2-NH2;
Determination of enzyme activity may be performed by moni-toring directly or indirectly the consumption of substrate or production of product at suitable pH and temperature using de-tection systems including colorimetry, spectrophotometry, fluoro-spectrophotometry gaseometry, thermometry (heat production), scintillation counting.
In order to increase the sensitivity of the system it is possible to use bioluminescence and enzymic cycling techni~ues, e.a. the techniques described by J. Lee et al in Liquid Scintillation ~ Counting:~ecent nevelopments, Stanley P.E. and Scoqqins, B.A., Academic Press, New York p. 403 and Lowry O.H. et al in J. Biol.
Chem 2_6, p. 2746-2755.
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1 In one embodiment of the present invention a labelled ligand can be used for the determination of the presence of a ligand in an unknown specimen by the simultaneous or sequential addition of the labelled ligand and the un~nown specimen to an aqueous medium, at appropriate pH, containing a receptor specific for the ligand and labelled ligand.
After a suitable incubation period the distribution of receptor bound to ligand and labelled ligand is determined by the addition of enzyme and substrates. The receptor which is specific for the ligand also binds the labelled ligand and, in so doing, reduces the interaction between the modifier and the enzyme thus decreasing the modification of enzyme activity.
Addition of ligand to the assay results in competition with the labelled ligand for binding with receptor and thereby increases the concentration of free labelled ligand in the assay. Interaction between ligand-modifier and enzyme is increased and the enzyme activity is once again affected. The modification of enzyme activity is therefore a function of l;gand concentration in the assay and is caused by unbound labelled ligand. Consequently the difference between the resulting enzyme activity and that obtained in the absence of ligand ;s indicative of the concentration Gf ligand in the unknown specimen.
One of the major advantages of this method is that separation of bound and free fractions in the procedure is unnecessary.
This does not preclude,however,the use of such a separation step in the assay after incubation of ligand and labelled ligand with receptor and prior to the estimation of enzyme activity.
Separation of fractions may be desirable in some instances for removal of substances in the specimen which may interfere with the enzyme assay. Separation may be achieved using any of the many techniques described for radioimmunoassay including gel filtration, adsorption and ion exchange chromatography, fractional precipitation, solid phase and electrophoresis.
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1 This assay is of course not limited to the determination of haptens and antigens as the ligand but can also be adapted to the identification and measurement of antibodies as the ligand.
This can e.g. be performed by labelling the antibody with an enzyme modifier and measuring the extent of enzy~e acti-vity modification following incubation of labelled antibody and specimen antibod,v with a limiting concentration of antiaen or hapten. The modification of enzyme activity is related to the specimen antibody concentration. If necessary the bound and free fractions may be separated before addition of the enzyme and the substrate.
The invention permits also for the determination of a ligand the use of a labelled receptor rather than that of a labelled ligand. Ligand in the unknown specimen reacts with excess receptor labelled with an enzyme modifier and, after incubation, excess solid phase insolubilized ligand is added and reacts with the free labelled receptor remaining. After separation of the solid phase, the modification of enz~me activity associated with the soluble ligand is measured and related to the concentration of ligand.
Z5 The reagents of the present invention can also be used in a 'sandwich' techni~ue provided the ligand has at least two binding sites. Ligand reacts with excess solid phase receptor and after incubation followed by washing, the solid phase receptor bound ligand is reacted with excess receptor labelled with an enzyme modifier. Free labelled receptor is removed by washing and the extent of enzyme modification in the separated fractions is determined. Tkis then gives an index of liqand concentration.
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The followin~ exam~les illustrate the invention.
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Example 1 Preparation of "Amino-digoxin"
To a suspension of 156 mg (0.2 mmol) digoxin in 5 ml absolute ethanol was added 10 ml 0.2 M sodium metaperiodate with stirring. The mixture became homogeneous after 10 m;nutes and then a precipitate slowly formed.
After 2 hours, 5 ml water plus 5 ml ethanol were added. 122 ~l (2.2 mmol) of ethylene glycol was added after a further 30 minutes and a dense white precipitate started to form immediately. After 80 minutes' stirring, 133 yl (2.0 mmol) ethylene diamine was added and the resultant pH of ,11.0 was adjusted to 9.5 with 0.1 M HCl and the ~ reaction mixture was allowed to stand at room temperature for 18 hou~s. The pH did not change during this time.
151.4 mg (4 0 mmol) sodium borohydride was then added and the mixture stirred for 3~ hours. The pH was then adjusted from 10.5 to 6.5 with IM formic acid (about 3 ml) and TLC of this mixture showed a single major spot with an Rf 0.15 (silica on aluminium developed in butanol:acetic acid:water/4:1:1). Digoxin had an Rf 0.7 when developed in the same system.
The solvents were evaporated to near dryness on a rotary evaporator under Yacuum using a water bath at 60. The last few ml of water - were removed by adding 95% ethanol (3 x 20 ml) and repeating the evaporation as above.
The resultant pale yellow solid was extracted three times with absolute ethanol and these combined extracts concentrated to about 4 ml and centrifuged to separate a small amount of salt which was discarded. The pale yellow supernatent solution was evaporated to dryness under a stream of dry nitrogen to give a yellow oil which showed the same Rf on TLC as the reaction mixture as described above. The product was shown to contain a free amino group b~ reacting it with "Fluram" (4-phenylspiro [fluran-2(3H), 1-phthalan]-3,~-dione) to form an intensely fluorescent compound.
The product exhibited a spectrum in concentrated sulphuric acid . ..
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similar to that of digoxin with absorption peaks at 385 and 495 m~.
The product also exhibited a strong affinity for antisera (rabbit) specific for digoxin. The most likely structure of the isolated product is as follows:
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Example 2 Preparat;on of methotrexate -aminod;qoxin conjugate 11 mg methotrexate was dissolved in 5 ml H20 and adjusted to pH 6.5.
10 mg "aminodigoxin" was dissolved in this solution and the volume adjusted to 20 ml with H20. The pH was readjusted to 6.5 and 484 mg N-ethyl-N"-(3-dimethyl am;no) propyl-carbodiimide hydrochloride, dissolved in 5 ml H20, was added to the reaction mixture. The pH
was maintained at 6.2 for 24 hours at room temperature. The conjugate - was purified on a silica gel column, using 3X ammonium citrate as a solvent. Fractions containing the desired product were combined and shown to have the dual capacity to bind strongly antisera (rabbit) specific for digoxin and also to inhibit strongly the enzyme dihydrofolate reductase (chicken liver). The most likely structure of the methotrexate-aminodigoxin conjugate is as follows:
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Example 3 Enzyme Inhib;tor Immunoassay for Digoxin 100 ~l serum was incubated at 30C for 15 minutes with 100 ~l antidigoxin antibody solution, 100 ~l NADPH solution, 100 ~l 2-mercaptoethanol solution and 550 ~l sodium phosphate buffer pH 7.5. 100 ~l Methotrexate-digoxin conjugate of example 2 (70 ~g/ml) was added and the mixture incubated for 15 minutes, after which, 100 ~l dihydro-folate reductase solution was added. The dihydrofolate reductase preparation employed was isolated from chicken liver by the method of Kaufman, B.T., & Gardiner, R.C., Journal of Biological Chemistry, Vol. 211, P 1319 (1966). The mixture was incubated for a further 3 minutes and the enzyme activity determined by the addition of 100 ~l dihydrofolate solution and monitored at 340 nm with a varian recording spectrophotometer. The results are shown in Table.
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-TABLE I
Reactants Enzyme Activity Digoxin Antidigoxin Methotrexate- Enzyme assayOD/min I Inhibition (sample conc.) antibody digoxin conjugate components (in assay) O absent O present 0.150 O
O absent 7 ng present 0.100 33 O present 7 ng present 0.145 3 _ 5 ng/ml present 7 ng present 0.125 16 10 ng/ml present 7 ng present 0.1l5 23 The reactants were added in the sequence described above.
OD = optical densitv It is evident from the above results that a few ng digoxin in serum can be de$ermined in the system described.
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:. ,, - .. ,. . . . ~ . -- 18 - i Example 4 Preparation of Methotrexate-Human Serum Albumin Conjugate 45 mg methotrexate was dissolved ;n 1.0 ml N,N-dimethyl formamide and 25 mg N-hydroxysuccinimide was added.
41 mg N,N-dicyclohexyl carbodiimide was dissolved and the mixture kept at room temperature for 13 hours. The insoluble urea byproduct was removed by filtration and 100 ~1 of the filtrate was added to a solution containing 5 mg human serum albumin in 800 ~1 O.lM sodium phosphate buffer pH 7.5 plus 100 ~1 dioxane.
,lO This reaction mixture was kept at room temperature for 30 minutes and then loaded onto a Sephadex G-25 column equilibrated with O.lM sodium phosphate buffer pH 7.5. The column was developed with this buffer and two elution peaks containing methotrexate were obtained, the first of which contained the methotrexate-human serum albumin conjugate.
Example 5 Enzyme Inhibitor Immunoassay for Human Serum Albumin.
1OOJU1 diluted serum was incubated at 30C for 15 minutes with 1OOJU1 antihuman serum albumin antibody (rabbit) solution, 100 ~1 NADPH solution, 100 ~1 2-mercaptoethanol solution and 550 yl sodium phosphate buffer pH 7.5. 100 ul methotrexate-human serum albumin conjugate (8Jug/ml) was added and the mixture incubated for 15 minutes, after which, 100Jul dihydrofolate reductase solution was added. The mixture was incubated for a further 3 minutes and the enzyme activity determined by the addition of lOO~ul dihydrofolate solution and monitored at 340 nm with a varian recording spectrophotometer.
The results are shown in Table II.
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TABLE I I
- ' . . . _ _ _ Reactants Enzyme Activity Human Serum Antihuman Methotrexate- Enzyme OD/min % Inhibition Albumin Serum albumin human serum Assay (Sample antibody albumin conjugate Component Conc.) (in assay) _ _ O absent O present 0.123 O
O absent 0.8 ~g present 0.068 45 O present 0.8 ~9 present 0.105 16 5~ug/ml present 0.8Jug present 0.092 25 10 ~g/ml present 0.8 ~9 present 0.085 31 .
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It is evident from the above results that a few ~9 human serum albumin can be determined in the system described.
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Synthesis of methotrexate-aminoethylmorphine conjugate.
a) Synthesis of 03-aminoethylmorphine In 10 ml of tetrahydrofuran (THF) freshly distilled from lithium aluminium hydride (LA~) was suspended 400 mg of LA~ un-der nitrogen. A solution of 400 mg of morphine and 400 mg of chloroacetonitrile in 4 ml of freshly distilled THF was added over 5 minutes, followed by refluxing for 1 hour. The mixture was allowed to cool and 0.6 ml of water was added followed by 0.6 ml of 10 weight % sodium hydroxide and 2 ml of water. After filtering the mixture, the salts were washed with THF, the THF
fractions combin~d, dried with magnesium sulphate under nitrogen, filte~-ed and the filtrate evaporated to yield 380 mg of 03-aminoethylmorphine.
b) Synthesis of N-t-Butoxycarboxyl-~-(03-aminoethylmorphine) glutamic aci*~ -benzylester.
A mixture of 03-aminoethylmorphine (50 mg prepared as ;, above), N-t-BOC-glutamic acid-~-benzyl ester(34 mg)and dicyclohexylcarbodiimide (25 mg) in dichloromethane (5 ml) was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate and washed with dilute sodium carbonate so-lution. The ethyl acetate solution was then extracted twice with 0.1 N hydrochloric acid and the combined!acidic extracts were .
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1 treated with sufficient 10 weight % sodium hydroxide solution to adjust the pH to 8Ø The solution was extracted twice with ethyl acetate and the combined ethyl acetate extracted were washed with brine, dried (anhydrous sodium sulphate) and evaporate~ to give a colourless oil (36 mg).
c) Synthesis of ~lutamic acid-~-(03-amidoethylmorphine) -~-benzyl ester-trifluoroacetic acid salt.
A solution of N-t-BOC-~lutamic acid-morphine derivative (36 mg, prepared as described above) in dichloromethane (3 ml) was stirred at room temperature and trifluoroacetic acid (1 ml) was added. The mixture was stirred for 15 minutes and then eva-porated to dryness. The residue was a colourless glass (40 mg).
d) Synthesis of methotrexate ~- (03- amidoethylmorphine) -~- benzyl ester.
A solution of 4-amino-4-deoxy~N10-methylpteroic acid (37 mg) in 3 ml di~ethylsulfoxide (DMSO) stirring at room tem-perature was treated with triethylamine (28 ~1) and i-butyl-chloroformate (25 ~1) and the mixture was stirred for 30 minu-tes. It was then added to a mixture of the morphine derivative (75 mg prepared as descrl~ed in ~) and triethylamine (28~1) in DMSO (2 ml) and the mixture was stirred at 60 for one hour.
The cooled mixture was diluted with water and extracted twice with ethylacetate. The combined ethyl acetate extracts were was-hed withwater and then extracted twice with O.IN hydrochloric acid. The combined acidic extracts were treated with sufficient 10 weight % sodium hydroxide to raise the pH to 8Ø The mix-ture was extracted three times with ethyl acetate and the com-. .. : . .. . : . - . - :
.
' - : ' 1 bined extracts washed with brine, dried (anhydrous sodium sul-phate) and evaporated to give a yellow oil (15 mg). This was purified using prepæative thin layer chromatography (TLC) on silica gel developing with chloroform~methanol, 4:1. The desired o~ound was obtained as a yellow solid (4 mg).
e) Synthesis of MethotreXate ~-(03-amidoethylmorphine).
The product prepared as described in d (~ mg) was mixed with O.INsodium hydroxide solution (5 ml) and the mix-ture was stirred at room temperature for 8 hours resulting in a clear yellow solution.
To this was added O.IN hydrochloric acid (5 ml) and the mixture was made upto 25 ml with sodium orthophosphate buffer (0.05 M, pH 7.4) to give a solution of the desired compound suitable for use in the enzyme assay.
Enzyme inhibitor immunoassay for morphine A mixture of 10~1 of morphine solution of suitable con-centration, 50~1 of methotrexat~-(03-amidoethylmorphine) solution (4 ng/25 ml), 50~1 of antimorphine antibody solution, 10~1 NADPH solution, 10~1 2-mercaptoethanol solution, 50~1 po-tassium chloride solution, 150~1 Tris-HCl,buffer solution (p~
7.5)containing EDTA and 10~1 of dihydrofolate reductase (E.~
casei) solution was incubated for 20 minutes at 37. The enzyme activity in the mixture was determined after the addition of 10~1 dihydrofolate solution by monitoring the change in extin-z~
ction of the solution at 340 nm using a Centrifichem centrifugal analyser. The results are shown in Table III
TABLE III
__ ~CIA~S ENZYME ACTIVITY
~)~PHINE ANTI~ PHINE ~PHINE- ENZY~!IE ASSAY . __ ME~OT~IE DD/ITin ~61NHIBITION
pg/assay ANTIBODY CONJUG~TE ~llENr3 __ _ O absent absent present O.54 O
O ~sent 3xlO M present 0.14 76 O ~resent 3xlO 8 M present 0.41 29 0.04 present 3xlO 8 M present 0.37 36 0.40 present 3xlO 8 M present 0.35 40 4.0 present 3xlO 8 M present 0.31 46 present 3xlO 8 M present 0.27 54 ¦ presen~ ¦ 3xlO a M ¦ presen~ 10.23 ¦ 60 ' ,~
g 1 The above table shows that with increasing concentration of morphine, there is decreasing activity of dihydrofolate reduc-tase. In this manner solutions of morkhine containing from 4~g per ml to 4 mg per ml of morphine could be assayed where only 10~1 of the solution was available. This is not intended to in-dicate however, that this is the range required, but rather only that it can be successfully emploved.
.
Synthesis of ferritin-methotrexate conjugate.
A solution of huma~ liver ferritin (1.3 mg) in sodium phospate buffer (0.05 M, pH 8.0, 5 ml) and 1 ml dimethyl formamide (DMF) was stirred at room temperature and 100~1 of a solution obtained by treating methotrexate (23 mg) in DMF (2 ml) with triethylamine (21~1) and i-butylchloroformate (15~1) at room temperature for 30 minutes, was added. The mixture was stirred at room temperature for 2 hours and then dialysed against 2x2 litres of sodium orthophosphate buffer (0.05 M, pH 7.4 con-taining sodium chloride O.IM and sod;'~m azide 0.05 weight %) for 24 hours. The solution was then passed through a column of Sephadex G-25 equilibrated with the same buffer and the ferritin containlng fractions combined and made upto 10 ml with the buffer.
The resulting solution is suitable for use in an enzyme immunoassay for the determination of ferritin .
.. . .~ ~ . : :
In the last years many analytical systems based on competitive protein binding analysis (also called saturation analysis) have been developed.
The terms 'saturation analysis' and 'competitive protein binding analysis', which are synonymous, refer to analytical systems used for the determination of immunologically active materials (ligands). The results of these determinations, `
in biological fluids, are emplo~ed in medical and veterinary diagnosis. The dia~nosis is dependent upon the level of the determined substance being normal or pathological. The analytical principle is e.g. based upon the competition between a ligand and a labelled ligand for a common specific binding agent (receptor), as illustrated in the following equation:-. , , _ , .
Label Label Lalel Ligand ~ Ligand + Receptor _ Ligand + Ligand + Ligand + Ligand Equation 1 Receptor Receptor (1) (2) (3) (4) .
Hen/ 7.2.1978 ~ .
The primary reaction is a combination of one molecule of ligand with one molecule of receptor to form a bimolecular ligand/receptor complex (1). A secondary reaction is caused by addition of labelled ligand which similarly combines with receptor to form a labelled ligand/receptor complex (2). In saturation analysis the concentrations of the receptor and of the labelled ligand are constant. The concentration of receptor is limited so that the labelled ligand is in excess relative to the receptor. Under these conditions the addition of ligand causes competition between ligand and labelled ligand for binding with receptor.
Hence an increase in ligand concentration lowers the amount of labelled ligand/receptor complex. The assay principle is based on the determination of the percentage of the total labelled ligand bound to the receptor.
This percentage is inversely proportional to the amount of ligand added to the reaction mixture from the specimen to be measured or from the standard used in the assay. The decrease in the concentration of labelled ligand/receptor complex or the increase in the concentration of labelled ligand in the reaction mixture can be used to determine the ligand concentration.
The sensitivity of saturation analysis depends upon the use of a receptor which has a very high affinity for the ligand and labelled ligand. Secondly, the sensitivity also depends upon the use of a label which can be detected at very low concentration.
The specificity of saturation analysis is dependent upon the capacity of the receptor to bind exclusively the ligand and labelled ligand in a complex mixture of different molecules.
Saturation analysis has been employed using many different techniques, the differences primarily being related to the type of label used.
Generally these techniques are classified by using the broad titles 'radioassay' or 'non-radioassay', depending upon whether or not a radioactive tracer is used as the label.
Radioassay has been utilised to a greater extent than non-radioassays.
Radioassay can be further classified as radioimmunoassay or radio-receptor .
.
. . , .. , :
_ 4 _ ~L~l~3 2 7 ~
assay, depending upon the type of receptor employed in the assay. In radioimmunoassay an antibody is used which specifically binds the ligand and labelled ligand. Alternatively, radio-receptor analysis refers to the use of any other type of biological receptor which will similarly specifically bind the ligand and labelled ligand.
In all radioassay techniques it is essential to physically separate the bound fraction (1 and 2 of Equation 1) from the unbound fraction (3 and 4 of Equation 1) of the reaction mixture. An index of the ligand concentration can subsequently be obtained by counting these fractions in a radioactivity counter and comparing the counts obtained for the unknowns specimens with those obtained for appropriate standard ligand samples subjected to the same assay. Many different and diverse methods have been described for the separation of the bound and free fractions of the radioassay reaction mixture utilising techniques including gel fitration, absorption and ion exchange chrom~tography, fractional precipitation, solid phase or electrophoresis.
Subsequent to the development of radioassay techniques in saturation analysis, methods employiny non-radioactive labels have been developed.
Methods utilising enzymes as the label have been demonstrated. These methods can have the advantage that physical separation of the bound (1 + 2) and free (3 + 4) fractions of labelled - ligand is not necessary ;n the assay procedure.
When antibody binds ligand labelled with an enzyme, the activity of the enzyme is modified. The degree of modification of the enzyme activity is indicative of the concentration of the labelled ligand in the bound fraction and hence gives an index of the concentration of ligand in the reaction mixture.
The chemical structure of the ligand-enzyme complex is extremely difficult to assess and this is a major disadvantage of enzyme-~0 immunoassay. This is undoubtedly due to the great multiplicity of amino acid side chains which are available on the enzyme surface for ` - 5 ~ 7~
complexing with the ligand. This causes great difficulty in reproducing the ligand-enzyme in different preparations of the complex.
The general lack of control of the complexing reaction results in the attachment of many ligand molecules to one enzyme molecule, although it is likely that the binding of only a few of these ligand molecules by antibody is involved in the inhibition of enzyme activity. Hence not all antibody/labelled antigen interactions result in a modification of the enzyme activity, thereby lowering the sensitivity of the technique.
Protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the multiplicity of ligand molecules on the enzyme surface. Protein-protein interaction is further increased if the ligand is also a polypeptide.
Hence the enzyme molecule induces a localised micro-environment of high protein concentration. In such situations it has been demonstrated that protein precipitation occurs, thereby causing a loss of one of the main advantages of enzymeimmunoassay by necessitating separation of antibody bound and free fractions.
A modification of enzymeimmunoassay has been described which partially overcomes these problems in that the ligand is labelled with a detector molecule which is ~f low molecular weight. In ~ this assay antibody binding of labelled ligand sterically hinders binding of detector molecule by another antibody specific for the detector molecule. The degree of hindrance is determined by competition for binding of the free ligand-detector molecule and detector molecule labelled enzyme with detector-molecule antibody.
The degree of modification of enzyme activity, as determined in normal enzymeimmunoassay, is indicative of the concentration of free ligand-detector molecule which is likewise indicative of the concentration of ligand in the reaction mixture. The advantage of this technique is that a small molecule rather than an enzyme is attached to the ligand, thereby allowing the chemical structure of the labelled ligand to be determined and thus overcoming many of the disadvantages of enzymeimmunoassay described previously. However, -, . . , - ~ .
.. . .
.
.
.
- 6 - ~ ~'Q2~
this system only overcomes the disadvantages of the primary blnding reaction involving the ligand and labelled ligand and transfers them to the detector system for determining the degree of antibody bound and antibody free fractions of the labelled ligand.
The disadvantages of the prior art methods are overcome by the present invention according to which an enzyme modifier is used as the label.
More particularlv the present invention relates to a reaaent for the determination of an immunologically active material, comprising the immunologically active material or a receptor which can specifically bind said immunologically active material,labelled with a substance capable of modifying the extent or the mode of activitv of an enzvme.
Furthermore the present invention relates to a method for the determination of an immunologically active material in a sample wherein the sample is contacted with a receptor which can specifically bind the immunologically active material, the immunologically active material labelled with a substance capable of modifyina the activity of an enzyme, an enz~me and an enzyme substrate, and wherein the extent or the mode of the resultina en-- zy~e activit~v is measured and ccmpared with that obtained with a standard.
Furthermore the present invention relates to a method for the determination of an immunologically active material in a sample wherein the sample is contacted with a receptor which can specifically bind the immunologically active material and which is labelled with a substance capable of modifyins the activity of an enzyme, with the immunologically active material in insolubilized form, and with an enzyme and an enzyme sllhstrate, and wherein the extent or the mode of the enzyme activitv, after the separation of the solid phase, is measured and compared with that obtained with a standard.
.
.
', . : - :
. . : - - .
- 7 ~
The terms 'immunologically active material' or 'ligand' within this specification refer to any immunologically active substance or part thereof capable of being immunologically determined e.g. by using saturation analysis techniques. The essential requirement is that there be a receptor which will specifically bind the ligand. When the receptor is an antibody the ligand will be haptenic or ahtigenic such that specific antibody can be formed. A ligand is referred to as a hapten when it will only elicit antibody formation when attached to a compound having antigenic properties. Alternatively, a ligand is referred to as an antigen when it will elicit antibody formation without chemical modification. The ligand may vary widely in molecular weight ranging from approximately 100 - 1,000,000.
This range of molecular weight is not limiting in the assay providing receptor is available which will specifically bind the-ligand.
The ligand may be either polymeric or non-polymeric in structure. When polymeric, the ligand will usually be biological in origin and be classified as a nucleic acid polysaccharide and/or polypeptide. Alternatively, when the ligand is non-polymeric, it will generally have a molecular weight of less than 2,000 and may have a wide variety of structures, functionalities and physiological properties.
Ligands of ~articular i~portance which mav be used in the ~resent invention are amines, aminoacids, peptides, proteins, lipoproteins, gl~copr~teins, ~ sterols, steroids, lipoids, nucleic acids, mono- and polvsaccharides, alka-loids, vitamins, druas, narcotics, antibiotics, metabolités, pesticides, toxins industrial pollutants, flavourinq agents, hormDnes, enzymes, coenzymes, cellul~r or extracellular ccmponents of tissues and isolated antibodies from humans or animals. The assay is,however,not limited to only these li~ands.
1 Components of immediate potential for analysis with the system are hepatitis B-surface antigen; ferritin;tumour antigens like CEA; a-feto protein; rheumatoid factor; C-reactive proteins;
imnunoglobulin classes IgC-, IgM or IgP.;myoglobin, th~roi~ hormones includina T3 and T4, insulin; steroid horniones including testosterone or estra-diol; drugs of abuse including narcotic analgesics, like morphine;
barbiturates; stimulants like amphetamine; drugs for the treat-ment of epilepsy including diphenyl hydantoin and phenobarbjtal;
cardiac glycocytes like digoxin; vitamines like vitamine Bl?~nd folic acid. Furthermore, antibodies which have immediate po-tential for analysis with the system are those associated with infection in syphilis, gonorrhoea, brucellosis, rubella and rheumatism.
The term 'labelled immunologically active material' or 'labelled receptor' in this specification refers to a ligand, analog of a ligand or part ther~of or to a receptor which is labelled with an enzyme modifier. One, more than one, and generally less than lOO molecules of label, can be attached to a ligand or receptor molecule. Similarly, one, more than one and usually less than 5 ligand or receptor molecules can be attached to a label molecule. The attachment of extra label molecules to a ligand or receptor molecule generally increases the sensitivity of the assay provided the extra labels do not affect binding.
Attachment of a modifier molecule to a ligand or receptor molecule involves the formation of intermolecular bonds which in most cases, but not necessarily, are covalent in nature. Attachment may be accomplished in some cases in the presence of a coupling agent by the insertion of a linking group between the label and the ligand or receptor.
The mcdifier molecule can be attached directly to the liqand or receptor molecule. However it may be desirable to insert chemical bridges of various length between the mcdifier rolecule and the ligand or receptor molecules depending on the speciic assay - 9 - ~
1 en~isa~ed. In some cases it may even be advanta~eous to attach themodifier molecule and the ligand or receptor molecules sepa-rately to the same carrier molecule e.~. a macromolecule like a polypeptide or a polysacch~ride.
The term'receptor'in this specification refers to any substance which can specifically bind ligand and labelled ligand or part thereof. Generally the receptor used in the assay is a specific antibody to the ligand formed in the blood of vertebrates following injection of appropriate hapten or antigen.
Alternatively, receptors which occur naturally can also be used in the assay. This latter group includes,but is not limited to, proteins, nucleic acids and cellular membranes. Such receptors have been used in radioassay techniques for thyroxine, insulin, angiotensin and various steroid hormones.
In case the ligand is an antibody the receptor may be the antigen utilized for inducing said antibody in a host animal.
In an another embodiment the receptor can be an antibodv against the antibody to be determined.
It is not possible to ascertain with certainty the mode of receptor -action which, on binding of the labelled ligand, reduces interaction between enzyme and modifier. The most likely explanation is that the affinity of the enzyme for the modifier is reduced as a result of a change in size and net charge of the receptor/ligand-modifier complex compared to that of the ligand-modifier alone.
The tërm'modifier'within this specification refers to any substance which is capable of interacting with an enzyme such that the extent or the mode of enzyme activity is modified.
This modification may result in an inhibition, activation or a change in specificity or any other property of the enzyme which is detectable either directly or indirectly by a change in enzyme activitv or in the mode of the activity e.g. a chanae in reaction conditions like co-factor requirements or pH-opti-mum, in kinetic properties, or in activation energy. The :
1 modifier can range in size from a small molecule to a macromolecule, and its interaction w;th an enzyme molecule can be either reversible or irreversible depending whether t~e inter-molecular association is ionic or covalent in nature.
Sensitivity of the assay is dependent amon~ others upon the affinity of the receptor for the ligand and the ability of the modifier to cause a change in the extent or the mode of enzyme activity. Preferably, modification of the extent or the mode of enzyme activity is achieved with a minimal con-centration of modifier. The closer this concentration is to that of the enzyme on a molecular basis, the ~reater will be the sensitivity of the assay.
Preferably, the modifier will be an enzyme inhibitor which, on interaction with an enzyme, will inhibit its activitv. The mode of action of the inhibitor may be competitive, non-competitive, uncompetitive,allosteroic or a combination of two or more of these modes. Preferably the inhibitor should ha~e an inhibition z~ constant (concentration of inhibitor necessary for 50% inhibition of the enzyme system) below lO ~ moles/l, dependin~ on the assay performed. ~ost preferably the inhibition constant is between lO 5 and lO 5.
Any enzyme ~an be used in the assay provided there exists a modifier which will specifically modify the enzyme activity in the manner described above Enzymes of choice are stable, easily obtained at low cost and have a high turnover number and a simply performed assay system. Preferably the turnover number (molecules of product formed per molecule of enzyme in one minute) is superior to lO0 dependinq on the specific test performed. Most preferably, the turnover number is as hi~h as possible and at least 200.
Enzyme modifier systems which are particularly suitable for the present invention are dihydrofolate reductase/
methotrexate, dihydrofolate reductase/4-aminopterin, .
.
, .
.
8~3 1 dihydrofolate reductase/other specific inhibitors of this enzyme;
~-glucoronidase/4-deoxy-5-amino glucarlc acid and its derivatives;
biotin containing enzymes/avidin like carboxylase/avidin;
chymotrypsin/TPCK CH2-Ph ~M3 ~ H 0 ~-cystathionase/propargylalycine; alanine racemase/tri-fluoroalanine; trvptophanase/trifluoroalanine; tryptophan svnthetase/trifluoroalanine; ~-cystathionase/trifluoro-alanine; pyruvate-glutamate transaminase/trifluoro-alanine; lactic acid oxidase/2-hydroxy-3-butynoic acid;
monoamine oxidase/N,N-dimethyl nropargyl amine; and diamine oxidase/~2N-CH2-C_CCH2-NH2;
Determination of enzyme activity may be performed by moni-toring directly or indirectly the consumption of substrate or production of product at suitable pH and temperature using de-tection systems including colorimetry, spectrophotometry, fluoro-spectrophotometry gaseometry, thermometry (heat production), scintillation counting.
In order to increase the sensitivity of the system it is possible to use bioluminescence and enzymic cycling techni~ues, e.a. the techniques described by J. Lee et al in Liquid Scintillation ~ Counting:~ecent nevelopments, Stanley P.E. and Scoqqins, B.A., Academic Press, New York p. 403 and Lowry O.H. et al in J. Biol.
Chem 2_6, p. 2746-2755.
' - 12 - ~ t7~
1 In one embodiment of the present invention a labelled ligand can be used for the determination of the presence of a ligand in an unknown specimen by the simultaneous or sequential addition of the labelled ligand and the un~nown specimen to an aqueous medium, at appropriate pH, containing a receptor specific for the ligand and labelled ligand.
After a suitable incubation period the distribution of receptor bound to ligand and labelled ligand is determined by the addition of enzyme and substrates. The receptor which is specific for the ligand also binds the labelled ligand and, in so doing, reduces the interaction between the modifier and the enzyme thus decreasing the modification of enzyme activity.
Addition of ligand to the assay results in competition with the labelled ligand for binding with receptor and thereby increases the concentration of free labelled ligand in the assay. Interaction between ligand-modifier and enzyme is increased and the enzyme activity is once again affected. The modification of enzyme activity is therefore a function of l;gand concentration in the assay and is caused by unbound labelled ligand. Consequently the difference between the resulting enzyme activity and that obtained in the absence of ligand ;s indicative of the concentration Gf ligand in the unknown specimen.
One of the major advantages of this method is that separation of bound and free fractions in the procedure is unnecessary.
This does not preclude,however,the use of such a separation step in the assay after incubation of ligand and labelled ligand with receptor and prior to the estimation of enzyme activity.
Separation of fractions may be desirable in some instances for removal of substances in the specimen which may interfere with the enzyme assay. Separation may be achieved using any of the many techniques described for radioimmunoassay including gel filtration, adsorption and ion exchange chromatography, fractional precipitation, solid phase and electrophoresis.
.
- 13 - ~ ~Z7~
1 This assay is of course not limited to the determination of haptens and antigens as the ligand but can also be adapted to the identification and measurement of antibodies as the ligand.
This can e.g. be performed by labelling the antibody with an enzyme modifier and measuring the extent of enzy~e acti-vity modification following incubation of labelled antibody and specimen antibod,v with a limiting concentration of antiaen or hapten. The modification of enzyme activity is related to the specimen antibody concentration. If necessary the bound and free fractions may be separated before addition of the enzyme and the substrate.
The invention permits also for the determination of a ligand the use of a labelled receptor rather than that of a labelled ligand. Ligand in the unknown specimen reacts with excess receptor labelled with an enzyme modifier and, after incubation, excess solid phase insolubilized ligand is added and reacts with the free labelled receptor remaining. After separation of the solid phase, the modification of enz~me activity associated with the soluble ligand is measured and related to the concentration of ligand.
Z5 The reagents of the present invention can also be used in a 'sandwich' techni~ue provided the ligand has at least two binding sites. Ligand reacts with excess solid phase receptor and after incubation followed by washing, the solid phase receptor bound ligand is reacted with excess receptor labelled with an enzyme modifier. Free labelled receptor is removed by washing and the extent of enzyme modification in the separated fractions is determined. Tkis then gives an index of liqand concentration.
-.
~7~
The followin~ exam~les illustrate the invention.
.
Example 1 Preparation of "Amino-digoxin"
To a suspension of 156 mg (0.2 mmol) digoxin in 5 ml absolute ethanol was added 10 ml 0.2 M sodium metaperiodate with stirring. The mixture became homogeneous after 10 m;nutes and then a precipitate slowly formed.
After 2 hours, 5 ml water plus 5 ml ethanol were added. 122 ~l (2.2 mmol) of ethylene glycol was added after a further 30 minutes and a dense white precipitate started to form immediately. After 80 minutes' stirring, 133 yl (2.0 mmol) ethylene diamine was added and the resultant pH of ,11.0 was adjusted to 9.5 with 0.1 M HCl and the ~ reaction mixture was allowed to stand at room temperature for 18 hou~s. The pH did not change during this time.
151.4 mg (4 0 mmol) sodium borohydride was then added and the mixture stirred for 3~ hours. The pH was then adjusted from 10.5 to 6.5 with IM formic acid (about 3 ml) and TLC of this mixture showed a single major spot with an Rf 0.15 (silica on aluminium developed in butanol:acetic acid:water/4:1:1). Digoxin had an Rf 0.7 when developed in the same system.
The solvents were evaporated to near dryness on a rotary evaporator under Yacuum using a water bath at 60. The last few ml of water - were removed by adding 95% ethanol (3 x 20 ml) and repeating the evaporation as above.
The resultant pale yellow solid was extracted three times with absolute ethanol and these combined extracts concentrated to about 4 ml and centrifuged to separate a small amount of salt which was discarded. The pale yellow supernatent solution was evaporated to dryness under a stream of dry nitrogen to give a yellow oil which showed the same Rf on TLC as the reaction mixture as described above. The product was shown to contain a free amino group b~ reacting it with "Fluram" (4-phenylspiro [fluran-2(3H), 1-phthalan]-3,~-dione) to form an intensely fluorescent compound.
The product exhibited a spectrum in concentrated sulphuric acid . ..
27~3~
similar to that of digoxin with absorption peaks at 385 and 495 m~.
The product also exhibited a strong affinity for antisera (rabbit) specific for digoxin. The most likely structure of the isolated product is as follows:
O~
c~C~
H2~_c"2cR2~
.. _ .
.
Example 2 Preparat;on of methotrexate -aminod;qoxin conjugate 11 mg methotrexate was dissolved in 5 ml H20 and adjusted to pH 6.5.
10 mg "aminodigoxin" was dissolved in this solution and the volume adjusted to 20 ml with H20. The pH was readjusted to 6.5 and 484 mg N-ethyl-N"-(3-dimethyl am;no) propyl-carbodiimide hydrochloride, dissolved in 5 ml H20, was added to the reaction mixture. The pH
was maintained at 6.2 for 24 hours at room temperature. The conjugate - was purified on a silica gel column, using 3X ammonium citrate as a solvent. Fractions containing the desired product were combined and shown to have the dual capacity to bind strongly antisera (rabbit) specific for digoxin and also to inhibit strongly the enzyme dihydrofolate reductase (chicken liver). The most likely structure of the methotrexate-aminodigoxin conjugate is as follows:
`
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.' ~j-CIl~c~ ,~o~
~F
o~
Ht~'--C112C112--~ ~\
Example 3 Enzyme Inhib;tor Immunoassay for Digoxin 100 ~l serum was incubated at 30C for 15 minutes with 100 ~l antidigoxin antibody solution, 100 ~l NADPH solution, 100 ~l 2-mercaptoethanol solution and 550 ~l sodium phosphate buffer pH 7.5. 100 ~l Methotrexate-digoxin conjugate of example 2 (70 ~g/ml) was added and the mixture incubated for 15 minutes, after which, 100 ~l dihydro-folate reductase solution was added. The dihydrofolate reductase preparation employed was isolated from chicken liver by the method of Kaufman, B.T., & Gardiner, R.C., Journal of Biological Chemistry, Vol. 211, P 1319 (1966). The mixture was incubated for a further 3 minutes and the enzyme activity determined by the addition of 100 ~l dihydrofolate solution and monitored at 340 nm with a varian recording spectrophotometer. The results are shown in Table.
- . ~. , . , . ~
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-TABLE I
Reactants Enzyme Activity Digoxin Antidigoxin Methotrexate- Enzyme assayOD/min I Inhibition (sample conc.) antibody digoxin conjugate components (in assay) O absent O present 0.150 O
O absent 7 ng present 0.100 33 O present 7 ng present 0.145 3 _ 5 ng/ml present 7 ng present 0.125 16 10 ng/ml present 7 ng present 0.1l5 23 The reactants were added in the sequence described above.
OD = optical densitv It is evident from the above results that a few ng digoxin in serum can be de$ermined in the system described.
_ .
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:. ,, - .. ,. . . . ~ . -- 18 - i Example 4 Preparation of Methotrexate-Human Serum Albumin Conjugate 45 mg methotrexate was dissolved ;n 1.0 ml N,N-dimethyl formamide and 25 mg N-hydroxysuccinimide was added.
41 mg N,N-dicyclohexyl carbodiimide was dissolved and the mixture kept at room temperature for 13 hours. The insoluble urea byproduct was removed by filtration and 100 ~1 of the filtrate was added to a solution containing 5 mg human serum albumin in 800 ~1 O.lM sodium phosphate buffer pH 7.5 plus 100 ~1 dioxane.
,lO This reaction mixture was kept at room temperature for 30 minutes and then loaded onto a Sephadex G-25 column equilibrated with O.lM sodium phosphate buffer pH 7.5. The column was developed with this buffer and two elution peaks containing methotrexate were obtained, the first of which contained the methotrexate-human serum albumin conjugate.
Example 5 Enzyme Inhibitor Immunoassay for Human Serum Albumin.
1OOJU1 diluted serum was incubated at 30C for 15 minutes with 1OOJU1 antihuman serum albumin antibody (rabbit) solution, 100 ~1 NADPH solution, 100 ~1 2-mercaptoethanol solution and 550 yl sodium phosphate buffer pH 7.5. 100 ul methotrexate-human serum albumin conjugate (8Jug/ml) was added and the mixture incubated for 15 minutes, after which, 100Jul dihydrofolate reductase solution was added. The mixture was incubated for a further 3 minutes and the enzyme activity determined by the addition of lOO~ul dihydrofolate solution and monitored at 340 nm with a varian recording spectrophotometer.
The results are shown in Table II.
*Trade Mark ~.~,. . .
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TABLE I I
- ' . . . _ _ _ Reactants Enzyme Activity Human Serum Antihuman Methotrexate- Enzyme OD/min % Inhibition Albumin Serum albumin human serum Assay (Sample antibody albumin conjugate Component Conc.) (in assay) _ _ O absent O present 0.123 O
O absent 0.8 ~g present 0.068 45 O present 0.8 ~9 present 0.105 16 5~ug/ml present 0.8Jug present 0.092 25 10 ~g/ml present 0.8 ~9 present 0.085 31 .
.
It is evident from the above results that a few ~9 human serum albumin can be determined in the system described.
- . . - ~ -- . -. : ,: :
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Synthesis of methotrexate-aminoethylmorphine conjugate.
a) Synthesis of 03-aminoethylmorphine In 10 ml of tetrahydrofuran (THF) freshly distilled from lithium aluminium hydride (LA~) was suspended 400 mg of LA~ un-der nitrogen. A solution of 400 mg of morphine and 400 mg of chloroacetonitrile in 4 ml of freshly distilled THF was added over 5 minutes, followed by refluxing for 1 hour. The mixture was allowed to cool and 0.6 ml of water was added followed by 0.6 ml of 10 weight % sodium hydroxide and 2 ml of water. After filtering the mixture, the salts were washed with THF, the THF
fractions combin~d, dried with magnesium sulphate under nitrogen, filte~-ed and the filtrate evaporated to yield 380 mg of 03-aminoethylmorphine.
b) Synthesis of N-t-Butoxycarboxyl-~-(03-aminoethylmorphine) glutamic aci*~ -benzylester.
A mixture of 03-aminoethylmorphine (50 mg prepared as ;, above), N-t-BOC-glutamic acid-~-benzyl ester(34 mg)and dicyclohexylcarbodiimide (25 mg) in dichloromethane (5 ml) was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate and washed with dilute sodium carbonate so-lution. The ethyl acetate solution was then extracted twice with 0.1 N hydrochloric acid and the combined!acidic extracts were .
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.:
. ~
- 21 - ~Z7~
1 treated with sufficient 10 weight % sodium hydroxide solution to adjust the pH to 8Ø The solution was extracted twice with ethyl acetate and the combined ethyl acetate extracted were washed with brine, dried (anhydrous sodium sulphate) and evaporate~ to give a colourless oil (36 mg).
c) Synthesis of ~lutamic acid-~-(03-amidoethylmorphine) -~-benzyl ester-trifluoroacetic acid salt.
A solution of N-t-BOC-~lutamic acid-morphine derivative (36 mg, prepared as described above) in dichloromethane (3 ml) was stirred at room temperature and trifluoroacetic acid (1 ml) was added. The mixture was stirred for 15 minutes and then eva-porated to dryness. The residue was a colourless glass (40 mg).
d) Synthesis of methotrexate ~- (03- amidoethylmorphine) -~- benzyl ester.
A solution of 4-amino-4-deoxy~N10-methylpteroic acid (37 mg) in 3 ml di~ethylsulfoxide (DMSO) stirring at room tem-perature was treated with triethylamine (28 ~1) and i-butyl-chloroformate (25 ~1) and the mixture was stirred for 30 minu-tes. It was then added to a mixture of the morphine derivative (75 mg prepared as descrl~ed in ~) and triethylamine (28~1) in DMSO (2 ml) and the mixture was stirred at 60 for one hour.
The cooled mixture was diluted with water and extracted twice with ethylacetate. The combined ethyl acetate extracts were was-hed withwater and then extracted twice with O.IN hydrochloric acid. The combined acidic extracts were treated with sufficient 10 weight % sodium hydroxide to raise the pH to 8Ø The mix-ture was extracted three times with ethyl acetate and the com-. .. : . .. . : . - . - :
.
' - : ' 1 bined extracts washed with brine, dried (anhydrous sodium sul-phate) and evaporated to give a yellow oil (15 mg). This was purified using prepæative thin layer chromatography (TLC) on silica gel developing with chloroform~methanol, 4:1. The desired o~ound was obtained as a yellow solid (4 mg).
e) Synthesis of MethotreXate ~-(03-amidoethylmorphine).
The product prepared as described in d (~ mg) was mixed with O.INsodium hydroxide solution (5 ml) and the mix-ture was stirred at room temperature for 8 hours resulting in a clear yellow solution.
To this was added O.IN hydrochloric acid (5 ml) and the mixture was made upto 25 ml with sodium orthophosphate buffer (0.05 M, pH 7.4) to give a solution of the desired compound suitable for use in the enzyme assay.
Enzyme inhibitor immunoassay for morphine A mixture of 10~1 of morphine solution of suitable con-centration, 50~1 of methotrexat~-(03-amidoethylmorphine) solution (4 ng/25 ml), 50~1 of antimorphine antibody solution, 10~1 NADPH solution, 10~1 2-mercaptoethanol solution, 50~1 po-tassium chloride solution, 150~1 Tris-HCl,buffer solution (p~
7.5)containing EDTA and 10~1 of dihydrofolate reductase (E.~
casei) solution was incubated for 20 minutes at 37. The enzyme activity in the mixture was determined after the addition of 10~1 dihydrofolate solution by monitoring the change in extin-z~
ction of the solution at 340 nm using a Centrifichem centrifugal analyser. The results are shown in Table III
TABLE III
__ ~CIA~S ENZYME ACTIVITY
~)~PHINE ANTI~ PHINE ~PHINE- ENZY~!IE ASSAY . __ ME~OT~IE DD/ITin ~61NHIBITION
pg/assay ANTIBODY CONJUG~TE ~llENr3 __ _ O absent absent present O.54 O
O ~sent 3xlO M present 0.14 76 O ~resent 3xlO 8 M present 0.41 29 0.04 present 3xlO 8 M present 0.37 36 0.40 present 3xlO 8 M present 0.35 40 4.0 present 3xlO 8 M present 0.31 46 present 3xlO 8 M present 0.27 54 ¦ presen~ ¦ 3xlO a M ¦ presen~ 10.23 ¦ 60 ' ,~
g 1 The above table shows that with increasing concentration of morphine, there is decreasing activity of dihydrofolate reduc-tase. In this manner solutions of morkhine containing from 4~g per ml to 4 mg per ml of morphine could be assayed where only 10~1 of the solution was available. This is not intended to in-dicate however, that this is the range required, but rather only that it can be successfully emploved.
.
Synthesis of ferritin-methotrexate conjugate.
A solution of huma~ liver ferritin (1.3 mg) in sodium phospate buffer (0.05 M, pH 8.0, 5 ml) and 1 ml dimethyl formamide (DMF) was stirred at room temperature and 100~1 of a solution obtained by treating methotrexate (23 mg) in DMF (2 ml) with triethylamine (21~1) and i-butylchloroformate (15~1) at room temperature for 30 minutes, was added. The mixture was stirred at room temperature for 2 hours and then dialysed against 2x2 litres of sodium orthophosphate buffer (0.05 M, pH 7.4 con-taining sodium chloride O.IM and sod;'~m azide 0.05 weight %) for 24 hours. The solution was then passed through a column of Sephadex G-25 equilibrated with the same buffer and the ferritin containlng fractions combined and made upto 10 ml with the buffer.
The resulting solution is suitable for use in an enzyme immunoassay for the determination of ferritin .
.. . .~ ~ . : :
Claims (37)
1. Reagent for the determination of an immunologically active material, comprising the immunologically active material ox a receptor which can specifically bind said immunologically active material, labelled with a substance capable of modifying the extent or the mode of activity of an enzyme.
2. Reagent according to claim 1 wherein the immunologically active material is labelled with a substance capable of modifying the extent or the mode of activity of an enzyme.
3. Reagent according to claim 1, wherein a receptor which can specifically bind the immunologically active material is labelled with a substance capable of modifying the extent of the mode of activity of an enzyme.
4. Reagent according to claim 3, wherein the receptor is an antibody specific for the immunologically active material to be determined.
5. Reagent according to claim 1 wherein the substance capable of modifying the activity of an enzyme is an enzyme inhibitor.
6. Reagent according to claim 5, wherein the enzyme inhibitor has an inhibition constant below 10-3 moles/l.
7. Reagent according to claim 6, wherein the enzyme inhibi-tor has an inhibition constant between 10-5 and 10-15 moles/l.
8. Reagent according to claim 7, wherein the inhibitor is methotrexate and the enzyme dihydrofolate reductase.
9. Reagent according to claim 1, wherein the substance capable of modifying the activity of an enzyme is an enzyme activator.
10. Reagent according to any one of claims 1, 5 or 9, wherein the immunologically active material is digoxin.
11. Reagent according to any one of claims 1, 5 or 9, wherein the immunologically active material is human serum albumin.
12. Reagent according to any one of claims 1, 5 or 9, wherein the immunologically active material is morphine.
13. Reagent according to any one of claims 1, 5 or 9, 10, wherein the immunologically active material is ferritin.
14. Reagent according to any one of claims 1, 5 or 9, wherein the immunologically active material or the receptor which can specifically bind said immunologically active material is covalently linked to the substance capable of modifying the extent or the mode of activity of an enzyme.
15. Conjugate of digoxin and methotrexate, a reagent of Claim 1.
16. Conjugate of human serum albumin and methotrexate, a reagent of Claim 1.
17. Conjugate of morphine and methotrexate, a reagent of Claim 1.
18. Conjugate of ferritin and methotrexate, a reagent of Claim 1.
19. Process for the manufacture of a reagent according to claim 1, comprising reacting an immunologically active material or a receptor which can specifically bind said immunologically active material with a substance capable of modifying the extent or the mode of the activity of an enzyme in the presence of a coupling agent.
20. Process according to claim 19, wherein the coupling agent is a carbodiimide.
21. Process according to claim 19, wherein the coupling agent is i-butylchloroformate.
22. Method for the determination of an immunologically active material in a sample, wherein the sample is contacted with a receptor which can specifically bind the immunologically active material, with the immunologically active material labelled with a substance capable of modifying the extent or the mode of the activity of an enzyme, and with an enzyme and an enzyme substrate, and wherein extent or the mode of the resul-ting enzyme activity is measured and compared with that ob-tained with a standard.
23. Method for the determination of an immunologically active material in a sample wherein the sample is contacted with a receptor which can specifically bind the immunologically active material and which is labelled with a substance capable of modifying the extent or the mode of activity of an enzyme, with the immunologically active material in insolubilized form, and with an enzyme and an enzyme substrate, and wherein the extent or the mode of enzyme activity after the separation of the solid phase is measured and compared with that obtained with a standard.
24. Method according to claim 22 or claim 23 in which the substance capable of modifying the activity of an enzyme is an enzyme inhibitor.
25. Method according to any one of claims 22 or 23 in which the receptor is an antibody.
26. Method according to any one of claims 22 or 23 in which the inhibitor is an inhibitor of the enzyme dihydrofolate reductase.
27. Method according to any one of claims 22 or 23 in which the inhibitor is methotrexate and the enzyme is dihydrofolate
28. Method according to any one of claims 22 or 23 in which the immunologically active material is a hapten.
29. Method according to claim 22 or 23 in which the immunologically active material is digoxin and the receptor is an antibody to digoxin.
30. Method according to claim 22 or 23 in which the immunologically active material is morphine and the receptor is an antibody to morphine.
31. Method according to any one of claims 22 or 23 in which the immunologically active material is a protein.
32. Method according to claim 22 or 23 in which the immunologically active material is human serum albumin and the receptor is an antibody to human serum albumin.
33. Reagent according to claim 2 or 3 wherein the substance capable of modifying the activity of an enzyme is an enzyme inhibitor.
34. Reagent according to claim 2, 3 or 4, wherein the substance capable of modifying the activity of an enzyme is an enzyme activator.
35. Process for the manufacture of a reagent according to claim 2, 3 or 4, comprising reacting an immunologically active material or a receptor which can specifically bind said immunologically active material with a substance capable of modifying the extent or the mode of the activity of an enzyme in the presence of a coupling agent.
36. Process for the manufacture of a reagent according to claim 9, comprising reacting an immunologically active material or a receptor which can specifically bind said immunologically active material with a substance capable of modifying the extent or the mode of the activity of an enzyme in the presence of a coupling agent.
37. Method according to claim 22 or claim 23 in which the substance capable of modifying the activity of an enzyme is an enzyme inhibitor, in which the receptor is an antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB10.859/77 | 1977-03-15 | ||
| GB10859/77A GB1595101A (en) | 1977-03-15 | 1977-03-15 | Enzyme modifier immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1102789A true CA1102789A (en) | 1981-06-09 |
Family
ID=9975639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA297,676A Expired CA1102789A (en) | 1977-03-15 | 1978-02-24 | Immunological determination |
Country Status (16)
| Country | Link |
|---|---|
| JP (1) | JPS53115814A (en) |
| AT (1) | AT367203B (en) |
| AU (1) | AU519326B2 (en) |
| BE (1) | BE864856A (en) |
| CA (1) | CA1102789A (en) |
| CH (1) | CH641570A5 (en) |
| DE (1) | DE2811257A1 (en) |
| DK (1) | DK152313C (en) |
| ES (2) | ES467831A1 (en) |
| FR (1) | FR2384262A1 (en) |
| GB (1) | GB1595101A (en) |
| IL (1) | IL54234A0 (en) |
| IT (1) | IT1158665B (en) |
| NL (1) | NL7802845A (en) |
| NO (2) | NO152955C (en) |
| SE (1) | SE447026B (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4273866A (en) | 1979-02-05 | 1981-06-16 | Abbott Laboratories | Ligand analog-irreversible enzyme inhibitor conjugates and methods for use |
| GB2059421A (en) * | 1979-10-03 | 1981-04-23 | Self C H | Assay method and reagents therefor |
| DE3006709A1 (en) * | 1980-02-22 | 1981-08-27 | Hans A. Dipl.-Chem. Dr. 8000 München Thoma | HOMOGENEOUS METHOD FOR COMPETITIVE DETERMINATION OF LIGANDS |
| US4341865A (en) | 1980-07-21 | 1982-07-27 | Abbott Laboratories | Determination of thyroxine binding globulin |
| FR2502786B1 (en) * | 1981-03-24 | 1985-06-21 | Stallergenes Laboratoire | METHOD FOR FIXING ANTIGENS AND ANTIBODIES TO POLYSACCHARIDE SUPPORT, AND USE OF THE PRODUCT OBTAINED THEREFOR FOR IMMUNOASSAYS |
| GB2116979B (en) * | 1982-02-25 | 1985-05-15 | Ward Page Faulk | Conjugates of proteins with anti-tumour agents |
| AU574646B2 (en) * | 1982-07-19 | 1988-07-14 | Cooperbiomedical Inc. | Enzyme assay method |
| GB2135773B (en) * | 1983-01-31 | 1985-12-04 | Boots Celltech Diagnostics | Enzyme inhibitor labelled immunoassay |
| US4650751A (en) * | 1983-04-29 | 1987-03-17 | Technicon Instruments Corporation | Protected binding assay avoiding non-specific protein interference |
| JPS607362A (en) * | 1983-06-27 | 1985-01-16 | Fujirebio Inc | Measurement of antigen determinant-containing substance using enzyme |
| US4837395A (en) * | 1985-05-10 | 1989-06-06 | Syntex (U.S.A.) Inc. | Single step heterogeneous assay |
| CA1330378C (en) * | 1986-05-08 | 1994-06-21 | Daniel J. Coughlin | Amine derivatives of folic acid analogs |
| US4939264A (en) * | 1986-07-14 | 1990-07-03 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
| IL85596A (en) * | 1987-05-18 | 1992-06-21 | Technicon Instr | Method for a specific binding enzyme immunoassay |
| US5972630A (en) * | 1991-08-19 | 1999-10-26 | Dade Behring Marburg Gmbh | Homogeneous immunoassays using enzyme inhibitors |
| AU663582B2 (en) * | 1992-03-04 | 1995-10-12 | Akzo N.V. | In vivo binding pair pretargeting |
| US5965106A (en) * | 1992-03-04 | 1999-10-12 | Perimmune Holdings, Inc. | In vivo binding pair pretargeting |
| CA2331587A1 (en) * | 1998-05-15 | 1999-11-25 | Katsumi Yoshikawa | Immunoassay reagents and immunoassay method |
| US6811998B2 (en) | 1999-06-25 | 2004-11-02 | Roche Diagnostics Operations, Inc. | Conjugates of uncompetitive inhibitors of inosine monophosphate dehydrogenase |
| US8394813B2 (en) * | 2000-11-14 | 2013-03-12 | Shire Llc | Active agent delivery systems and methods for protecting and administering active agents |
| EP2131866B1 (en) * | 2007-02-16 | 2015-08-26 | KTB Tumorforschungsgesellschaft mbH | Dual acting prodrugs |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2308005A1 (en) * | 1975-04-15 | 1976-11-12 | Hadaway Robert | Drive in wall plug fastener - has body provided with indentations and covered with pliable externally smooth coating |
-
1977
- 1977-03-15 GB GB10859/77A patent/GB1595101A/en not_active Expired
-
1978
- 1978-02-24 CA CA297,676A patent/CA1102789A/en not_active Expired
- 1978-03-07 CH CH246378A patent/CH641570A5/en not_active IP Right Cessation
- 1978-03-08 AU AU33965/78A patent/AU519326B2/en not_active Expired
- 1978-03-08 IL IL54234A patent/IL54234A0/en unknown
- 1978-03-14 ES ES467831A patent/ES467831A1/en not_active Expired
- 1978-03-14 DK DK114778A patent/DK152313C/en not_active IP Right Cessation
- 1978-03-14 SE SE7802923A patent/SE447026B/en not_active IP Right Cessation
- 1978-03-14 AT AT0182178A patent/AT367203B/en not_active IP Right Cessation
- 1978-03-14 NO NO780902A patent/NO152955C/en unknown
- 1978-03-14 FR FR7807271A patent/FR2384262A1/en active Granted
- 1978-03-14 BE BE185901A patent/BE864856A/en not_active IP Right Cessation
- 1978-03-15 JP JP2976778A patent/JPS53115814A/en active Pending
- 1978-03-15 NL NL7802845A patent/NL7802845A/en not_active Application Discontinuation
- 1978-03-15 IT IT21256/78A patent/IT1158665B/en active
- 1978-03-15 DE DE19782811257 patent/DE2811257A1/en not_active Ceased
- 1978-12-01 ES ES475984A patent/ES475984A1/en not_active Expired
-
1984
- 1984-11-29 NO NO84844764A patent/NO154814C/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO154814B (en) | 1986-09-15 |
| ES467831A1 (en) | 1979-09-01 |
| FR2384262A1 (en) | 1978-10-13 |
| AT367203B (en) | 1982-06-11 |
| NO152955B (en) | 1985-09-09 |
| ATA182178A (en) | 1981-10-15 |
| CH641570A5 (en) | 1984-02-29 |
| NO844764L (en) | 1978-09-18 |
| JPS53115814A (en) | 1978-10-09 |
| AU519326B2 (en) | 1981-11-26 |
| NL7802845A (en) | 1978-09-19 |
| FR2384262B1 (en) | 1983-04-08 |
| SE447026B (en) | 1986-10-20 |
| IT7821256A0 (en) | 1978-03-15 |
| DK152313B (en) | 1988-02-15 |
| DK152313C (en) | 1988-09-26 |
| DE2811257A1 (en) | 1978-09-21 |
| NO780902L (en) | 1978-09-18 |
| AU3396578A (en) | 1979-09-13 |
| BE864856A (en) | 1978-09-14 |
| NO154814C (en) | 1986-12-29 |
| NO152955C (en) | 1985-12-18 |
| GB1595101A (en) | 1981-08-05 |
| IT1158665B (en) | 1987-02-25 |
| IL54234A0 (en) | 1978-06-15 |
| SE7802923L (en) | 1978-09-16 |
| ES475984A1 (en) | 1979-06-16 |
| DK114778A (en) | 1978-09-16 |
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