DK152313B - REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL - Google Patents

REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL Download PDF

Info

Publication number
DK152313B
DK152313B DK114778AA DK114778A DK152313B DK 152313 B DK152313 B DK 152313B DK 114778A A DK114778A A DK 114778AA DK 114778 A DK114778 A DK 114778A DK 152313 B DK152313 B DK 152313B
Authority
DK
Denmark
Prior art keywords
ligand
enzyme
receptor
solution
labeled
Prior art date
Application number
DK114778AA
Other languages
Danish (da)
Other versions
DK152313C (en
DK114778A (en
Inventor
Douglas E Hawley
Peter G Tonkes
Original Assignee
Hoffmann La Roche
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hoffmann La Roche filed Critical Hoffmann La Roche
Publication of DK114778A publication Critical patent/DK114778A/en
Publication of DK152313B publication Critical patent/DK152313B/en
Application granted granted Critical
Publication of DK152313C publication Critical patent/DK152313C/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D475/00Heterocyclic compounds containing pteridine ring systems
    • C07D475/06Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
    • C07D475/08Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D489/00Heterocyclic 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:
    • C07D489/02Heterocyclic 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
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J43/00Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Description

DK 152313 BDK 152313 B

Den foreliggende opfindelse angår et reagens til bestemmelse af et immunologisk aktivt materiale, hvilket reagens består af en receptor, som specifikt binder det immunologisk aktive materiale, et enzym, et enzymsubstrat og det immunologisk aktive materiale, der er mærket med en forbindelse, som er i stand til at modificere aktiviteten af enzymet.The present invention relates to a reagent for the determination of an immunologically active material, which reagent consists of a receptor that specifically binds the immunologically active material, an enzyme, an enzyme substrate and the immunologically active material labeled with a compound which is in capable of modifying the activity of the enzyme.

Der er i de sidste år udviklet mange analytiske systemer, som byg- ger på konkurrerende proteinbindingsanalyse (også kaldet mætningsanalyse) .Many analytical systems have been developed in recent years, which are based on competing protein binding analysis (also called saturation analysis).

Udtrykkene "mætningsanalyse" og "konkurrerende proteinbindingsanalyse", som er synonyme, dækker analytiske systemer, som anvendes til bestemmelse af immunologisk aktive materialer (ligander). Resultaterne af disse bestemmelser, udført i biologiske væsker, anvendes til medicinske og veterinære diagnoser. Diagnosen er afhængig af, om niveauet afThe terms "saturation assay" and "competing protein binding assay", which are synonymous, cover analytical systems used to determine immunologically active materials (ligands). The results of these assays, performed in biological fluids, are used for medical and veterinary diagnoses. The diagnosis depends on whether the level of

2 DK 152313B2 DK 152313B

den bestemte substans er normalt eller patologisk. Det analytiske princip bygger f.eks. på en konkurrence mellem en ligand og en mærket ligand om et fælles specifikt bindemiddel (receptor), som det belyses ved nedenstående ligning: mærke ”“fke ”“ykethe particular substance is normal or pathological. The analytical principle builds, for example. on a competition between a ligand and a labeled ligand for a common specific binder (receptor), as illustrated by the following equation: brand "" fke "" yke

I III II

ligand + ligand + receptor —^ ligand + ligand + ligand + ligandligand + ligand + receptor - ^ ligand + ligand + ligand + ligand

I II I

receptor receptor (1) (2) (3) (4) ligning 1receptor receptor (1) (2) (3) (4) Equation 1

Den primære reaktion er en kombination af 1 molekyle ligand med 1 molekyle receptor til dannelse af et bimolekylært ligand/recep-tor-complex (1). En anden reaktion affødes af tilsætningen af mærket ligand, som på lignende måde kombineres med receptoren til dannelse af mærket ligand/receptor-complex (2). I mætningsanalyse er koncentrationerne af receptoren og af den mærkede ligand konstante. Koncentrationen af receptor er begrænset, så at den mærkede ligand er i overskud i forhold til receptoren. Under disse betingelser forårsager tilsætningen af ligand en konkurrence mellem ligand og mærket ligand om binding med receptoren. En forøgelse af ligandkoncentrationen vil derfor formindske mængden af mærket ligand/receptor-complex. Forsøgsprincippet bygger på en bestemmelse af den procentdel af den totale mærkede ligandmængde, som er bundet til receptoren. Denne procentdel er omvendt proportional med den mængde ligand, der sættes til reaktionsblandingen fra den prøve, som skal måles, eller fra den standard, som anvendes under forsøget. Reduktionen af koncentrationen af mærket ligand/receptor--complex eller forøgelsen af koncentrationen af mærket ligand i reaktionsblandingen kan anvendes til at bestemme ligandkoncentrationen.The primary reaction is a combination of 1 molecule ligand with 1 molecule receptor to form a bimolecular ligand / receptor complex (1). Another reaction is caused by the addition of labeled ligand, which is similarly combined with the receptor to form labeled ligand / receptor complex (2). In saturation analysis, the concentrations of the receptor and of the labeled ligand are constant. The concentration of receptor is limited so that the labeled ligand is in excess of the receptor. Under these conditions, the addition of ligand causes competition between ligand and labeled ligand for binding to the receptor. Therefore, an increase in ligand concentration will decrease the amount of labeled ligand / receptor complex. The test principle is based on a determination of the percentage of the total labeled ligand amount bound to the receptor. This percentage is inversely proportional to the amount of ligand added to the reaction mixture from the sample to be measured or from the standard used during the test. The reduction in the concentration of labeled ligand / receptor complex or the increase in the concentration of labeled ligand in the reaction mixture can be used to determine the ligand concentration.

Følsomheden af mætningsanalyse afhænger af anvendelsen af en receptor, som har en meget høj affinitet til liganden og den mærkede ligand. Desuden afhænger følsomheden også af anvendelsen af et mærke, som kan påvises i meget lav koncentration.The sensitivity of saturation analysis depends on the use of a receptor which has a very high affinity for the ligand and the labeled ligand. Moreover, the sensitivity also depends on the use of a mark which can be detected at very low concentration.

3 DK 152313 B3 DK 152313 B

Mætningsanalysens specificitet afhænger af receptorens evne til kun at binde ligand og mærket ligand i en complex blanding af forskellige molekyler.The specificity of the saturation assay depends on the receptor's ability to bind only ligand and labeled ligand in a complex mixture of different molecules.

Mætningsanalyse har været anvendt i en række forskellige teknikker, hvis forskel primært hidrører fra den anvendte type mærke. Sædvanligvis klassificeres disse teknikker ved de brede benævnelser "radioforsøg" eller "ikke-radioforsøg" afhængigt af, om der anvendes et radioaktiv sporstof som mærke.Saturation analysis has been used in a variety of techniques, the difference of which is primarily due to the type of brand used. Usually, these techniques are classified by the broad terms "radio test" or "non-radio test" depending on whether a radioactive tracer is used as a label.

Radioforsøg er anvendt i større udstrækning end ikke-radioforsøg. Radioforsøg kan yderligere klassificeres som radioimmunoforsøg eller radio-receptorforsøg afhængigt af den type receptor, som anvendes ved forsøget. I radioimmunoforsøg anvendes et antistof, som specifikt binder liganden og den mærkede ligand. Ved radio-re-ceptoranalyse anvendes derimod en anden type biologisk receptor, som på lignende måde specifikt binder liganden og den mærkede ligand.Radio experiments have been used to a greater extent than non-radio experiments. Radio experiments may further be classified as radioimmunoassays or radio-receptor assays depending on the type of receptor used in the assay. In radioimmunoassays, an antibody is used which specifically binds the ligand and the labeled ligand. In radio-receptor analysis, on the other hand, another type of biological receptor is used which similarly specifically binds the ligand and the labeled ligand.

Ved alle radioforsøgteknikker er det vigtigt fysisk at adskille den bundne fraktion (1 og 2 i ligning 1) fra den ubundne fraktion (3 og 4 i ligning 1) i reaktionsblandingen. Et indeks for ligandkoncentrationen kan derpå fås ved at tælle disse fraktioner i en radioaktivitetstæller og sammenligne tællingerne for de ukendte prøver med tællinger for hensigtsmæssige standardligandprøver testet ved samme forsøg. Der er beskrevet mange forskellige og afvigende metoder til adskillelse af de bundne og fri fraktioner i radioforsøgsreaktionsblandingen under anvendelse af teknikker som f.eks. gelfiltrering, absorptions- og ionbytterchromatografi, fraktioneret udfældning, fastfase eller elektroforese.In all radio test techniques, it is physically important to separate the bound fraction (1 and 2 in equation 1) from the unbound fraction (3 and 4 in equation 1) in the reaction mixture. An index of ligand concentration can then be obtained by counting these fractions in a radioactivity counter and comparing the counts of the unknown samples with counts of appropriate standard ligand samples tested in the same experiment. Many different and divergent methods have been described for separating the bound and free fractions in the radio test reaction mixture using techniques such as e.g. gel filtration, absorption and ion exchange chromatography, fractional precipitation, solid phase or electrophoresis.

Efter udviklingen af radioforsøgsteknikker i mætningsanalyse er der udviklet metoder, som anvender ikke-radioaktive mærker. Der er demonstreret metoder, som anvender enzymer som mærke. Disse metoder kan have den fordel, at en fysisk adskillelse af de bundne (1+2) og fri (3+4) fraktioner af mærket ligand ikke er nødvendig ved forsøgsproceduren hvis enzymaktiviteten ændres, når et antistofFollowing the development of radio test techniques in saturation analysis, methods have been developed using non-radioactive labels. Methods have been demonstrated that use enzymes as a label. These methods may have the advantage that a physical separation of the bound (1 + 2) and free (3 + 4) fractions of labeled ligand is not required in the experimental procedure if enzyme activity changes when an antibody is changed.

4 DK 152313B4 DK 152313B

binder en ligand, som er mærket med et enzym. Graden i ændringen af enzymets aktivitet er indikation for den mærkede ligands koncentration i den bundne fraktion og giver derfor et indeks for ligandkoncentrationen i reaktionsblandingen.binds a ligand which is labeled with an enzyme. The degree of change in the activity of the enzyme is indicative of the concentration of the labeled ligand in the bound fraction and therefore gives an index of the ligand concentration in the reaction mixture.

Den kemiske struktur af ligand-enzym-complexet er yderst svært at bestemme, og dette er hovedulempen ved enzymimmunoforsøg. Dette skyldes uden tvivl den store mængde aminosyresidekæder, som er til stede på enzymoverfladen til complexdannelse med liganden. Dette bevirker store problemer ved reproduktionen af ligand--enzymet ved forskellige præparater af complexet. Den generelle mangel på styring af den complexdannende reaktion resulteret i binding af mange ligandmolekyler til ét enzymmolekyle, selv om det er sandsynligt, at bindingen af kun nogle få af disse ligandmolekyler til antistoffet er involveret i inhiberingen af enzymaktiviteten. Derfor resulterer ikke alle antistof/mærket antigen“inter-aktioner i en modificering af enzymaktiviteten, og derved reduceres teknikkens følsomhed.The chemical structure of the ligand-enzyme complex is extremely difficult to determine, and this is the major disadvantage of enzyme immunoassays. This is undoubtedly due to the large amount of amino acid side chains present on the enzyme surface for complexation with the ligand. This causes major problems in the reproduction of the ligand enzyme by various preparations of the complex. The general lack of control of the complex-forming reaction resulted in the binding of many ligand molecules to one enzyme molecule, although it is likely that the binding of only a few of these ligand molecules to the antibody is involved in the inhibition of enzyme activity. Therefore, not all antibody / tagged antigen "interactions result in a modification of the enzyme activity, thereby reducing the sensitivity of the technique.

Protein-protein-vekselvirkning mellem forskellige antistofmolekyler og antistof- og enzymmolekyler er en anden følge af mængden af ligandmolekyler på enzymoverfladen. Protein-protein-vekselvirkningen forøges yderligere, hvis liganden også er et polypeptid. Enzymmolekylet inducerer derfor et lokaliseret mikro-område med høj proteinkoncentration. I sådanne situationer er det påvist, at der sker proteinudfældning, hvilket forårsager, at én af hovedfordelene ved enzymimmunoforsøg går tabt, idet det bliver nødvendigt at adskille antistofbundne og frie fraktioner.Protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the amount of ligand molecules on the enzyme surface. The protein-protein interaction is further enhanced if the ligand is also a polypeptide. Therefore, the enzyme molecule induces a localized micro region of high protein concentration. In such situations, it has been shown that protein precipitation occurs, causing one of the main benefits of enzyme immunoassay to be lost as it becomes necessary to separate antibody bound and free fractions.

Der er beskrevet en modifikation af enzymimmunoforsøget, ved hvilket disse problemer delvis løses, idet liganden mærkes med et detektormolekyle, som har lav molekylvægt. I dette forsøg bevirker antistof-bindingen af mærket ligand sterisk hindring af binding af detektormolekylet til et andet antistof, som er specifikt for detektormolekylet. Hindringsgraden bestemmes af konkurrence om binding af det fri ligand-detektormolekyle og detektormolekylmærket enzym med detektor molekyle-antistof . Graden af ændringen af enzymaktiviteten,A modification of the enzyme immunoassay has been described, in which these problems are partially solved, labeling the ligand with a low molecular weight detector molecule. In this experiment, the antibody binding of labeled ligand sterically inhibits binding of the detector molecule to another antibody specific to the detector molecule. The degree of inhibition is determined by competition for binding of the free ligand detector molecule and detector molecule labeled enzyme with detector molecule antibody. The degree of change in enzyme activity,

5 DK 152313B5 DK 152313B

som bestemmes ved normalt enzymimmunoforsøg, er indikation for koncentrationen af det fri ligand-detektormolekyle, som ligeledes er indikation for ligandkoncentrationen i reaktionsblandingen. Fordelen ved denne teknik er, at et lille molekyle snarere end et enzym bindes til liganden og derved tillader en bestemmelse af den mærkede ligands kemiske struktur, hvorved mange af de ovenfor beskrevne ulemper ved enzymimmunoforsøget overvindes. Ved dette system overvindes imidlertid kun ulemperne ved den primære bindingsreaktion, som involverer liganden og den mærkede ligand, og de overføres til detektorsystemet, hvor graden af antistofbundne og antistoffri fraktioner af mærket ligand bestemmes.which is determined by normal enzyme immunoassay is indication of the concentration of the free ligand detector molecule which is also indication of the ligand concentration in the reaction mixture. The advantage of this technique is that a small molecule, rather than an enzyme, binds to the ligand, thereby allowing a determination of the chemical structure of the labeled ligand, thereby overcoming many of the disadvantages of the enzyme immunoassay described above. However, in this system, only the disadvantages of the primary binding reaction involving the ligand and the labeled ligand are overcome and they are transferred to the detector system where the degree of antibody bound and antibody-free fractions of the labeled ligand is determined.

Ulemperne ved de hidtil kendte fremgangsmåder overvindes ved den foreliggende opfindelse, ifølge hvilken der anvendes en enzymmod :i -F i k a r o r .9 nm mæ r ke .The disadvantages of the known methods are overcome by the present invention, according to which an enzyme mode is used: i -F i k a r o r .9 nm label.

Nærmere bestemt angår den foreliggende opfindelse et reagens af den indledningsvis beskrevne art, hvilket reagens er ejendommeligt ved, at det mærkede immunologiske materiale er et antigen-enzyminhibitor- eller anti-gen-enzymaktivator-complex.More specifically, the present invention relates to a reagent of the type described initially, which is characterized in that the labeled immunological material is an antigen-enzyme inhibitor or anti-gene enzyme activator complex.

Udtrykket "immunologisk aktivt materiale" eller "ligand" betegne., her ethvert immunologisk aktivt stof eller en del deraf, som kan bestemmes immunologisk, f.eks. under anvendelse af mætningsanalyseteknik. Det væsentlige krav er, at der er en receptor, som specifikt kan binde liganden. Når receptoren er et antistof, er liganden hapte-nisk eller antigenisk, så at der kan dannes specifikt antistof. En ligand betegnes som en hapten, når den kun udløser antistofdannelse, når den bindes til en forbindelse med antigen-egenskaber. En ligand betegnes alternativt som antigen, når den udløser antistofdannelse uden kemisk modifikation. Ligandens molekylvægt kan variere i et bredt område fra ca. 100 til ca. 1.000.000. Dette molekylvægtsområde er ikke begrænsende for forsøget, forudsat at der findes en receptor, som specifikt kan binde liganden.The term "immunologically active material" or "ligand" refers to any immunologically active substance or part thereof which may be immunologically determined, e.g. using saturation analysis technique. The essential requirement is that there is a receptor that can specifically bind the ligand. When the receptor is an antibody, the ligand is haptic or antigenic so that specific antibody can be formed. A ligand is referred to as a hapten when it triggers antibody formation only when it binds to a compound having antigenic properties. Alternatively, a ligand is referred to as antigen when it triggers antibody formation without chemical modification. The molecular weight of the ligand can vary in a wide range from ca. 100 to approx. 1,000,000. This molecular weight range is not limiting the assay, provided that a receptor is found that can specifically bind the ligand.

Liganden kan have polymer- eller ikke-polymerstruktur. Når liganden har polymerstruktur, har den sædvanligvis biologisk oprindelse og kan klassificeres som en nucleinsyre, et polysaccharid og/eller polypeptid. Når liganden på den anden side er ikke-polymer, har den sædvanligvis en molekylvægt på mindre end 2.000 og kan have enThe ligand may have polymer or non-polymer structure. When the ligand has polymer structure, it usually has biological origin and can be classified as a nucleic acid, a polysaccharide and / or polypeptide. On the other hand, when the ligand is non-polymeric, it usually has a molecular weight of less than 2,000 and can have a

e DK 152313Be DK 152313B

Særligt vigtige ligander, som kan anvendes ifølge den foreliggende opfindelse, er aminer, aminosyrer, peptider, proteiner, lipoproteiner, glycoproteiner, steroler, steroider, lipoider, nucleinsyrer, mono-og polysaccharider, alkaloider, vitaminer, medikamenter, narkotika, antibiotika, metaboliter, pesticider, toxiner, industrielle forurenende stoffer, aromastoffer, hormoner, enzymer, co-enzymer, cellulære eller ekstracellulære vævsbestanddele og antistoffer isoleret fra mennesker eller dyr.Particularly important ligands which can be used in the present invention are amines, amino acids, peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, mono- and polysaccharides, alkaloids, vitamins, drugs, drugs, antibiotics, metabolites, pesticides, toxins, industrial pollutants, flavors, hormones, enzymes, co-enzymes, cellular or extracellular tissue components and antibodies isolated from humans or animals.

Umiddelbart potentielle bestanddele til analyse med det foreliggende system er hepatitis B-overfladeantigen, ferritin, tumorantigener såsom CEA, α-fetoproteiner, rheumatoidfaktor, C-reaktive proteiner, immunoglobulinklasserne IgG, IgM eller IgA, myoglobin, thyroidhormoner omfattende T3 og , insulin, steroidhormoner omfattende testosteron eller estradiol, vanedannende medikamenter, herunder narkotiske analgetika såsom morphin, barbiturater, stimulanser såsom amphetamin, medikamenter til behandling af epilepsi herunder diphenylhydantoin og phenobarbital, cardialglycocyter såsom digoxin, vitaminer såsom vitamin og folinsyre. Endvidere er antistoffer, som er umiddelbart potentielle til analyse med systemet, sådanne, som er forbundet med infektioner ved syfilis, gonorrhoae, brucellosis, rubella og rheumatisme.Immediately potential constituents for analysis with the present system are hepatitis B surface antigen, ferritin, tumor antigens such as CEA, α-fetoproteins, rheumatoid factor, C-reactive proteins, immunoglobulin classes IgG, IgM or IgA, myoglobin, thyroid hormones comprising T3 and, insulin, steroid hormones. testosterone or estradiol, addictive drugs, including narcotic analgesics such as morphine, barbiturates, stimulants such as amphetamine, drugs for the treatment of epilepsy including diphenylhydantoin and phenobarbital, cardiac glycocytes such as digoxin, vitamins such as vitamin and folic acid. Furthermore, antibodies which are immediately potential for analysis with the system are those associated with infections by syphilis, gonorrhea, brucellosis, rubella and rheumatism.

Udtrykket "mærket immunologisk aktivt materiale" eller "mærket receptor" betegner her en ligand, en ligandanalog eller en del deraf eller en receptor, som er mærket med en enzymmodifikator. Ét, mere end ét og sædvanligvis mindre end 100 mærkemolekyler kan bindes til et ligand- eller receptormolekyle. Ligeledes kan et, mere end ét og sædvanligvis mindre end 5 ligand- eller receptormolekyler bindes til et mærkemolekyle. Bindingen af ekstra mærkemolekyler til et ligand- eller receptormolekyle forøger sædvanligvis forsøgets følsomhed, forudsat at de ekstra mærker ikke påvirker binding.The term "labeled immunologically active material" or "labeled receptor" here refers to a ligand, a ligand analogue or a portion thereof, or a receptor labeled with an enzyme modifier. One, more than one, and usually less than 100 label molecules can be bound to a ligand or receptor molecule. Likewise, one, more than one, and usually less than 5 ligand or receptor molecules can be bound to a label molecule. The binding of extra labels to a ligand or receptor molecule usually increases the sensitivity of the experiment, provided that the extra labels do not affect binding.

Binding af et modifikatomolekyle til et ligand- eller receptormolekyle medfører dannelse af intermolekylære bindinger, som i de fleste tilfælde, men ikke nødvendigvis, er af covalent art. Bindingen kan i nogle tilfælde gennemføres i nærværelse af et koblingsmiddel ved indsættelse af en kædegruppe mellem mærket ocr licranden eller receptoren.Binding of a modifier atom to a ligand or receptor molecule results in the formation of intermolecular bonds, which in most cases, but not necessarily, are of a covalent nature. The binding may in some cases be carried out in the presence of a coupling agent by inserting a chain group between the label and the licorice or receptor.

7 DK 152313 B7 DK 152313 B

iin

Modifikatormolekylet kan bindes direkte til liganden eller receptormolekylet. Det kan imidlertid være ønskeligt at indsætte kemiske broer af forskellig længde mellem modifikatormolekylet og ligand- eller receptormolekylerne afhængigt af det tilsigtede specifikke forsøg. I nogle tilfælde kan det endog være fordelagtigt at binde modifikatormolekylet og ligand- eller receptormolekylerne separat til samme bærermolekyle, f.eks. et makromolekyle såsom et poly-peptid eller et polysaccharid.The modifier molecule can be directly linked to the ligand or receptor molecule. However, it may be desirable to insert different length chemical bridges between the modifier molecule and the ligand or receptor molecules, depending on the intended specific experiment. In some cases, it may even be advantageous to bind the modifier molecule and the ligand or receptor molecules separately to the same carrier molecule, e.g. a macromolecule such as a polypeptide or a polysaccharide.

Udtrykket "receptor" betegner her enhver substans, som specifikt kan binde ligand og mærket ligand eller en del deraf. Sædvanligvis er den i forsøget anvendte receptor et specifikt antistof for den ligand, som er dannet i blodet hos hvirveldyr efter injektion af en hensigtsmæssigt hapten eller et hensigtsmæssigt antigen. Der kan også anvendes naturligt forekommende receptorer i forsøget. Denne sidstnævnte gruppe omfatter proteiner, nucleinsyrer og cellulære membraner . Sådanne receptorer har været anvendt i radioforsøgsteknik-ker for thyroxin, insulin, angiotensin og forskellige steroidhormoner .The term "receptor" as used herein refers to any substance that can specifically bind ligand and labeled ligand or part thereof. Usually, the receptor used in the assay is a specific antibody for the ligand that is formed in the blood of vertebrates after injection of a suitable hapten or an appropriate antigen. Naturally occurring receptors may also be used in the experiment. This latter group comprises proteins, nucleic acids and cellular membranes. Such receptors have been used in radio test techniques for thyroxine, insulin, angiotensin and various steroid hormones.

Når liganden er et antistof, kan receptoren være det antigen, som anvendes til at inducere dette antistof i et værtsdyr. Ved en anden udførelsesform kan receptoren være et antistof mod det antistof, som skal bestemmes.When the ligand is an antibody, the receptor may be the antigen used to induce this antibody in a host animal. In another embodiment, the receptor may be an antibody to the antibody to be determined.

Det er ikke muligt med sikkerhed at bestemme den receptorvirkningsmåde, som, ved binding af den mærkede ligand, reducerer vekselvirkningen mellem enzym og modifikator. Den mest sandsynlige forklaring er, at enzymets affinitet til modifikatoren reduceres som et resultat af en ændring i størrelse og nettoladning af receptor/ligand--modifikatorcomplexet i sammenligning med værdierne for ligand--modifikator alene.It is not possible to determine for sure the receptor mode of action which, by binding of the labeled ligand, reduces the interaction between enzyme and modifier. The most likely explanation is that the affinity of the enzyme to the modifier is reduced as a result of a change in size and net charge of the receptor / ligand-modifier complex in comparison with the values of ligand-modifier alone.

Udtrykket "modifikator" betegner her en hvilken som helst substans, som er i stand til at vekselvirke med et enzym, så at enzymaktivitetsgraden og -måden modificeres. Denne modifikation kan resultere i en inhibering, aktivering eller ændring i specificitet eller en hvilken som helst anden af enzymets egenskaber, som kan detekte-The term "modifier" here means any substance capable of interacting with an enzyme so that the degree and manner of enzyme activity are modified. This modification may result in an inhibition, activation or change in specificity or any other of the enzyme's properties which can detect

8 DK 152313B8 DK 152313B

res enten direkte eller indirekte ved en ændring i enzymaktiviteten eller i aktivitetsmåden, f.eks. en ændring i reaktionsbetingelserne, f.eks. co-faktorkrav eller pH-værdi-optimum, i kinetiske egenskaber eller i aktiveringsenergi. Modifikatoren kan i størrelse variere fra et lille molekyle til et makromolekyle, og dets vekselvirkning med et enzymmolekyle kan enten være reversibel eller irreversibel afhængig af, om den inter-molekylære binding er ionisk eller covalent.is either directly or indirectly caused by a change in enzyme activity or in the mode of activity, e.g. a change in reaction conditions, e.g. co-factor requirements or pH value optimum, in kinetic properties or in activation energy. The modifier can range in size from a small molecule to a macromolecule, and its interaction with an enzyme molecule can be either reversible or irreversible depending on whether the inter-molecular bond is ionic or covalent.

Forsøgets følsomhed er bl.a. afhængig af receptorens affinitet til liganden og modifikatorens evne til at forårsage en ændring i enzymaktivitetsgraden eller -måden. Fortrinsvis opnås ændringen i enzymaktivitetsgraden eller -måden med en minimal modifikatorkoncentration. Jo nærmere denne koncentration ligger til enzymkoncentrationen på molekylår basis, jo større vil forsøgets følsomhed være.The sensitivity of the experiment is i.a. depending on the affinity of the receptor for the ligand and the ability of the modifier to cause a change in the degree or mode of enzyme activity. Preferably, the change in enzyme activity degree or mode is achieved with a minimal modifier concentration. The closer this concentration is to the enzyme concentration on a molecular year basis, the greater the sensitivity of the experiment.

Modifikatoren er fortrinsvis en enzyminhibitor, som, ved vekselvirkning med enzymet, inhiberer dets aktivitet. Inhibitorens virkemåde kan være konkurrerende, ikke-konkurrerende, aliosteroisk eller en kombination af to eller flere af disse måder. Inhibitoren bør fortrinsvis have en inhiberingskonstant (den inhibitorkoncentration, som er nødvendig til 50%'s inhibering af enzymsystemet) _3 på under 10 mol/liter, af hasngigt af det udførte forsøg. Inhibe- -15 -5 ringskonstanten ligger særligt foretrukket mellem 10 og 10The modifier is preferably an enzyme inhibitor which, upon interaction with the enzyme, inhibits its activity. The mode of action of the inhibitor may be competing, non-competing, aliosteroic, or a combination of two or more of these ways. Preferably, the inhibitor should have an inhibition constant (the inhibitor concentration required for 50% inhibition of the enzyme system) of less than 10 moles / liter of gout of the test performed. The inhibition constant is particularly preferred between 10 and 10

Et hvilket som helst enzym kan anvendes i forsøget, forudsat at der eksisterer en modifikator, som specifikt modificerer enzymaktiviteten på den ovenfor beskrevne måde. Foretrukne enzymer er stabile, let tilgængelige til lav pris og har et højt omsætningstal og et simpelt udførligt forsøgssystem. Omsætningstallet (antal produktmolekyler dannet pr. enzymmolekyle på 1 minut) ligger fortrinsvis over 100 afhængigt af den specifikke test. Mere foretrukket er omsætningstallet så højt som muligt og mindst 200.Any enzyme may be used in the assay, provided that a modifier exists which specifically modifies the enzyme activity in the manner described above. Preferred enzymes are stable, readily available at low cost and have a high turnover rate and a simple elaborate experimental system. The turnover rate (number of product molecules formed per enzyme molecule in 1 minute) is preferably above 100 depending on the specific test. More preferably, the turnover rate is as high as possible and at least 200.

Enzymmodifikatorsystemer, som er særligt egnede ved den foreliggende opfindelse , er dihydrofolatreduktase/methotrexat, dihydrofolatreduk-Enzyme modifier systems which are particularly useful in the present invention are dihydrofolate reductase / methotrexate, dihydrofolate reductase.

9 .....DK 152313 B9 ..... DK 152313 B

tase/4-aminopterin, dihydrofolatreduktase/andre specifikke inhibitorer af dette enzym; p-glucoronidase/4-deoxy-5-aminoglucarsyre og derivater deraf; biotin indeholdende enzymer/avidinlignende carboxylase/avidin; chymotrypsin/TPCKtase / 4-aminopterin, dihydrofolate reductase / other specific inhibitors of this enzyme; p-glucoronidase / 4-deoxy-5-aminoglucaric acid and its derivatives; biotin containing enzymes / avidin-like carboxylase / avidin; chymotrypsin / TPCK

CH2-Ph \ I TPCK = CH,-// A-S0o-NH-C-C-CH,Cl |CH2-Ph \ I TPCK = CH, - // A-SOO-NH-C-C-CH, Cl |

V \J I II JV \ J I II J

γ-cystathionase/propargylglycin; alaninracemase/trifluoralanin; tryptophanase/trifluoralanin; tryptophansynthetase/trifluoralanin; β-cystathionase/trifluoralanin; pyrovat-glutamattransaminase/tri-fluoralanin; mælkesyreoxidase/2-hydroxy-but-3-yn-syre; monoamin-OXidase/N,N-dimethylpropargylamin og diaminoxidase/H^N-CH^-CSCC^-N^.γ-cystathionase / propargylglycine; alanine racemase / trifluoroalanine; tryptophanase / trifluoroalanine; tryptophansynthetase / trifluoroalanine; β-cystathionase / trifluoroalanine; pyruvate-glutamattransaminase / tri-fluoroalanine; mælkesyreoxidase / 2-hydroxy-but-3-yn-acid; monoamine-Oxidase / N, N-dimethylpropargylamine and diamine oxidase / H ^ N-CH2 -CSCC ^ -N ^.

Enzymaktivitetsbestemmelsen kan udføres ved direkte eller indirekte at overvåge substratkonsumptionen eller produktdannelsen ved en egnet pH-værdi og temperatur under anvendelse af et detektorsystem, f.eks. colorimetri, spektrofotometri, fluorospektrofotometri, gaseometri, thermometri (varmeproduktion) eller scintillations-tælling.The enzyme activity assay can be performed by directly or indirectly monitoring the substrate consumption or product formation at a suitable pH and temperature using a detector system, e.g. colorimetry, spectrophotometry, fluorospectrophotometry, gasometry, thermometry (heat production) or scintillation counting.

For at forøge systemets følsomhed er det muligt at anvende bioluminescens og enzymcyklus-teknikker, f.eks. de teknikker, som er beskrevet af J. Lee et al. i Liquid Scintillation Counting:In order to increase the sensitivity of the system, it is possible to use bioluminescence and enzyme cycle techniques, e.g. the techniques described by J. Lee et al. in Liquid Scintillation Counting:

Recent Developments, Stanley P.E. og Scoggins, B.A., Academic Press, New York, side 403 og Lowry O.H. et al. i J. Biol. Chem.Recent Developments, Stanley P.E. and Scoggins, B.A., Academic Press, New York, page 403 and Lowry O.H. et al. in J. Biol. Chem.

236, side 2746 - 2755.236, pages 2746 - 2755.

Ved en særlig udførelsesform af den foreliggende opfindelse kan der anvendes en mærket ligand til bestemmelse af tilstedeværelsen af en ligand i en ukendt prøve ved simultan eller sekventiel tilsætning ved en hensigtsmæssig pH-værdi af den mærkede ligand og den ukendte prøve til et vandigt medium, som indeholder en receptor, som er specifik for liganden og den mærkede ligand.In a particular embodiment of the present invention, a labeled ligand can be used to determine the presence of a ligand in an unknown sample by simultaneous or sequential addition at an appropriate pH of the labeled ligand and the unknown sample to an aqueous medium which contains a receptor specific to the ligand and the labeled ligand.

Efter en passende inkubationsperiode bestemmes fordelingen af receptor bundet til ligand og mærket ligand ved tilsætning af enzym og substrater. Den receptor, som er specifik for liganden,After a suitable incubation period, the distribution of receptor bound to ligand and labeled ligand is determined by the addition of enzyme and substrates. The receptor specific for the ligand

10 DK 152313B10 DK 152313B

binder også den mærkede ligand, og derved reduceres vekselvirkningen mellem modifikatoren og enzymet, så at modificeringen af enzymaktiviteten formindskes. Tilsætning af ligand i forsøget resulterer i en konkurrence med den mærkede ligand om binding til receptoren, og derved forøges koncentrationen af fri mærket ligand i forsøget. Vekselvirkningen mellem ligand-modifikator og enzym forøges, og enzymaktiviteten påvirkes atter. Modificeringen af enzymaktiviteten er derfor en funktion af ligandkoncentrationen i forsøget og forårsages af ubundet mærket ligand. Derfor er forskellen mellem den resulterende enzymaktivitet og den aktivitet, der fås i fraværelse af ligand, indikativ for ligandkoncentrationen i den ukendte prøve.also binds the labeled ligand, thereby reducing the interaction between the modifier and the enzyme so as to reduce the modification of the enzyme activity. Addition of ligand in the experiment results in a competition with the labeled ligand for binding to the receptor, thereby increasing the concentration of free labeled ligand in the experiment. The interaction between ligand modifier and enzyme is increased and enzyme activity is affected again. Therefore, the modification of enzyme activity is a function of the ligand concentration in the assay and is caused by unbound labeled ligand. Therefore, the difference between the resulting enzyme activity and the activity obtained in the absence of ligand is indicative of the ligand concentration in the unknown sample.

En at hovedfordelene ved denne fremgangsmåde er, at adskillelse af bundne og fri fraktioner er unødvendig ved fremgansmåden. Dette udelukker imidlertid ikke anvendelsen af et sådant separationstrin i forsøget efter inkubering af liganden og mærket ligand med receptoren før enzymaktivitetsbestemmelsen. Adskillelse af fraktioner kan være ønskelig i nogle tilfælde til fjernelse af stoffer i prøven, som kan forstyrre enzymforsøget. Separationen kan udføres under anvendelse af en hvilken som helst af de teknikker, som er beskrevet ved radioimmunoforsøget, herunder gelfiltrering, adsorptions- og ionby tterchromatograf i, fraktioneret udfældning, fastfase og elektro-forese.One of the main advantages of this process is that separation of bound and free fractions is unnecessary in the process. However, this does not preclude the use of such a separation step in the experiment after incubating the ligand and labeled ligand with the receptor prior to the enzyme activity assay. Separation of fractions may be desirable in some cases for the removal of substances in the sample which may interfere with the enzyme assay. The separation can be carried out using any of the techniques described in the radioimmunoassay, including gel filtration, adsorption and ion exchange chromatograph, fractional precipitation, solid phase and electrophoresis.

Den foreliggende opfindelse belyses nærmere ved følgende eksempler. Eksempel 1.The present invention is further illustrated by the following examples. Example 1.

Fremstilling af "amino-digoxin".Preparation of "amino digoxin".

Til en suspension af 156 mg (0,2 millimol) digoxin i 5 ml absolut ethanol sættes under omrøring 10 ml 0,2M natriummetaperiodat.To a suspension of 156 mg (0.2 millimoles) of digoxin in 5 ml of absolute ethanol is added 10 ml of 0.2M sodium metaperiodate with stirring.

Blandingen bliver homogen efter 10 minutter, og derpå dannes langsomt et bundfald. Efter 2 timer tilsættes 5 ml vand + 5 ml ethanol. Efter yderligere 30 minutter tilsættes 122^ul (2,2 millimol) ethylenglycol, og der begynder straks at udfældes et tæt hvidt bundfald. Efter 80 minutters omrøring tilsættesThe mixture becomes homogeneous after 10 minutes, and then a precipitate is slowly formed. After 2 hours, add 5 ml of water + 5 ml of ethanol. After another 30 minutes, 122 µl (2.2 millimoles) of ethylene glycol is added and a dense white precipitate immediately begins to precipitate. After stirring for 80 minutes, add

11 DK 152313 B11 DK 152313 B

133^ul (2,0 millimol) ethylendiamin, og den resulterende pH-vær-di på 11,0 indstilles til 9,5 med 0,1M saltsyre, og reaktionsblandingen lades henstå ved stuetemperatur i 18 timer. pH-Værdien ændres ikke i denne periode.133 µl (2.0 millimoles) of ethylenediamine, and the resulting pH of 11.0 is adjusted to 9.5 with 0.1M hydrochloric acid and the reaction mixture is allowed to stand at room temperature for 18 hours. The pH value does not change during this period.

151,4 mg (4,0 millimol) natriumborhydrid tilsættes derpå, og blandingen omrøres i 3 1/2 time. pH-Værdien indstilles derpå fra 10,5 til 6,5 med 1M myresyre (ca. 3 ml), og tyndtlagschromatografi på denne blanding viste en enkelt stor plet med en R^-værdi på 0,15 (siliciumdioxid på aluminium fremkaldt i butanol:eddikesyre:vand (4:1:1))· Digoxin har en R^-værdi på 0,7, når det fremkaldes i samme system.151.4 mg (4.0 millimoles) of sodium borohydride is then added and the mixture is stirred for 3 1/2 hours. The pH was then adjusted from 10.5 to 6.5 with 1M formic acid (about 3 ml) and thin layer chromatography on this mixture showed a single large spot with an R R value of 0.15 (silica on aluminum developed in butanol : acetic acid: water (4: 1: 1)) · Digoxin has an R 2 value of 0.7 when developed in the same system.

Opløsningsmidlerne afdampes til næsten tørhed på en rotationsfordamper under vakuum med en vandbadtemperatur på 60°C. De sidste få ml vand fjernes ved at tilsætte 95% ethanol (3 x 20 ml) og at gentage inddampningen som ovenfor.The solvents are evaporated to near dryness on a rotary evaporator under vacuum with a water bath temperature of 60 ° C. The last few ml of water are removed by adding 95% ethanol (3 x 20 ml) and repeating the evaporation as above.

Det resulterende bleggule faste stof ekstraheres tre gange med absolut ethanol, og disse forenede ekstrakter inddampes til ca. 4 ml og centrifugeres til fraskillelse af en lille mængde salt, som kasseres. Den bleggule supernatantopløsning inddampes til tørhed under en strøm af tørt nitrogen, hvorved fås en gul olie, som har samme R^-værdi ved tyndtlagschromatografi som den ovenfor beskrevne reaktionsblanding. Det vises, at produktet indeholder en fri aminogruppe ved at omsætte det med Fluram® (4-phenylspiro[flu-ran-2(3H),11-phthalan]-3,31-dion) til dannelse af en intensivt fluorescerende forbindelse. Dette produkt har i koncentreret svovlsyre et spektrum, som ligner digoxins med absorptionstoppe ved 385 og 495 nm. Produktet udviser også stærk affinitet til antisera (kanin), som er specifikke for digoxin. Den mest sandsynlige struktur for det isolerede produkt er den nedenstående: ,ro ™ r°'« CH3 —p 1 li & & jTiCyit h2n-ch2ch?-n -The resulting pale yellow solid is extracted three times with absolute ethanol and these combined extracts are evaporated to ca. 4 ml and centrifuged to separate a small amount of salt, which is discarded. The pale yellow supernatant solution is evaporated to dryness under a stream of dry nitrogen to give a yellow oil having the same R R value by thin layer chromatography as the reaction mixture described above. It is shown that the product contains a free amino group by reacting it with Fluram® (4-phenylspiro [fluorane-2 (3H), 11-phthalane] -3,31-dione) to form an intensely fluorescent compound. This concentrated sulfuric acid product has a spectrum similar to digoxins with absorption peaks at 385 and 495 nm. The product also exhibits strong affinity for antisera (rabbit) specific for digoxin. The most likely structure of the isolated product is the following:, ro ™ r ° '' CH3 —p 1 li & & jTiCyit h2n-ch2ch? -N -

12 DK 152313B12 DK 152313B

Eksempel 2.Example 2.

Fremstilling af methotrexat-aminodigoxin-konjuat.Preparation of methotrexate-aminodigoxin conjuate.

11 mg methotrexat opløses i 5 ml vand, og pH-værdien indstilles til 6,5. 10 mg "aminodigoxin" opløses i denne opløsning, og volumenet indstilles til 20 ml med vand. pH-Værdien indstilles atter til 6,5, og til reaktionsblandingen sættes 484 mg N-ethyl-Nn-(3-dimethyl-amino)-propylcarbodiimid-hydrochlorid opløst i 5 ml vand. pH-Vær-dien holdes på 6,2 i 24 timer ved stuetemperatur. Konjugatet renses på en silicagelkolonne under anvendelse af 3%'s ammoniumcitrat som opløsningsmiddel. Fraktioner, som indeholder det ønskede produkt, forenes og vises at have den dobbelte kapacitet til stærkt at binde antisera (kanin), som er specifikke for digoxin, og også til stærkt at inhibere enzymet dihydrofolatreduktase (kyllingelever). Den mest sandsynlige struktur for methotrexat-aminodigoxin-konju-gatet er den nedenstående:Dissolve 11 mg of methotrexate in 5 ml of water and adjust the pH to 6.5. 10 mg of "aminodigoxin" is dissolved in this solution and the volume is adjusted to 20 ml with water. The pH is adjusted to 6.5 again and 484 mg of N-ethyl-Nn- (3-dimethylamino) propylcarbodiimide hydrochloride dissolved in 5 ml of water are added to the reaction mixture. The pH value is kept at 6.2 for 24 hours at room temperature. The conjugate is purified on a silica gel column using 3% ammonium citrate as solvent. Fractions containing the desired product are combined and shown to have the dual capacity to strongly bind antisera (rabbits) specific for digoxin and also to strongly inhibit the enzyme dihydrofolate reductase (chicken liver). The most likely structure of the methotrexate-aminodigoxin conjugate is the following:

ΠΟ 0NΠΟ 0N

ljH3 f-—?°ljH3 f -—? °

Qx~fQ x ~ f

æ7 ch3 ch3 0Hæ7 ch3 ch3 0H

HN-CH2CH2-n ~HN-CH2CH2-n ~

HH

O =C CH2 CH f' NH2 λ i 2 I il 1 HO __ I _ T CH3 |__j CHNHC ?CH2 NH2 / I I ch3.„ ?®3 rtjtr I 1 hn-ch2-ch2-n tiO = C CH2 CH f 'NH2 λ i 2 I il 1 HO __ I _ T CH3 | __j CHNHC? CH2 NH2 / I I ch3.'? ®3 rtjtr I 1 hn-ch2-ch2-n ti

DK 152313BDK 152313B

1313

Eksempel 3.Example 3

Enzyminhibitorimmunoforsøg for digoxin.Enzyme inhibitor immunoassay for digoxin.

100 μΙ serum inkuberes ved 30°C i 15 minutter sammen med 100 μΙ anti-digoxin-antistofopløsning, 100 μΙ NADPH-opløsning, 100 μΙ 2-mercaptoeth-anolopløsning og 500 μΙ natriumphosphatpuffer med pH-værdi 7,5. 100 μΙ methotrexat-digoxin-konjugat fra eksempel 2 (70 μg/ml) tilsættes, og blandingen inkuberes i 15 minutter, hvorefter der tilsættes 100 μΙ dihydrofolat-reduktaseopløsning. Det anvendte dihydrofolatreduktasepræparat isoleres fra kyllingelever ved fremgangsmåden ifølge Kaufman, B.T., & Gardiner, R.C., Journal of Biological Chemistry, bind 211, side 1319 (1966). Blandingen inkuberes i yderligere 3 minutter, og enzymaktiviteten bestemmes ved tilsætning af 100 μΙ dihydrofolatopløsning og måling ved 340 nm med et Varian® spektrofotometer med skriver. Resultaterne er anført i tabel I.100 μΙ serum is incubated at 30 ° C for 15 minutes together with 100 μΙ anti-digoxin antibody solution, 100 μΙ NADPH solution, 100 μΙ 2-mercaptoeth anole solution and 500 μΙ sodium phosphate buffer with pH 7.5. Add 100 μΙ of methotrexate digoxin conjugate from Example 2 (70 μg / ml) and incubate for 15 minutes, then add 100 μΙ dihydrofolate reductase solution. The dihydrofolate reductase used is isolated from chicken livers by the method of Kaufman, B. T., & Gardiner, R. C., Journal of Biological Chemistry, Vol. 211, page 1319 (1966). The mixture is incubated for an additional 3 minutes and the enzyme activity is determined by adding 100 μΙ of dihydrofolate solution and measuring at 340 nm with a Varian® spectrophotometer with printer. The results are listed in Table I.

Tabel I.Table I.

Reaktanter EnzymaktivitetReactants Enzyme activity

Digoxin Antidigoxin- Methotrexat- Enzym- DD/min Inhibering (prøvekon- -antistof -digoxin-kon- -for- i % centration) jugat (i for- søgs- søg) kompo- ____nenter___ 0 fraværende 0 til stede 0,150 0 0 fraværende 7 ng til stede 0,100 33 0 til stede 7 ng til stede 0,145 3 5 ng/ml til stede 7 ng til stede 0,125 16 10 ng/ml til stede 7 ng til stede 0,115 23Digoxin Antidigoxin- Methotrexate- Enzyme- DD / min Inhibition (sample con- antibody-digoxin con- -for- in% centration) jugate (in trial) Components _____ 0 absent 0 present 0.150 0 0 absent 7 present 0,100 33 0 present 7 ng present 0.145 3 5 ng / ml present 7 ng present 0.125 16 10 ng / ml present 7 ng present 0.115 23

Reaktanterne tilsættes i den ovenfor anførte rækkefølge.The reactants are added in the order listed above.

OD = optisk densitet.OD = optical density.

X4 DK 152313 BX4 DK 152313 B

Af ovenstående resultater fremgår det, at nogle få nanogram digoxin i serum kan bestemmes ved det beskrevne system.From the above results, it appears that a few nanograms of digoxin in serum can be determined by the system described.

Eksempel 4.Example 4

Fremstilling af methotrexat-humant serumalbumin-konjugat.Preparation of methotrexate-human serum albumin conjugate.

45 mg methotrexat opløses i 1,0 ml Ν,Ν-dimethylformamid, og der tilsættes 25 mg N-hydroxysuccinimid. 41 mg Ν,Ν-dicyclohexylcarbo-diimid opløses, og blandingen holdes ved stuetemperatur i 13 timer. Det som biprodukt fremkomne uopløselige urinstof frafiltreres, og lOOyUl af filtratet sættes til en opløsning, som indeholder 5 mg humant serumalbumin i 800/ul 0,1M natriumphosphatpuffer med pH-værdi 7,5 plus lOO^ul dioxan. Reaktionsblandingen holdes ved stuetemperatur i 30 minutter og hældes derpå på en Sephadex G-25-kolonne, som er ækvilibreret med 0,1M natriumphosphatpuffer med pH-værdi 7,5. Kolonnen elueres med denne puffer, og der fås to elueringstoppe, som indeholder methotrexat, og den første indeholder methotrexat-humant serumalbumin-konjugatet.Dissolve 45 mg of methotrexate in 1.0 ml of Ν, Ν-dimethylformamide and 25 mg of N-hydroxysuccinimide is added. Dissolve 41 mg of Ν, Ν-dicyclohexylcarbo-diimide and keep the mixture at room temperature for 13 hours. The by-product insoluble urea is filtered off and 100 µl of the filtrate is added to a solution containing 5 mg of human serum albumin in 800 µl 0.1 M sodium phosphate buffer with pH 7.5 plus 100 µl dioxane. The reaction mixture is kept at room temperature for 30 minutes and then poured onto a Sephadex G-25 column equilibrated with 0.1M sodium phosphate buffer of pH 7.5. The column is eluted with this buffer and two elution peaks containing methotrexate are obtained and the first one contains the methotrexate human serum albumin conjugate.

Eksempel 5.Example 5

Enzyminhibitor-immunoforsøg for humant serumaibumin.Enzyme inhibitor immunoassay for human serum albumin.

100 yl fortyndet serum inkuberes ved 30°C i 15 minutter sammen med 100 yl antihuman serumalbuminantistofopløsning (kanin), 100 yl NADPH-opløsning, 100 yl 2-mercaptoethanol-opløsning og 550 yl natriumphosphatpuffer med pH-værdi 7,5. Derpå tilsættes 100 yl methotrexat-humant serumalbumin-konjugat (8 yg/ml), og blandingen inkuberes i 15 minutter, hvorefter der tilsættes 100 yl dihydrofolatreduktaseopløsning. Blandingen inkuberes i yderligere 3 minutter, og enzymaktiviteten bestemmes ved tilsætning af 100 yl dihydrofolatopløsning og måling ved 340 nm med et Varian® spektrofoto-meter med skriver. Resultaterne er anført i tabel II.100 µl of diluted serum is incubated at 30 ° C for 15 minutes together with 100 µl of anti-human serum albumin antibody (rabbit), 100 µl of NADPH solution, 100 µl of 2-mercaptoethanol solution, and 550 µl of sodium phosphate buffer with pH 7.5. Then 100 µl methotrexate-human serum albumin conjugate (8 µg / ml) is added and the mixture is incubated for 15 minutes, then 100 µl dihydrofolate reductase solution is added. The mixture is incubated for a further 3 minutes and the enzyme activity is determined by adding 100 µl of dihydrofolate solution and measuring at 340 nm with a Varian® spectrophotometer with printer. The results are listed in Table II.

1515

Tabel II DK 152313 BTable II DK 152313 B

Reaktanter EnzymaktivitetReactants Enzyme activity

Humant serum- Antihumant Methotrexat- Enzym-for- OD/min Inhibering albumin (prø- serumalbu- -humant se- søgskompo- i % vekoncentra- min-anti- rumalbumin- nenter tion) stof -konjugat (i forsøg) 0 fraværende 0 til stede 0,123 0 0 fraværende 0,8yug til stede 0,068 45 0 til stede 0,8^ug til stede 0,105 16 S^ug/ml til stede 0,8^ug til stede 0,092 25 10^ug/ml til stede 0,8^,ug til stede 0,085 31Human Serum Antihuman Methotrexate Enzyme-for-OD / min Inhibition Albumin (Trial Serum Album-Humane Sequence Component in% Concentrate Anti-Rumum Albumin) Substance Conjugate (in Experiment) 0 absent 0 to present 0.123 0 0 absent 0.8yug present 0.068 45 0 present 0.8 ^ ug present 0.105 16 S ^ ug / ml present 0.8 ^ ug present 0.092 25 10 ^ ug / ml present 0.8 ^, ug present 0.085 31

Det fremgår af de ovennævnte resultater, at nogle fåyug humant serumalbumin kan bestemmes i det beskrevne system.It is apparent from the above results that some few human serum albumin can be determined in the system described.

Eksempel 6.Example 6

Syntese af methotrexat-aminoethylmorphin-konjugat.Synthesis of methotrexate-aminoethylmorphine conjugate.

3 a) Syntese af O -aminoethylmorphin.3 a) Synthesis of O -aminoethylmorphine.

400 mg lithiumaluminiumhydrid suspenderes under nitrogenatmosfære i 10 ml tetrahydrofuran, som er frisk destilleret fra lithiumaluminiumhydrid. I løbet af 5 minutter tilsættes en opløsning af 400 mg morphin og 400 mg chloracetonitril i 4 ml frisk destilleret tetrahydrofuran, hvorefter der tilbagesvales i 1 time. Blandingen lades afkøle, og der tilsættes 0,6 ml vand efterfulgt af 0,6 ml 10 vægtprocent natriumhydroxidopløsning og 2 ml vand. Blandingen filtreres, og saltene vaskes med tetrahydrofuran, tetrahydrofuranfraktionerne kombineres, tørres med magnesiumsulfat under nitrogen og 3 filtreres, og filtratet inddampes, hvorved fås 380 mg O -aminoethylmorphin .400 mg of lithium aluminum hydride is suspended under a nitrogen atmosphere in 10 ml of tetrahydrofuran freshly distilled from lithium aluminum hydride. Over 5 minutes, a solution of 400 mg of morphine and 400 mg of chloroacetonitrile in 4 ml of freshly distilled tetrahydrofuran is added and then refluxed for 1 hour. The mixture is allowed to cool and 0.6 ml of water is added followed by 0.6 ml of 10% by weight sodium hydroxide solution and 2 ml of water. The mixture is filtered and the salts are washed with tetrahydrofuran, the tetrahydrofuran fractions combined, dried with magnesium sulfate under nitrogen and filtered and the filtrate is evaporated to give 380 mg of O-aminoethylmorphine.

16 DK 152313B16 DK 152313B

3 b) Syntese af N-tert.butoxycarbonyl-γ-(O -aminoethylmorphin)glutamin-syre-a-benzylester.3 b) Synthesis of N-tert-butoxycarbonyl-γ- (O-aminoethylmorphine) glutamic acid α-benzyl ester.

33

En blanding af 50 mg O -aminoethylmorphin (fremstillet som ovenfor), 34 mg N-tert.butoxycarbonyl-glutaminsyre-a-benzylester og 25 mg dicyclohexylcarbodiimid i 5 ml dichlormethan omrøres i 4 timer ved stuetemperatur. Blandingen fortyndes med ethylacetat og vaskes med fortyndet natriumcarbonatopløsning. Ethylacetatopløsningen ekstraheres derpå to gange med 0,1M saltsyre, og de forenede sure ekstrakter behandles med tilstrækkeligt 10 vægtprocents natriumhydroxidopløsning til indstilling af pH-værdien til 8,0. Opløsningen ekstraheres to gange med ethylacetat, og de forenede ethylacetat-ekstrakter vaskes med natriumchloridopløsning, tørres over vandfri natriumsulfat og inddampes, hvorved fås 36 mg af en farveløs olie.A mixture of 50 mg of O-aminoethylmorphine (prepared as above), 34 mg of N-tert-butoxycarbonyl-glutamic acid α-benzyl ester and 25 mg of dicyclohexylcarbodiimide in 5 ml of dichloromethane is stirred for 4 hours at room temperature. The mixture is diluted with ethyl acetate and washed with dilute sodium carbonate solution. The ethyl acetate solution is then extracted twice with 0.1M hydrochloric acid and the combined acidic extracts are treated with a sufficient 10% by weight sodium hydroxide solution to adjust the pH to 8.0. The solution is extracted twice with ethyl acetate and the combined ethyl acetate extracts are washed with sodium chloride solution, dried over anhydrous sodium sulfate and evaporated to give 36 mg of a colorless oil.

c) Syntese af glutaminsyre-γ-(O -amidoethylmorphin)-a-benzylester-tri-fluoreddikesyresalt.c) Synthesis of glutamic acid γ- (0-amidoethylmorphine) -α-benzyl ester trifluoroacetic acid salt.

En opløsning af 36 mg N-tert.butoxycarbonyl-glutaminsyre-morphin-derivat (fremstillet som ovenfor) i 3 ml dichlormethan omrøres ved stuetemperatur, og der tilsættes 1 ml trifluoreddikesyre. Blandingen omrøres i 15 minutter og inddampes derpå til tørhed. Som remanens fås 40 mg af et farveløst glasagtigt materiale.A solution of 36 mg of N-tert-butoxycarbonyl-glutamic acid morphine derivative (prepared as above) in 3 ml of dichloromethane is stirred at room temperature and 1 ml of trifluoroacetic acid is added. The mixture is stirred for 15 minutes and then evaporated to dryness. As the residue, 40 mg of a colorless glassy material is obtained.

d) Syntese af methotrexat-γ-(0 -amidoethylmorphin)-a-benzylester.d) Synthesis of methotrexate-γ- (O-amidoethylmorphine) -α-benzyl ester.

En opløsning af 37 mg 4-amino-4-desoxy-N^-methylpteroinsyre i 3 ml dimethylsulfoxid behandles under omrøring ved stuetemperatur med 28^ul triethylamin og 25 ^ul isobutylchlorformiat, og blandingen omrøres i 30 minutter. Den sættes derpå til en blanding af 75 mg morphinderivat fremstillet som beskrevet under c) og 28^ul triethylamin i 2 ml dimethylsulfoxid, og blandingen omrøres ved 60°C i 1 time. Blandingen afkøles, fortyndes med vand og ekstraheres to gange med ethylacetat. De forenede ethylacetat-ekstrakter vaskes med vand og ekstraheres derpå to gange sammen med 0,1M saltsyre. De forenede sure ekstrakter behandles med tilstrækkeligt 10 vægtprocents natriumhydroxidopløsning til at hæve pH-værdienA solution of 37 mg of 4-amino-4-deoxy-N 2 -methylpteroic acid in 3 ml of dimethyl sulfoxide is treated with stirring at room temperature with 28 µl of triethylamine and 25 µl of isobutyl chloroformate and the mixture is stirred for 30 minutes. It is then added to a mixture of 75 mg of morphine derivative prepared as described under c) and 28 µl of triethylamine in 2 ml of dimethyl sulfoxide, and the mixture is stirred at 60 ° C for 1 hour. The mixture is cooled, diluted with water and extracted twice with ethyl acetate. The combined ethyl acetate extracts are washed with water and then extracted twice with 0.1M hydrochloric acid. The combined acidic extracts are treated with a sufficient 10% by weight sodium hydroxide solution to raise the pH

DK 152313BDK 152313B

til 8,0. Blandingen ekstraheres tre gange med ethylacetat, og de forenede ekstrakter vaskes med natriumchloridopløsning, tørres over vandig natriumsulfat og inddampes, hvorved fås 15 mg af en gul olie. Denne renses ved præparativ tyndtlagschromatografi på silicagel (fremkaldelse med chloroform/methanol (4:1)). Den ønskede forbindelse vindes i form af 4 mg gult fast stof.to 8.0. The mixture is extracted three times with ethyl acetate and the combined extracts are washed with sodium chloride solution, dried over aqueous sodium sulfate and evaporated to give 15 mg of a yellow oil. This is purified by preparative thin layer chromatography on silica gel (developing with chloroform / methanol (4: 1)). The desired compound is obtained in the form of 4 mg of yellow solid.

3 e) Syntese af methotrexat-γ-(0 -amidoethylmorphin).E) Synthesis of methotrexate-γ- (0-amidoethylmorphine).

Produktet fremstillet som beskrevet under d), 4 mg, blandes med 5 ml 0,1M natriumhydroxidopløsning, og blandingen omrøres ved stuetemperatur i 8 timer, hvilket resulterer i en klar gul opløsning.The product prepared as described under d), 4 mg, is mixed with 5 ml of 0.1M sodium hydroxide solution and the mixture is stirred at room temperature for 8 hours, resulting in a clear yellow solution.

Til denne sættes 5 ml 0,1M saltsyre, og blandingen fortyndes til 25 ml med 0,05M natriumorthophosphatpuffer med pH-værdi 7,4, og der fås en til enzymforsøget egnet opløsning af den ønskede forbindelse.To this is added 5 ml of 0.1M hydrochloric acid and the mixture is diluted to 25 ml with 0.05M sodium orthophosphate buffer pH 7.4 and a solution of the desired compound suitable for the enzyme experiment is obtained.

Eksempel 7.Example 7

Enzyminhibitor-immunoforsøg for morphin.Enzyme inhibitor immunoassay for morphine.

En blanding af 10 μΙ morphinopløsning med passende koncentration, 50 μΙ methotrexat-y-(0 -amidoethylmorphin)-opløsning (4 ng/25 ml), 50 yl anti-morphin-antistofopløsning, 10 μΙ NADPH-opløsning, 10 μΙ 2-mercaptoethan-olopløsning, 50 μΙ kaliumchloridopløsning, 150 μΙ tris-saltsyrepufferopløsning (pH-værdi 7,5) indeholdende EDTA og 10 μΙ dihydrofolatreduktaseopløsning (E. casei) inkuberes i 20 minutter ved 37°C. Blandingens enzymaktivitet bestemmes efter tilsætning af 10 μΙ dihydrofolatopløsning ved at måle ændringen i opløsningens ekstinktion ved 340 nm under anvendelse af et Centrifichem® centrifugal-analyseapparat. Resultaterne er anført i nedenstående tabel III.A mixture of 10 μΙ morphine solution of appropriate concentration, 50 μΙ methotrexate-γ (0-amidoethylmorphine) solution (4 ng / 25 ml), 50 μl anti-morphine antibody solution, 10 μΙ NADPH solution, 10 μΙ 2-mercaptoethane -ol solution, 50 μΙ potassium chloride solution, 150 μΙ tris-hydrochloric acid buffer solution (pH 7.5) containing EDTA and 10 μΙ dihydrofolate reductase solution (E. casei) are incubated for 20 minutes at 37 ° C. The enzyme activity of the mixture is determined after addition of 10 μΙ dihydrofolate solution by measuring the change in extinction of the solution at 340 nm using a Centrifichem® centrifugal analyzer. The results are given in Table III below.

18 DK 152313B18 DK 152313B

Tabel XIITable XII

Reaktanter EnzymaktivitetReactants Enzyme activity

Morphin Antimorphin- Morphin- Enzym-for- OD/min Inhibering ,ug/forsøg -antistof -methotre- søgskompo- i % 7 xat-konju- nenter gat 0 fraværende fraværende til stede 0,54 0 —8 0 fraværende 3 x 10 M til stede 0,14 76 0 til stede 3 x 10 ^ M til stede 0,41 29 0,04 til stede 3 x 10 ® M til stede 0,37 36 0,40 til stede 3 x 10 ^ M til stede 0,35 40 4,0 til stede 3 x 10 ^ M til stede 0,31 46 20 til stede 3 x 10 ® M til stede 0,27 54 40 til stede 3 x 10 ® M til stede 0,23 60Morphine Antimorphine- Morphine-Enzyme-for-OD / min Inhibition, µg / test antibody -methotreatment compo- in% 7 xate conjugates hole 0 absent absent present 0.54 0 -8 0 absent 3 x 10 M present 0.14 76 0 present 3 x 10 ^ M present 0.41 29 0.04 present 3 x 10 ® M present 0.37 36 0.40 present 3 x 10 ^ M present 0, 35 40 4.0 present 3 x 10 ^ M present 0.31 46 20 present 3 x 10 ® M present 0.27 54 40 present 3 x 10 ® M present 0.23 60

Af ovenstående tabel fremgår det, at med voksende morphinkoncentra-tion er der en reduktion i dihydrofolatreduktaseaktiviteten. På denne måde kan morphinopløsninger indeholdende fra 4^ug pr. ml til 4 mg pr. ml morphin undersøges, blot der er lO^ul af opløsningen. Dette skal imidlertid ikke indicere, at dette· er det krævede område, men snarere blot, at det kan anvendes i praksis.From the above table it can be seen that with increasing morphine concentration there is a reduction in dihydrofolate reductase activity. In this way, morphine solutions containing from 4 µg per ml to 4 mg per ml. ml of morphine is examined, provided there is 10 µl of the solution. However, this should not indicate that this is the required area, but rather that it can be used in practice.

Eksempel 8.Example 8.

Syntese af ferritin-methotrexat-konjugat.Synthesis of ferritin-methotrexate conjugate.

En opløsning af 1,3 mg humant leverferritin i 5 ml 0,05M natrium-phosphatpuffer med pH-værdi 8,0 og 1 ml dimethylformamid omrøres ved stuetemperatur, og der tilsættes lOO^ul af en opløsning fremstillet ved at behandle 23 mg methotrexat i 2 ml dimethylformamid med 21^ul triethylamin og 15 ^ul isobutylchlor-formiat ved stuetemperatur i 30 minutter. Blandingen omrøres vedA solution of 1.3 mg of human liver ferritin in 5 ml of 0.05 M sodium phosphate buffer pH 8.0 and 1 ml of dimethylformamide is stirred at room temperature and 100 µl of a solution prepared by treating 23 mg of methotrexate in 2 ml of dimethylformamide with 21 µl of triethylamine and 15 µl of isobutyl chloroformate at room temperature for 30 minutes. The mixture is stirred

Claims (1)

19 DK 152313B stuetemperatur i 2 timer og dialyseres derpå mod 2x2 liter 0,05M natriumorthophosphatpuffer (med pH-værdi 7,4, indeholdende 0,1M natriumchlorid og 0,05 vægtprocent natriumazid) i 24 timer. Opløsningen lades derpå løbe gennem en kolonne af Sephadex G-25, som er ækvilibreret med samme puffer, og de ferritinholdige fraktioner forenes og fyldes op til 10 ml med pufferen. Den resulterende opløsning er egnet til anvendelse i et enzymimmunoforsøg til bestemmelse af ferritin. Reagens til bestemmelse af et immunologisk aktivt materiale, hvilket reagens består af en receptor, som specifikt kan binde det immunologisk aktive materiale, et enzym, et enzymsubstrat og det immunologisk aktive materiale, der er mærket med en forbindelse, som er i stand til at modificere aktiviteten af enzymet, kendetegnet ved, at det mærkede immunologiske materiale er et antigen-enzyminhibitor- eller antigen-enzymaktivator-complex.At room temperature for 2 hours, then dialyzed against 2x2 liters of 0.05M sodium orthophosphate buffer (pH 7.4 containing 0.1M sodium chloride and 0.05% by weight sodium azide) for 24 hours. The solution is then passed through a column of Sephadex G-25 which is equilibrated with the same buffer and the ferritin-containing fractions are combined and filled up to 10 ml with the buffer. The resulting solution is suitable for use in an enzyme immunoassay to determine ferritin. Reagent for determination of an immunologically active material, which reagent consists of a receptor that can specifically bind the immunologically active material, an enzyme, enzyme substrate and the immunologically active material labeled with a compound capable of modifying the activity of the enzyme, characterized in that the labeled immunological material is an antigen-enzyme inhibitor or antigen-enzyme activator complex.
DK114778A 1977-03-15 1978-03-14 REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL DK152313C (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB10859/77A GB1595101A (en) 1977-03-15 1977-03-15 Enzyme modifier immunoassay
GB1085977 1977-03-15

Publications (3)

Publication Number Publication Date
DK114778A DK114778A (en) 1978-09-16
DK152313B true DK152313B (en) 1988-02-15
DK152313C DK152313C (en) 1988-09-26

Family

ID=9975639

Family Applications (1)

Application Number Title Priority Date Filing Date
DK114778A DK152313C (en) 1977-03-15 1978-03-14 REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL

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)

* Cited by examiner, † Cited by third party
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
WO1993017707A1 (en) * 1992-03-04 1993-09-16 Akzo N.V. In vivo binding pair pretargeting
US5965106A (en) * 1992-03-04 1999-10-12 Perimmune Holdings, Inc. In vivo binding pair pretargeting
AU751938B2 (en) * 1998-05-15 2002-08-29 Sekisui Chemical Co., Ltd. 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
JP2010518135A (en) * 2007-02-16 2010-05-27 ケイテーベー ツモルフォルシュングスゲゼルシャフト ミット ベシュレンクテル ハフツング Dual action prodrug

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2518511A1 (en) * 1975-04-15 1976-11-04 Charters John Dumergue FASTENING ELEMENT

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2518511A1 (en) * 1975-04-15 1976-11-04 Charters John Dumergue FASTENING ELEMENT

Also Published As

Publication number Publication date
NO154814B (en) 1986-09-15
AT367203B (en) 1982-06-11
FR2384262A1 (en) 1978-10-13
JPS53115814A (en) 1978-10-09
DK152313C (en) 1988-09-26
ES467831A1 (en) 1979-09-01
NO780902L (en) 1978-09-18
CH641570A5 (en) 1984-02-29
NO152955B (en) 1985-09-09
SE447026B (en) 1986-10-20
NO152955C (en) 1985-12-18
ATA182178A (en) 1981-10-15
NO154814C (en) 1986-12-29
AU3396578A (en) 1979-09-13
GB1595101A (en) 1981-08-05
DK114778A (en) 1978-09-16
DE2811257A1 (en) 1978-09-21
ES475984A1 (en) 1979-06-16
NO844764L (en) 1978-09-18
BE864856A (en) 1978-09-14
NL7802845A (en) 1978-09-19
CA1102789A (en) 1981-06-09
SE7802923L (en) 1978-09-16
IT1158665B (en) 1987-02-25
IT7821256A0 (en) 1978-03-15
IL54234A0 (en) 1978-06-15
FR2384262B1 (en) 1983-04-08
AU519326B2 (en) 1981-11-26

Similar Documents

Publication Publication Date Title
DK152313B (en) REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL
US4235960A (en) Competitive enzyme-linked immunoassay
US4476229A (en) Substituted carboxyfluoresceins
US5538901A (en) Nucleophilic polysubstituted aryl acridinium ester conjugates uses thereof
CA2111467C (en) Reagents and methods for the quantification of total doxepins in biological fluids
JPS6240662B2 (en)
JP7398366B2 (en) Detection of symmetrical dimethylarginine
CA1199576A (en) Chromogenic tracers for use in an assay
Kabakoff Chemical aspects of enzyme-immunoassay
JPH06508214A (en) Methods and reagents for performing ion capture digoxin assays
JPH04224583A (en) Hapten-biotin complex and immunoassay
JPH08508301A (en) Photoactivatable biotin derivative and its use to reduce interference in immunoassays
JPH06313768A (en) Composition for free ligand assay
JPH06508211A (en) Reagents and methods for performing two-step ion capture binding assays
US4801726A (en) Repetitive hit-and-run immunoassay and stable support-analyte conjugates; applied to T-2 toxin
US5780243A (en) Methods for the quantitative analysis of organic compounds
US5227311A (en) Intrinsic factor to determine B12
JP3360174B2 (en) A homobifunctional reagent that binds enzymes, etc., to antibodies, etc.
US5939332A (en) Phencyclidine analogs for immunoassay
JP2682716B2 (en) Gem-diphenyl derivative and use for immunoassay
JP2023521886A (en) Fluorescence quenching immunoassay
JP3257793B2 (en) Reagents and methods for the determination of amitriptyline or nortriptyline in body fluids
AU661013B2 (en) ddI immunoassays, derivatives, conjugates and antibodies
JP3828891B2 (en) Water-soluble derivatives of lipophilic drugs
CA1272193A (en) Divalent hapten derivatives, process for preparing and use of them

Legal Events

Date Code Title Description
PBP Patent lapsed